OA20296A - Optimized strategy for exon skipping modifications using CRISPR/CAS9 with triple guide sequences. - Google Patents

Abstract

CRISPR/Cas9-mediated genome editing holds clinical potential for treating genetic diseases, such as Duchenne muscular dystrophy (DMD), which is caused by mutations in the dystrophin gene. Here, using three promoters to drive expression of the same DMD guide RNA, a more robust and safe form of genome editing was achieved in a humanized mouse model for DMD with a deletion in exon 50, and in a ΔEx50-MD Dog.

Description

DESCRIPTION

OPTIMIZED STRATEGY FOR EXON SKIPPING MODIFICATIONS USING CRISPR/CAS9 WITH TRIPLE GUIDE SEQUENCES

PRIORITY CLAIM

The présent application daims benefit of priority to U.S. Provisional Application Serial No. 62/596,298, filed December 8, 2017, U.S. Provisional Application Serial No. 62/544,499, filed Aug. 11, 2017, and U.S. Provisional Application Serial No. 62/442,606, filed January 5, 2017, the entire contents of which are hereby incorporated by reference in 10 their entireties.

FEDERAL FUNDING SUPPORT CLAUSE

This invention was made with government support under grant no. U54 HD 087351 awarded by National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 5, 2018, is named UTFD_P3178WO.txt and is 1,316,974 bytes in size.

FIELD OF THE DISCLOSURE

The présent disclosure relates to the fields of molecular biology, medicine and genetics. More particularly, the disclosure relates to compositions and uses thereof for genome editing to correct mutations in vivo using an exon-skipping approach.

BACKGROUND

Muscular dystrophies (MD) are a group of more than 30 genetic diseases characterized by progressive weakness and degeneration of the skeletal muscles that control movement. Duchenne muscular dystrophy (DMD) is one of the most severe forms of MD that affects approximately I in 5000 boys and is characterized by progressive muscle weakness and 30 prématuré death. Cardiomyopathy and heart failure are common, incurable and léthal features of DMD. The disease is caused by mutations in the gene encoding dystrophin (DMD), a large intracellular protein that links the dystroglycan complex at the cell surface with the underlying cytoskeleton, thereby maintaining integrity of the muscle cell membrane during contraction. Mutations in the dystrophin gene resuit in loss of expression of dystrophin, causing muscle membrane fragility and progressive muscle wasting.

SUMMARY

Despite intense efforts to find cures through a variety of approaches, including myoblast transfer, viral delivery, and oligonucleotide-mediated exon skipping, there remains no cure for any type of muscular dystrophy. The présent inventors recently used clustered regularly interspaced short palindromie repeat/Cas9 (CRISPR/Cas9)-mediated genome editing to correct the dystrophin gene (DMD) mutation in postnatal mdx mice, a mode! for DMD. In vivo AAVmediated delivery of gene-editing components successfully removed the mutant genomic sequence by exon skipping in the cardiac and skeletal muscle cells ofmdx mice. Using different modes of AAV9 delivery, the inventors restored dystrophin protein expression in cardiac and skeletal muscle of mdx mice. The mdx mouse model and the correction exon 23 using AAV delivery of myoediting machinery has been useful to show proof-of concept of exon skipping approach using several cuts in the genomic région encompassing the mutation in vivo. However, more efficient and safe approaches to genome editing and DMD would provide a more powerful means to intervene in this disease.

In some embodiments, a nucleic acid comprises a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a sequence encoding a first promoter wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and a sequence encoding a second promoter wherein the first promoter drives expression of the sequence encoding the second DMD guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a dystrophin splice accepter site. In some embodiments, the sequence encoding the first promoter and the sequence encoding the second promoter are identical. In some embodiments, the sequence encoding the first promoter and the sequence encoding the second promoter not identical. In some embodiments, the sequence encoding the first promoter and the sequence encoding the second promoter share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity. In some embodiments, the first genomic target sequence and the second genomic target sequence are identical. In some embodiments, the first genomic target sequence and the second genomic target sequence are not identical. In some embodiments, the first genomic target sequence and the second genomic target sequence share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity. In some embodiments, the first genomic target sequence and the second genomic target sequence are complementary. In some embodiments, the nucleic acid further comprises a sequence encoding a third DMD guide RNA targeting a third genomic target sequence, and a sequence encoding a third promoter wherein the third promoter drives expression of the sequence encoding the third DMD guide RNA, and wherein the third genomic target sequence comprises a dystrophin splice acceptor site. In some embodiments, at least two of the sequences encoding the first promoter, the sequence encoding the second promoter, and the sequence encoding the third promoter are identical. In some embodiments, at least two of the sequence encoding the first promoter, the sequence encoding the second promoter, and the sequence encoding the third promoter are not identical. In some embodiments, at least two of the sequences encoding the first promoter, the sequence encoding the second promoter, and the sequence encoding the third promoter share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, and the third genomic target sequence are identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, and the third genomic target sequence are not identical. In some embodiments, at least two of the the first genomic target sequence, the second genomic target sequence, and the third genomic target sequence share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, and the third genomic target sequence are complementary. In some embodiments, the nucleic acid further comprises a sequence encoding a fourth DMD guide RNA targeting a fourth genomic target sequence, and a sequence encoding a fourth promoter, wherein the fourth promoter drives expression of the fourth sequence encoding a DMD guide RNA, wherein the fourth genomic target sequence comprises a dystrophin splice acceptor site. In some embodiments, at least two of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, and the sequence encoding the fourth promoter are identical. In some embodiments, at least two of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, and the sequence encoding the fourth promoter are not identical. In some embodiments, at least two of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, and the sequence encoding the fourth promoter share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, and the fourth genomic target 5 sequence are identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, and the fourth genomic target sequence are not identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, and the fourth genomic target sequence share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence 10 identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, and the fourth genomic target sequence are complementary. In some embodiments, the nucleic acid further comprises a sequence encoding a fifth DMD guide RNA targeting a fifth genomic target sequence, and a sequence encoding a fifth promoter, wherein the fifth promoter drives expression of the 15 sequence encoding the fifth DMD guide RNA, wherein the fifth genomic target sequence comprises a dystrophin splice acceptor site. In some embodiments, at least two of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter are identical. In some embodiments, at least two of the sequence 20 encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter are not identical. In some embodiments, at least two of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the 25 sequence encoding the fifth promoter share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, the fourth genomic target sequence, and the fifth genomic target sequence are identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic 30 target sequence, the fourth genomic target sequence, and the fifth genomic target sequence are not identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, the fourth genomic target sequence, and the fifth genomic target sequence share at least 50%, 60%, 70%, 80%, 90%, or

95% sequence identity. In some embodiments, at least two ofthe first genomic target sequence, the second genomic target sequence, the third genomic target sequence, the fourth genomic target sequence, and the fifth genomic target sequence are complementary. In some embodiments, the nucleic acid further comprises at least one sequence encoding an additional DMD guide RNA targeting a genomic target sequence, and at least one additional promoter, wherein the additional promoter drives expression of the sequence encoding the additional DMD guide RNA, wherein the additional genomic target sequence comprises a dystrophin splice acceptor site. In some embodiments, the dystrophin splice acceptor site comprises the 5’ splice acceptor site of exon 51. In some embodiments, the sequence encoding the first promoter or the sequence encoding the second promoter comprises a sequence encoding a constitutive promoter. In some embodiments, the first promoter or the second promoter comprises a constitutive promoter. In some embodiments, at least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter comprises a sequence encoding a constitutive promoter. Jn some embodiments, at least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises a constitutive promoter. In some embodiments, the sequence encoding the first promoter or the sequence encoding the second promoter comprises a sequence encoding an inducible promoter. In some embodiments, at least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises an inducible promoter. In some embodiments, the sequence encoding the first promoter or the sequence encoding the second promoter comprises a sequence encoding a cell-type spécifie promoter. In some embodiments, at least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter comprises a cell-type spécifie promoter. In some embodiments, the cell type spécifie promoter comprises a muscle-specific promoter. In some embodiments, the sequence encoding the first promoter or the sequence encoding the second promoter comprises a sequence encoding a U6 promoter, an H1 promoter, or a 7SK promoter. In some embodiments, at least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter comprises a U6 promoter, an H1 promoter, or a 7SK. promoter. In some embodiments, at least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter comprises a U6 promoter. In some embodiments, at least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter comprises an H1 promoter. In some embodiments, at least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter comprises a 7SK promoter. In some embodiments, the sequence encoding the first DMD guide RNA, the sequence encoding the second DMD guide RNA, and sequence encoding the third DMD guide RNA are identical, and the 5’ splice acceptor site comprises a 5’ splice accepter site of exon 51. In some embodiments, the sequence encoding the first promoter comprises a sequence encoding a U6 promoter, the sequence encoding the second promoter comprises a sequence encoding an H1 promoter, and the sequence encoding the third promoter comprises a 7SK promoter. In some embodiments, the nucleic acid comprises a DNA sequence. In some embodiments, the nucleic acid comprises an RNA sequence. In some embodiments, the nucleic acid further comprises one or more sequences encoding an inverted terminal repeat (ITR). In some embodiments, the nucleic acid further comprises a sequence encoding a 5’ inverted terminal repeat (ITR) and a sequence encoding a 3’ ITR. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated virus (AAV). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated virus (AAV) of serotype 2 (AAV2). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) and the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an AAV2. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated virus (AAV) of serotype 4 (AAV4). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) and the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an AAV4. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises or consists of 145 nucléotides. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises or consists of 115 nucléotides. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises or consists of 141 nucléotides. In some embodiments, the nucleic acid further comprises a polyadenosine (poly A) sequence. In some embodiments, the poly A sequence is a mini poly A sequence. In some embodiments, the sequence encoding the first DMD guide RNA or the sequence encoding the second DMD guide RNA comprises the sequence of any one of SEQ ID NOs. 60-382, 706-708 and 712-789. In some embodiments, the sequence encoding the first DMD guide RNA or the sequence encoding the second DMD guide RNA comprises the sequence of SEQ ID NO. 714.

Also provided is a vector comprising a nucleic acid comprising a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a sequence encoding a first promoter wherein the first promoter drives expression ofthe sequence encoding the first DMD guide RNA, and a sequence encoding a second promoter wherein the first promoter drives expression of the sequence encoding the second DMD guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a dystrophin splice accepter site. In some embodiments, the vector further comprises a sequence encoding an inverted terminal repeat of a transposable element. In some embodiments, the transposable element is a transposon. In some embodiments, the transposon is a Tn7 transposon. In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a plasmid. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector. In some embodiments, the AAV vector is replication-defective or conditionally réplication defective. In some embodiments, the AAV vector is a recombinant AAV vector. In some embodiments, the AAV vector comprises a sequence isolated orderived from an AAV vector of serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6), 7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV 10), 11 (AAV11) or any combination thereof. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 9 (AAV9). In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 2 (AAV2). In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV2 and a sequence isolated or derived from an AAV9. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV4 and a sequence isolated or derived from an AAV9. In some embodiments, the vector is optimized for expression in mammalian cens. In some embodiments, the vector is optimized for expression in human cells. In some embodiments, the vector comprises the sequence of SEQ ID NO. 914, SEQ ID NO. 915, SEQ ID NO. 916, or SEQ ID NO. 917.

Also provided is a nucleic acid comprising a sequence encoding a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the sequence encoding the promoter comprises a sequence encoding a muscle-specific promoter. In some embodiments, the sequence encoding the muscle-specific promoter comprises a sequence encoding a CK8 promoter. In some embodiments, the sequence encoding the muscle-specific promoter comprises a sequence encoding a CK8e promoter. In some embodiments, the sequence encoding the Cas9 or the nuclease domain thereof is isolated or derived from a sequence encoding an S. pyogenes Cas9 or a nuclease domain thereof. In some embodiments, the sequence encoding the Cas9 or the nuclease domain thereof is isolated or derived from a sequence encoding S. aureus Cas9 or a nuclease domain thereof. In some embodiments, the sequence encoding the Cas9 or the nuclease domain thereof is codon optimized for expression in a mammal. In some embodiments, the sequence encoding the Cas9 or the nuclease domain thereof is codon optimized for expression in a human. In some embodiments, the nucleic acid further comprises a polyA sequence. In some embodiments, the polyA sequence is a mini polyA sequence. In some embodiments, the nucleic acid further comprises one or more sequences encoding an inverted terminal repeat (ITR). In some embodiments, the nucleic acid further comprises a sequence encoding a 5’ inverted terminal repeat (ITR) and a sequence encoding a 3’ ITR. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated virus (AAV). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated virus (AAV) of serotype 2 (AAV2). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) and the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an AAV2. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated virus (AAV) of serotype 4 (AAV4). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) and the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an AAV4. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises or consists of 145 nucléotides, 1 15 nucléotides, or 141 nucléotides. In some embodiments, the nucleic acid further comprises a nuclear localization signal. In some embodiments, the nucleic acid is optimized for expression in mammalian cells. In some embodiments, the nucleic acid is optimized for expression in human cells.

Also provided is a vector comprising a nucleic acid comprising a sequence encoding a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the sequence encoding the promoter comprises a sequence encoding a muscle-specific promoter such as the CK8 or CK8e promoter. In some embodiments, the vector further comprises a sequence encoding an inverted terminal repeat (ITR) of a transposable element. In some embodiments, the transposable element is a transposon. In some embodiments, the transposon is a Tn7 transposon. In some embodiments, the vector further comprises a sequence encoding a 5’ ITR of a T7 transposon and a sequence encoding a 3’ ITR of a T7 transposon. In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a plasmid. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector. In some embodiments, the AAV vector is replication-defective or conditionally réplication defective. In some embodiments, the AAV vector is a recombinant AAV vector. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6), 7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV 10), 11 (AAV11) or any combination thereof. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 9 (AAV9). In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 2 (AAV2). In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV2 and a sequence isolated or derived from an AAV9. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 4 (AAV4). In some embodiments, the AAV vector comprises a sequence isolated or derived , from an AAV4 and a sequence isolated or derived from an AAV9. In some embodiments, wherein the vector is optimized for expression in mammalian cells. In some embodiments, the vector is optimized for expression in human cells. In some embodiments, the vector comprises the nucleic acid sequence of SEQ ID NO. 899, SEQ ID NO. 900, SEQ ID NO. 901, or SEQ ID NO. 902.

Also provided is a cell comprising one or more nucleic acids of the disclosure. In some embodiments, the cell is a human cell. In some embodiments, the cell is a muscle cell or satellite cell. In some embodiments, the cell is an induced pluripotent stem (iPS) cell.

Also provided is a composition comprising one or more nucleic acids of the disclosure. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

Also provided is a composition comprising a cell comprising one or more nucleic acids of the disclosure. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

Also provided is a composition comprising one or more vectors of the disclosure. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

Also provided is a cell comprising a composition comprising one or more vectors of the disclosure. In some embodiments, the cell is a human cell. In some embodiments, the cell is a muscle cell or satellite cell. In some embodiments, the cell is an induced pluripotent stem (iPS) cell.

In some embodiments, a composition comprises (i) a first nucleic acid sequence comprising a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a sequence encoding a first promoter wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and a sequence encoding a second promoter wherein the first promoter drives expression of the sequence encoding the second DMD guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a dystrophin splice acceptor and (ii) a second nucleic acid sequence comprising a sequence encoding a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the sequence encoding the promoter comprises a sequence encoding a muscle-specific promoter such as the CK8 or CK8e promoter. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

In some embodiments, a composition comprises (i) a first vector comprising a nucleic acid sequence comprising a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a sequence encoding a first promoter wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and a sequence encoding a second promoter wherein the first promoter drives expression of the sequence encoding the second DMD guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a dystrophin splice accepter and (ii) a second vector comprising a nucleic acid sequence comprising a sequence encoding a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the sequence encoding the promoter comprises a sequence encoding a muscle-specific promoter such as the CK8 or CK8e promoter. In some embodiments, at least one of the first vector and the second vectors are AAVs. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

Also provided is a method for correcting a dystrophin defect, the method comprising contacting a cell with one or more compositions of the disclosure under conditions suitable for expression of the first DMD guide RNA, the second DMD guide RNA and the Cas9 protein or a nuclease domain thereof, wherein at least one of first DMD guide RNA or the second DMD guide RNA forms a complex with the Cas9 protein or the nuclease domain thereof to form at least one DMD guide RNA-Cas9 complex, wherein the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and induces sélective skipping of a DMD exon.

Also provided is a method for correcting a dystrophin defect, the method comprising contacting a cell with one or more compositions of the disclosure under conditions suitable for expression of the first DMD guide RNA, the second DMD guide RNA and the Cas9 protein or a nuclease domain thereof, wherein at least one of first DMD guide RNA or the second DMD guide RNA forms a complex with the Cas9 protein or the nuclease domain thereof to form at least one DMD guide RNA-Cas9 complex, wherein the at least one DMD guide RNA-Cas9 complex induces a reframing of a dystrophin reading frame. In some embodiments, the refraining of a dystrophin reading frame induces an insertion. In some embodiments, the insertion comprises or consists of a single adenosine nucléotide.

Also provided is a method for inducing sélective skipping of a DMD exon, the method comprising contacting a cell with one or more compositions of the disclosure under conditions suitable for expression of the first DMD guide RNA, the second DMD guide RNA and the Cas9 protein or a nuclease domain thereof, wherein at least one of first DMD guide RNA or the second DMD guide RNA forms a complex with the Cas9 protein or the nuclease domain thereof to form at least one DMD guide RNA-Cas9 complex, wherein the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and induces sélective skipping of a DMD exon.

Also provided is a method for inducing a reframing event in the dystrophin reading frame, the method comprising contacting a cell with one or more compositions of the disclosure under conditions suitable for expression of the first DMD guide RNA, the second DMD guide RNA and the Cas9 protein or a nuclease domain thereof, wherein at least one of first DMD guide RNA or the second DMD guide RNA forms a complex with the Cas9 protein or the nuclease domain thereof to form at least one DMD guide RNA-Cas9 complex, wherein the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and induces sélective skipping of a DMD exon. In some embodiments, the at least one DMD guide RNACas9 complex disrupts a dystrophin splice site and induces sélective skipping of exon 51 of a human DMD gene.

Also provided is a method of treating muscular dystrophy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of one or more compositions of the disclosure. In some embodiments, the composition is administered locally. In some embodiments, the composition is administered directly to a muscle tissue. In some embodiments, the composition is administered by an intramuscular infusion or injection. In some embodiments, the muscle tissue comprises a tibialis anterior tissue, a quadriceps tissue, a soleus tissue, a diaphragm tissue, or a heart tissue. In some embodiments, the composition is administered by an intra-cardiac injection. In some embodiments, the composition is administered systemically. In some embodiments, the composition is administered by an intravenous infusion or injection. In some embodiments, following administration of the composition, the subject exhibits normal dystrophin-positive myofibers, and mosaic dystrophin-positive myofibers containing centralized nuclei, or a combination thereof. In some embodiments, following administration of the composition, the subject exhibits an emergence or an increase in a level of abundance of normal dystrophin-positive myofibers when compared to an absence or a level of abundance of normal dystrophin-positive myofibers prior to administration of the composition. In some embodiments, following administration of the composition, the subject exhibits an emergence or an increase in a level of abundance of mosaic dystrophin-positive myofibers containing centralized nuclei when compared to an absence or an level of abundance of mosaic dystrophin-positive myofibers containing centralized nuclei prior to administration of the composition. In some embodiments, following administration of the composition, the subject exhibits a decreased sérum CK level when compared to a sérum CK level prior to administration of the composition. In some embodiment, following administration of the composition, the subject exhibits improved grip strength when compared to a gnp strength prior to administration of the composition. In some embodiments, the subject is a neonate, an infant, a child, a young adult, or an adult. In some embodiments, the subject has muscular dystrophy. In some embodiments, the subject is a genetic carrier for muscular dystrophy. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject appears to be asymptomatic and wherein a genetic diagnosis reveals a mutation in one or both copies of a DMD gene that impairs function of the DMD gene product. In some embodiments, the subject présents an early sign or symptom of muscular dystrophy. In some embodiments, the early sign or symptom of muscular dystrophy comprises loss of muscle mass or proximal muscle weakness. In some embodiments, the loss of muscle mass or proximal muscle weakness occurs in one or both leg(s) and/or a pelvis, followed by one or more upper body muscle(s). In some embodiments, the early sign or symptom of muscular dystrophy further comprises pseudohypertrophy, low endurance, difficulty standing, difficulty walking, difficulty ascending a staircase or a combination thereof. In some embodiments, the subject présents a progressive sign or symptom of muscular dystrophy. In some embodiments, the progressive sign or symptom of muscular dystrophy comprises muscle tissue wasting, replacement of muscle tissue with fat, or replacement of muscle tissue with fibrotic tissue. In some embodiments, the subject présents a later sign or symptom of muscular dystrophy. In some embodiments, the later sign or symptom of muscular dystrophy comprises abnormal bone development, curvature of the spine, loss of movement, and paralysis. In some embodiments, the subject présents a neurological sign or symptom of muscular dystrophy. In some embodiments, the neurological sign or symptom of muscular dystrophy comprises intellectual impairment and paralysis. In some embodiments, administration of the composition occurs prior to the subject presenting one or more progressive, later or neurological signs or symptoms of muscular dystrophy. In some embodiments, the subject is less than 10 years old, less than 5 years old, or less than 2 years old.

Also provided is the use of a therapeutically-effective amount of one or more compositions of the disclosure for treating muscular dystrophy in a subject in need thereof.

As used herein the spécification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a définition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that a value includes the inhérent variation of error for the device, for the method being employed to détermine the value, or that exists among the study subjects. Such an inhérent variation may be a variation of ±10% of the stated value.

Throughout this application, nucléotide sequences are listed in the 5’ to 3’ direction, 10 and amino acid sequences are listed in the N-terminal to C-terminal direction, unless indicated otherwise.

Other objects, features and advantages of the présent invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the spécifie examples, while indicating preferred embodiments of the 15 invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

The following drawings form part of the présent spécification and are included to further demonstrate certain aspects of the présent disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of spécifie embodiments presented herein.

FIGS. 1A-F. “Humanized”-AEx50 mouse model. (FIG. IA) Strategy showing CRlSPR/Cas9-mediated genome editing approach to generate a humanized mouse model. (FIG. IB) RT-PCR analysis to validate délétion of exon 50 (ΔΕχ50). (FIG. IC) Sequence of RT-PCR product to validate exon 50 délétion and génération of an out-of-frame sequence (nucleic acid sequence = SEQ ID NO: 1; amino acid sequence = SEQ 1D NO: 2). (FIG. 1E) Western blot analysis of dystrophin and vinculin expression in tibialis anterior and heart tissues. (FIG. 1F) Levels of sérum CK, a marker of muscle dystrophy that reflects muscle damage and membrane leakage were measured in wildtype (WT), ΔΕχ50 and mdx mice. (FIG. 1D) Histochemistry of cardiac and skeletal muscle by hematoxylin and eosin (H&E) staining, and immunohistochemistry using dystrophin antibody. Scale bar: 50 pm.

FIG. 2A-B. Exon 51 skipping. (FIG. 2A) RT-PCR of RNA from ΔΕχ50 mice 3 weeks after intramuscular injection indicates délétion of exon 51 (termed ΔΕχ50-51, lower band). (FIG. 2B) Sequence of the RT-PCR products of ΔΕχ50-51 band confirmed that exon 49 spliced directly to exon 52, excluding exon 51 (nucleic acid sequence = SEQ ID NO: 3; amino acid sequence = SEQ ID NO: 4).

FIG. 3A-G. Correction of dystrophin expression using triplicate gRNA strategy 3 weeks after intra-muscular injection. (FIG. 3A) Strategy showing CRISPR/Cas9-mediated genome editing approach to correct the reading frame in ΔΕχ50 mouse model. (FIG. 3B) sgRNA targeting the splice acceptor site (sgRNA-ex51-SA) sequence and schematic illustration of sgRNA binding position. Fig. 3B discloses SEQ ID NOS 954-957, respectively, in order of appearance. (FIG. 3C) Schematic illustration of AAV injection plasmids and strategy. Double guide strategy using rAAV9-sgRNA plasmid for sgRNA-ex51-SA and sgRNA-ex51-SD. Triplicate using rAAV9-sgRNA plasmid containing 3 copies of sgRNA20296 ¹⁶ ex51-SA. Muscle creatine kinase 8 (CK8) promoter to express SpCas9. U6, H1 and 7SK promoter for RNA polymerase III to express sgRNA. (FIG. 3D) Dystrophin immunohistochemistry staining of tibialis anterior muscle. (FIG. 3E) Quantification of dystrophin positive fibers normalized by area. (FIG. 3F) Western blot analysis of dystrophin 5 (DMD) and vinculin (VCL) expression 3 weeks after intramuscular injection. (FIG. 3G) Quantification of dystrophin expression after normalization to vinculin. Data are represented as mean ± SEM. **P<0.005. Scale bar: 50 pm

FIG. 4A-B. Histological improvement of injected muscle after 3 weeks. (FIG. 4A) Histochemistry of tibialis anterior muscle by hematoxylin and eosin (H&E) staining. (FIG. 4B) 10 Quantification of fiber size and percentage of frequency. Data are represented as mean ± SEM. Scale bar: 50 μm. Each set of four data points for a given fiber size are A50-CTL, WT-CTL, A50-AAV-TriSA and A50-AAV-SA+SD left to right.

FIG. 5. Screen of sgRNA in human 293 cells and mouse 10T cells. Undigested PCR products (upper panel) and T7E1 digestion (lower panel) on a 2% agarose gel. M dénotés size 15 marker lane. bp indicates the length of the marker bands.

FIG. 6. Dystrophin immunohistochemistry staining of entire tibialis anterior muscle.

FIGS. 7A-B. Strategy for CRISPR/Cas9-mediated genome editing in AExSO mice.

(FIG. 7A) Strategy showing CRISPR/Cas9-mediated genome editing approach to correct the 20 reading frame in ΔΕχ50 mouse model. (FIG. 7B) sgRNA targeting the splice accepter site (sgRNA-ex51-SA2) sequence and schematic illustration of sgRNA binding position. Fig. 7B discloses SEQ ID NOS 958-959, respectively, in order of appearance.

FIG. 8A-F. sgRNA genomic analysis in mouse and human cells (FIG. 8A) Cas9 was expressed in the presence or absence of mouse sgRNA-sgRNA-51-SA2 in 10T1/2 cells and 25 gene editing was monitored by T7E1 assay in fluorescence-based cell sorted (FACS) (+) and non-sorted cells (-). GFP was used as a control. Undigested PCR products (upper panel) and T7E1 digestion (lower panel) are shown on a 2% agarose gel. Black arrowhead indicates the undigested 771 bp PCR band. Green arrowheads in the lower panel indicate the eut bands by T7E1 assay. M dénotés size marker lane. bp indicates the length of the marker bands. (FIG. 30 8B) Genomic deep sequencing analysis of PCR amplicons generated across the exon 51 target site in 10T1/2 cells. Sequence of représentative indels aligned with sgRNA sequence (indicated in blue) revealing insertions (highlighted in green) and délétions (highlighted in red) (SEQ 1D

NOS 960-966, respectively, in order of appearance). The line indicates the predicted exon splicing enhancers (ESEs) sequence located at the site of sgRNA. Black arrow indicates the cleavage site. (FIG. 8C) Mouse Exon 51 sequence (SEQ ID NO: 967) with the predicted exon splicing enhancers (ESEs) located at the site of sgRNA is indicated in red. Human Exon 51 sequence (SEQ ID NO: 968) with the predicted exon splicing enhancers (ESEs) located at the site of sgRNA is indicated in red. (FIG. 8D) Mouse and human ESE sites of exon 51 predicted using ESEfinder3. (FIG. 8E) Cas9 was expressed in the presence or absence of mouse sgRNAsgRNA-51-SA2 in 293 T human cells and gene editing was monitored by T7E1 assay in fluorescence-based cell sorted (FACS) (+) and non-sorted cells (-). GFP was used as a control. Undigested PCR products (upper panel) and T7E1 digestion (lower panel) are shown on a 2% agarose gel. Black arrowhead indicates the undigested 771bp PCR band. Green arrowheads in the lower panel indicate the eut bands by T7E1 assay. M dénotés size marker lane. bp indicates the length of the marker bands. (FIG. 8F) Sequence of représentative indels aligned with sgRNA sequence (indicated in blue) revealing délétions and insertions (SEQ ID NOS 969-978, respectively, in order of appearance). Black arrowhead indicates the cleavage site.

FIG. 9A-B. Schematic illustration of AAV injection plasmids and strategy. (FIG. 9A) Muscle creatine kinase 8 (CK8) promoter to express SpCas9. (FIG. 9B) Triplicate using rAAV9-sgRNA plasmid containing 3 copies of sgRNA-ex51-SA2. U6, H1 and 7SK promoter for RNA polymerase III to express sgRNA.

FIG. 10A-B. In vivo Dmd gene editing. (FIG. 10A) Undigested PCR products (upper panel) and T7E1 digestion (lower panel) are shown on a 2% agarose gel of TA (tibialis anterior) muscle samples from WT and ΔΕχ50 mice 3 weeks after intramuscular injection with AAV9sgRNA-SA2 and AAV9-Cas9 expression vectors. Controls were injected with only AAV9Cas9 not AAV9-sgRNA-SA2. Black arrowhead in the upper panel indicates the 771 bp PCR band. Green arrowheads in the lower panel indicate the eut bands by T7E1 assay. M dénotés size marker lane. bp indicates the length of the marker bands. N=4. (FIG. 10B) Genomic deep sequencing analysis of PCR amplicons generated across the exon 51 target site in AEx50 mice injected with AAV9-sgRNA-51-SA2 and AAV9-Cas9. Sequence of représentative indels aligned with sgRNA sequence (indicated in blue) revealing insertions (highlighted in green) and délétions (highlighted in red). (SEQ ID NOS 979-1014, respectively, in order of appearance). Black arrowheads indicate the cleavage site. n=3.

FIG. 11A-D. RT-PCR analysis of correction of reading frame. (FIG. 11 A) RT-PCR of RNA from tibialis anterior muscles of wildtype (WT) and AEx50 mice 3 weeks after intramuscular injection of the sgRNA-51-SA2 and Cas9 expression vectors. Lower dystrophin bands indicate délétion of exon 51. Primer positions in exons 48 and 53 are indicated (Fw, Rv). (FIG. 1 IB) Percentage of events detected at exon 51 after AAV9-sgRNA-51-SA2 treatment using RT-PCR sequence analysis of TOPO-TA generated clones. For each of 4 different samples, we generated and sequenced 40 clones. RT-PCR products were divided into 4 groups: not-edited (NE), exon51 -skipped (SK), reframed (RF) and out of frame (OF). (FIG. 11 C) Sequence of the RT-PCR products of the AEx50-51 mouse dystrophin lower band confirmed that exon 49 spliced directly to exon 52, excluding exon 51. Sequence of RT-PCR products of AEx50 reframed (AEx50-RF). Fig. 1 IC discloses SEQ ID NOS 1015-1022, respectively, in order ofappearance. (FIG. HD) Deep sequencinganalysis of RT-PCR products from the upper band containing AEx50 not-edited (NE) and AEx50-RF. Sequence of RT-PCR products revealing insertions (highlighted in green) and délétions (highlighted in red). n=4. Data are represented as mean ± SEM. Fig. HD discloses SEQ ID NOS 1023-1026, respectively, in order appearance.

FIG. 12A-D. Intramuscular injection of AAV9-Cas9 and AAV9-sgRNA-51-SA2 corrects dystrophin expression. (FIG. 12A) Tibialis anterior muscles of ΔΕχ50 mice were injected with AAV9 vector encoding sgRNA and Cas9 and analyzed 3 weeks later by immunostaining for dystrophin. Wildtype control (WT-CTL) mice and AEx50 mice control (AEx50-CTL) were injected with AAV9-Cas9 alone without sgRNAs. Percentages of dystrophin-positive myofibers in AEx50-CTL mice and in treated AEx50 mice (AEx50-AAV9sgRNA-51-SA2 and AAV9-Cas9) compared to WT-CTL are indicated in each panel. (FIG. I2B) Hematoxylin and eosin (H&E) staining of tibialis anterior muscles. (FIG. 12C) Western blot analysis of dystrophin (DMD) and vinculin (VCL) expression in tibialis anterior muscles 3 weeks after intramuscular injection. (FIG. 12D) Quantification of dystrophin expression from blots after normalization to vinculin. Asterisk indicates non-specific immunoreactive bands. n=5 for AAV9-sgRNA-51-SA2. Scale bar: 50μιη

FIG. 13. Rescue of dystrophin expression following intramuscular injections of AAV9-Cas9 and AAV9-sgRNA-51-SA2 in AEx50 mouse model. Dystrophin immunohistochemistry of entire tibialis anterior muscle. CTL mice were injected with AAV9Cas9 alone without AAV9-sgRNA-51-SA2. n=5

FIG. 14. Histological improvement of injected muscle after 3 weeks. Histochemistry of tibialis anterior muscle by hematoxylin and eosin (H&E) staining.

FIG. 15A-B. Rescue of dystrophin expression following intramuscular injections of AAV9-Cas9 combined with different AAV9s expressing single copy or triple copy of sgRNA in AEx50 mouse model. (FIG. 15A) The U6, H1 and 7SK promoters for RNA polymerase 111 were each individually used to express sgRNA in a single copy (AAV9-U6sgRNA-51-SA2; AAV9-Hl-sgRNA-51-SA2; AAV9-7SK-sgRNA-51-SA2) or triple copy. (FIG. 15B) Dystrophin immunohistochemistry of entire tibialis anterior muscle. Control (CTL) mice were injected with AAV9-Cas9 alone without AAV9-sgRNA-51-SA2.

FIG. 16A-B. Rescue of dystrophin expression 4 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51-SA2 in AExSO mice. (FIG. 16A) Dystrophin immunostaining of tibialis anterior (TA), triceps, diaphragm and cardiac muscles 8 weeks after systemic injection of AAV9-sgRNA-51. (FIG. 16B) Western blot analysis of dystrophin (DMD) and vinculin (VCL) expression in TA, triceps, diaphragm muscles and heart. n=5 for each group. Scale bar: 50pm.

FIG. 17A-B. Rescue of dystrophin expression 8 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51-SA2 in AExSO mice. (FIG. 17A) Dystrophin immunostaining of tibialis anterior (TA), triceps, diaphragm and cardiac muscles 8 weeks after systemic injection of AAV9-sgRNA-51. (FIG. 17B) Western blot analysis of dystrophin (DMD) and vinculin (VCL) expression in TA, triceps, diaphragm muscles and heart. n=5 for each group. Scale bar: 50pm.

FIG. 18A-B. Functional improvement 4 weeks after systemic delivery of AAV9Cas9 and AAV9-sgRNA-51-SA2 in AExSO mice. (FIG. 18A) Wildtype (WT) mice, control AEx50 mice and AEx50 mice treated with AAV9-sgRNA-51 (AEx50-AAV9-sgRNA-51) were subjected to grip strength testing to measure muscle performance (grams of force). (FIG. 18B) Sérum creatine kinase (CK) was measured in WT, AEx50 and AEx50-AAV9-sgRNA-51 mice. n=5. Asterisk indicates non-specific immunoreactive bands. Data are represented as mean ± SEM.

FIG. 19. Correction of dystrophin expression 6 weeks after intra-muscular injection in AEx50-MD Dog. Dystrophin immunohistochemistry staining of cranial tibialis muscle of a wild-type dog (Nathan), a AEx50-MD Dog untreated, and two AEx50-MD Dogs (Norman and Newton) contralatéral uninjected and AAV9-Cas9-sgRNA injected cramai tibialis muscle. Scale bar: 50pm.

FIGS. 20A-B. Correction of dystrophin expression 6 weeks after intra-muscular injection in AEx50-MD Dog. (FIG. 20A) Western blot analysis of dystrophin (DMD) and v incul in (VCL) of cranial tibialis muscle of a wild-type dog (Nathan), a AEx50-MD Dog untreated, and two AEx50-MD Dogs (Norman and Newton) contralatéral uninjected and AAV9-Cas9-sgRNA injected cranial tibialis muscle. (FIG. 20B) Quantification of dystrophin expression from two independent blots after normalization to vinculin.

FIG. 21. Histological improvement of injected muscle after 6 weeks in AEx50-MD Dog. Histochemistry by hematoxylin and eosin (H&E) staining of cranial tibialis muscle of wild-type dog (Nathan), AEx50-MD Dogs (Norman and Newton) contralatéral uninjected and AAV9-Cas9-sgRNA injected cranial tibialis muscle. Scale bar: 50 pm.

DETAILED DESCRIPTION

DMD is a new mutation syndrome with more than 4,000 independent mutations that hâve been identified in humans (world-wide web at dmd.nl). The majority of patient mutations include délétions that cluster in a hotspot, and thus a therapeutic approach for skipping certain exon applies to large group of patients. The rationale of the exon skipping approach is based on the genetic différence between DMD and Becker muscular dystrophy (BMD) patients. In DMD patients, the reading frame of dystrophin mRNA is disrupted resulting in prematurely truncated, non-functional dystrophin proteins. BMD patients hâve mutations in the DMD gene that maintain the reading frame allowing the production of intemally deleted, but partially functional dystrophins leading to much milder disease symptoms compared to DMD patients.

Compositions and methods for treating DMD are provided herein. In some embodiments, an AAV construct is provided, wherein the AAV construct comprises a nucleic acid encoding three promoters that each drive expression of a DMD guide RNA. Using compositions and methods disclosed herein, a more robust and safe form of genome editing was achieved in a humanized mouse model for DMD with a délétion in exon 50, and in a AEx50-MD Dog. These and other aspects of the disclosure are reproduced below.

I. Duchenne Muscular Dystrophy

A. Background

Duchenne muscular dystrophy (DMD) is a récessive X-linked form of muscular dystrophy, affecting around 1 in 5000 boys, which results in muscle degeneration and prématuré death. The disorder is caused by a mutation in the gene dystrophin (see GenBank Accession NO. NC_000023.11), located on the human X chromosome, which codes for the protein dystrophin (GenBank Accession No. AAA53189; SEQ 1D NO: 5), the sequence of which is reproduced below:

mlwweevedc yeredvqkkt ftkwvnaqfs kfgkqhienl fsdlqdgrrl Idllegltgq klpkekgstr vhalnnvnka Irvlqnnnvd Ivnigstdiv dgnhkltlgl iwniilhwqv

121 knvmknimag Iqqtnsekil Iswvrqstrn ypqvnvinft tswsdglaln alihshrpdl

181 fdwnsvvcqq satqrlehaf niaryqlgie klldpedvdt typdkksilm yitslfqvlp

241 qqvsieaiqe vemlprppkv tkeehfqlhh qmhysqqitv slaqgyerts spkprfksya

301 ytqaayvtts dptrspfpsq hleapedksf gsslmesevn Idryqtalee vlswllsaed

361 tlqaqgeisn dvevvkdqfh thegymmdlt ahqgrvgnil qIgskligtg klsedeetev

421 qeqmnllnsr weclrvasme kqsnlhrvlm dlqnqklkel ndwltkteer trkmeeeplg

481 pdledlkrqv qqhkvlqedl eqeqvrvnsl thmvvvvdes sgdhataale eqlkvlgdrw

541 anicrwtedr wvllqdillk wqrlteeqcl fsawlseked avnkihttgf kdqnemlssl

601 qklavlkadl ekkkqsmgkl yslkqdllst Iknksvtqkt eawldnfarc wdnlvqklek

661 staqisqavt ttqpsltqtt vmetvttvtt reqilvkhaq eelpppppqk krqitvdsei

721 rkrldvdite Ihswitrsea vlqspefaif rkegnfsdlk ekvnaierek aekfrklqda

781 srsaqalveq mvnegvnads ikqaseqlns rwiefcqlls erlnwleyqn niiafynqlq

841 qleqmtttae nwlkiqpttp septaiksql kickdevnrl sglqpqierl kiqsialkek

901 gqgpmfldad fvaftnhfkq vfsdvqarek elqtifdtlp pmryqetmsa irtwvqqset

961 klsipqlsvt dyeimeqrlg elqalqsslq eqqsglyyls ttvkemskka pseisrkyqs

1021 efeeiegrwk klssqlvehc qkleeqmnkl rkiqnhiqtl kkwmaevdvf Ikeewpalgd

1081 seilkkqlkq crllvsdiqt iqpslnsvne ggqkikneae pefasrlete Ikelntqwdh

1141 mcqqvyarke alkgglektv slqkdlsemh ewmtqaeeey lerdfeyktp delqkaveem

1201 krakeeaqqk eakvklltes vnsviaqapp vaqealkkel etlttnyqwl ctrlngkckt

1261 leevwacwhe llsylekank wlnevefklk ttenipggae eisevldsle nlmrhsednp

1321 nqirilaqtl tdggvmdeli neeletfnsr wrelheeavr rqklleqsiq saqetekslh

1381 liqesltfid kqlaayiadk vdaaqmpqea qkiqsdltsh eisleemkkh nqgkeaaqrv

1441 Isqidvaqkk Iqdvsmkfrl fqkpanfelr Iqeskmilde vkmhlpalet ksveqevvqs

1501 qlnhcvnlyk slsevkseve mviktgrqiv qkkqtenpke Idervtalkl hynelgakvt

1561 erkqqlekcl klsrkmrkem nvltewlaat dmeltkrsav egmpsnldse vawgkatqke

1621 iekqkvhlks itevgealkt vlgkketlve dklsllnsnw iavtsraeew Inllleyqkh

1681 metfdqnvdh itkwiiqadt lldesekkkp qqkedvlkrl kaelndirpk vdstrdqaan

1741 Imanrgdhcr klvepqisel nhrfaaishr iktgkasipl keleqfnsdi qkllepleae

1801 iqqgvnlkee dfnkdmnedn egtvkellqr gdnlqqritd erkreeikik qqllqtkhna

1861 Ikdlrsqrrk kaleishqwy qykrqaddll kclddiekkl aslpeprder kikeidrelq

1921 kkkeelnavr rqaeglsedg aamaveptqi qlskrwreie skfaqfrrln faqihtvree

1981 tmmvmtedmp leisyvpsty Iteithvsqa lleveqllna pdlcakdfed Ifkqeeslkn

2041 ikdslqqssg ridiihskkt aalqsatpve rvklqealsq Idfqwekvnk mykdrqgrfd

2101 rsvekwrrfh ydikifnqwl teaeqflrkt qipenwehak ykwylkelqd gigqrqtvvr

2161 tlnatgeeii qqssktdasi Iqeklgslnl rwqevckqls drkkrleeqk nilsefqrdl

2221 nefvlwleea dniasiplep gkeqqlkekl eqvkllveel plrqgilkql netggpvlvs

2281 apispeeqdk lenklkqtnl qwikvsralp ekqgeieaqi kdlgqlekkl edleeqlnhl

2341 llwlspirnq leiynqpnqe gpfdvqetei avqakqpdve eilskgqhly kekpatqpvk

2401 rkledlssew kavnrllqel rakqpdlapg Ittigasptq tvtlvtqpvv tketaiskle

2461 mpsslmlevp aladfnrawt eltdwlslld qviksqrvmv gdledinemi ikqkatmqdl

2521 eqrrpqleel itaaqnlknk tsnqeartii tdrieriqnq wdevqehlqn rrqqlnemlk

2581 dstqwleake eaeqvlgqar akleswkegp ytvdaiqkki tetkqlakdl rqwqtnvdva

2641 ndlalkllrd ysaddtrkvh miteninasw rsihkrvser eaaleethrl Iqqfpldlek

2701 flawlteaet tanvlqdatr kerlledskg vkelmkqwqd Iqgeieahtd vyhnldensq

2761 kilrslegsd davllqrrld nmnfkwselr kkslnirshl eassdqwkrl hlslqellvw

2821 Iqlkddelsr qapiggdfpa vqkqndvhra fkrelktkep vimstletvr iflteqpleg

2881 leklyqepre Ippeeraqnv trllrkqaee vnteweklnl hsadwqrkid etlerlqelq

2941 eatdeldlkl rqaevikgsw qpvgdllids Iqdhlekvka Irgeiaplke nvshvndlar

3001 qlttlgiqls pynlstledl ntrwkllqva vedrvrqlhe ahrdfgpasq hflstsvqgp

3061 weraispnkv pyyinhetqt tcwdhpkmte lyqsladlnn vrfsayrtam klrrlqkalc

3121 Idllslsaac daldqhnlkq ndqpmdilqi inclttiydr leqehnnlvn vplcvdmcln

3181 wllnvydtgr tgrirvlsfk tgiislckah ledkyrylfk qvasstgfcd qrrlglllhd

3241 siqiprqlge vasfggsnie psvrscfqfa nnkpeieaal fldwmrlepq smvwlpvlhr

3301 vaaaetakhq akcnickecp iigfryrslk hfnydicqsc ffsgrvakgh kmhypmveyc

3361 tpttsgedvr dfakvlknkf rtkryfakhp rmgylpvqtv legdnmetpv tlinfwpvds

3421 apasspqlsh ddthsriehy asrlaemens ngsylndsis pnesiddehl liqhycqsln

3481 qdsplsqprs paqilisles eergeleril adleeenrnl qaeydrlkqq hehkglsplp

3541 sppemmptsp qsprdaelia eakllrqhkg rlearmqile dhnkqlesql hrlrqlleqp

3601 qaeakvngtt vsspstslqr sdssqpmllr vvgsqtsdsm geedllsppq dtstgleevm

3661 eqlnnsfpss rgrntpgkpm redtm.

In humans, dystrophin mRNA contains 79 exons. Dystrophin mRNA is known to be alternatively spliced, resulting in varions isoforms. Exemplary dystrophin isoforms are listed in Table 1.

Table 1: Dystrophin isoforms

Sequence Name

Nucleic Acid Accession No.

Nuclei c Acid SEQ ID NO:

Protein Accession No.

Protei n SEQ ID NO:

Description

DMD Genomic Sequence	NC_000023.ll (positions 31119219 to 33339609)	None	None	None	Sequence from Human X Chromosome ( at positions Xp21.2 to p21.1 ) from Assembly GRCh38.p7 (GCF_000001405.33 )
Dystrophi n Dp427c isoform	NM_000109.3	6	NP_000100.2	7	Transcript Variant: transcript Dp427c is expressed predominantly in neurons of the cortex and the CA régions of the hippocampus. It uses a unique promoter/exon 1 located about 130 kb upstream of the Dp427m transcript promoter. The transcript includes the common exon 2 of transcript Dp427m and has a similar length of 14 kb. The Dp427c isoform contains a unique Nterminal MED sequence, instead of the MLWWEEVEDCY sequence (SEQ ID NO: 949) of isoform Dp427m. The remainder of isoform Dp427c is identical to isoform Dp427m.
Dystrophi n Dp427m isoform	NM_004006.2	8	NP_003 997.1	9	Transcript Variant: transcript Dp427m encodes the main dystrophin protein found in muscle. As a resuit of alternative promoter use, exon 1 encodes a unique Nterminal MLWWEEVEDCY

					(SEQ ID NO: 949) aa sequence.
Dystrophi η Dp427pl isoform	NM_004009.3	10	NP_004000.1	11	Transcript Variant: transcript Dp427pl initiâtes from a unique promoter/exon 1 located in what corresponds to the first intron of transcript Dp427m. The transcript adds the common exon 2 of Dp427m and has a similar length (14 kb). The Dp427pl isoform replaces the MLWWEEVEDCY (SEQ ID NO: 949) start of Dp427m with a unique N-terminal MSEVSSD (SEQ ID NO: 950) aa sequence.
Dystrophi n Dp2601 isoform	NM 004011.3	12	NP_004002.2	13	Transcript Variant: transcript Dp260-l uses exons 30-79, and originates from a promoter/exon 1 sequence located in intron 29 of the dystrophin gene. As a resuit, Dp260-1 contains a 95 bp exon 1 encoding a unique N-terminal 16 aa MTEIILLIFFPAYFL N-sequence (SEQ ID NO: 951) that replaces amino acids 1-1357 of the fulllength dystrophin product (Dp427m isoform).
Dystrophi n Dp2602 isoform	NM_004012.3	14	NP_004003.1	15	Transcript Variant: transcript Dp260-2 uses exons 30-79, starting from a promoter/exon 1

					sequence located in intron 29 of the dystrophin gene that is alternatively spliced and lacks Nterminal amino acids 1-1357 of the full length dystrophin (Dp427m isoform). The Dp260-2 transcript encodes a unique N-terminal MSARKLRNLSYK K sequence (SEQ ID NO: 952).
Dystrophi n Dpl40 isoform	NM_004013.2	16	NP_004004.1	17	Transcript Variant: Dp 140 transcripts use exons 45-79, starting at a promoter/exon 1 located in intron 44. Dp 140 transcripts hâve a long (1 kb) 5' UTR since translation is initiated in exon 51 (corresponding to aa 2461 of dystrophin). In addition to the alternative promoter and exon 1, differential splicing of exons 71-74 and 78 produces at least five Dp 140 isoforms. Of these, this transcript (Dp 140) contains ail of the exons.
Dystrophi n Dpi 16 isoform	NM_004014.2	18	NP_004005.1	19	Transcript Variant: transcript Dpi 16 uses exons 56-79, starting from a promoter/exon 1 within intron 55. As a resuit, the Dpi 16 isoform contains a unique N-terminal MLHRKTYHVK aa sequence (SEQ ID

					NO: 953), instead of aa 1-2739 of dystrophin. Differential splicing produces several Dpi 16-subtypes. The Dpi 16 isoform is also known as Sdystrophin or apodystrophin-2.
Dystrophi n Dp71 isoform	NM_004015.2	20	NP_004006.1	21	Transcript Variant: Dp71 transcripts use exons 63-79 with a novel 80- to 100-nt exon containing an ATG start site for a new coding sequence of 17 nt. The short coding sequence is in-frame with the consecutive dystrophin sequence from exon 63. Differential splicing of exons 71 and 78 produces at least four Dp71 isoforms. Of these, this transcript (Dp71) includes both exons 71 and 78.
Dystrophi n Dp71b isoform	NM_004016.2	22	NP_004007.1	23	Transcript Variant: Dp71 transcripts use exons 63-79 with a novel 80- to 100-nt exon containing an ATG start site for a new coding sequence of 17 nt. The short coding sequence is in-frame with the consecutive dystrophin sequence from exon 63. Differential splicing of exons 71 and 78 produces at least four Dp71 isoforms. Of these, this transcript (Dp71 b) lacks exon 78 and encodes a

					protein with a different C-terminus than Dp71 and Dp71a isoforms.
Dystrophi n Dp71a isoform	NM_004017.2	24	NP_004008.1	25	Transcript Variant: Dp71 transcripts use exons 63-79 with a novel 80- to 100-nt exon containing an ATG start site for a new coding sequence of 17 nt. The short coding sequence is in-frame with the consecutive dystrophin sequence from exon 63. Differential splicing of exons 71 and 78 produces at least four Dp71 isoforms. Of these, this transcript (Dp71a) lacks exon 71.
Dystrophi n Dp71ab isoform	NM_004018.2	26	NP_004009.1	27	Transcript Variant: Dp71 transcripts use exons 63-79 with a novel 80- to 1 OO-nt exon containing an ATG start site for a new coding sequence of 17 nt. The short coding sequence is in-frame with the consecutive dystrophin sequence from exon 63. Differential splicing of exons 71 and 78 produces at least four Dp71 isoforms. Of these, this transcript (Dp71 ab) lacks both exons 71 and 78 and encodes a protein with a C-terminus like isoform Dp71b.
Dystrophi n Dp40 isoform	NM_004019.2	28	NP_004010.1	29	Transcript Variant: transcript Dp40 uses exons 63-70. The 5'

					UTR and encoded first 7 aa are identical to that in transcript Dp71, but the stop codon lies at the splice junction ofthe exon/intron 70. The 3' UTR includes nt from intron 70 which includes an alternative polyadenylation site. The Dp40 isoform lacks the normal Cterminal end of fulllength dystrophin (aa 3409-3685).
Dystrophi n Dp 140c isoform	NM_004020.3	30	NP_004011.2	31	Transcript Variant: Dp 140 transcripts use exons 45-79, starting at a promoter/exon 1 located in intron 44. Dp 140 transcripts hâve a long (1 kb) 5' UTR since translation is initiated in exon 51 (corresponding to aa 2461 of dystrophin). In addition to the alternative promoter and exon 1, differential splicing of exons 71-74 and 78 produces at least five Dp 140 isoforms. Of these, this transcript (Dp 140c) lacks exons 71-74.
Dystrophi n Dp 140b isoform	NM_004021.2	32	NP_004012.1	33	Transcript Variant: Dp 140 transcripts use exons 45-79, starting at a promoter/exon 1 located in intron 44. Dp 140 transcripts hâve a long (1 kb) 5' UTR since translation is initiated

					in exon 51 (corresponding to aa 2461 of dystrophin). In addition to the alternative promoter and exon 1, differential splicing of exons 71-74 and 78 produces at least five Dp 140 isoforms. Of these, this transcript (Dp 140b) lacks exon 78 and encodes a protein with a unique Cterminus.
Dystrophi η Dpl40ab isoform	NM_004022.2	34	NP_004013.1	35	Transcript Variant: Dp 140 transcripts use exons 45-79, starting at a promoter/exon 1 located in intron 44. Dpl40 transcripts hâve a long (1 kb) 5' UTR since translation is initiated in exon 51 (corresponding to aa 2461 of dystrophin). In addition to the alternative promoter and exon 1, differential splicing of exons 71-74 and 78 produces at least five Dp 140 isoforms. Of these, this transcript (Dpl40ab) lacks exons 71 and 78 and encodes a protein with a unique C-terminus.
Dystrophi n Dpl40bc isoform	NM_004023.2	36	NP_004014.1	37	Transcript Variant: Dp 140 transcripts use exons 45-79, starting at a promoter/exon 1 located in intron 44. Dp 140 transcripts hâve a long (1 kb) 5'

					UTR since translation is initiated in exon 51 (corresponding to aa 2461 of dystrophin). In addition to the alternative promoter and exon 1, differential splicing of exons 71-74 and 78 produces at least five Dp 140 isoforms. Of these, this transcript (Dpl40bc) lacks exons 71-74 and 78 and encodes a protein with a unique C-terminus.
Dystrophi n isoform X2	XM_006724469 .3	38	XP_006724532. 1	39
Dystrophi n isoform X5	XM_011545467 .1	40	XP_011543769. 1 ·	41
Dystrophi n isoform X6	XM_006724473 .2	42	XP_006724536. 1	43
Dystrophi n isoform X8	XM_006724475 .2	44	XP_006724538. 1	45
Dystrophi n isoform X4	XM_017029328 .1	46	XP_016884817. 1	47
Dystrophi n isoform XI	XM_006724468 .2	48	XP_006724531. 1	49
Dystrophi n isoform X13	XM_017029331 .1	50	XP_016884820. 1	51
Dystrophi n isoform X3	XM_006724470 .3	52	XP_006724533. 1	53
Dystrophi n isoform X7	XM_006724474 .3	54	XP_006724537. 1	55
Dystrophi n isoform X9	XM_011545468 .2	56	XP_011543770. 1	57

Dystrophi n isoform XI1	XM_017029330 .1	58	XP_016884819. 1	59
Dystrophi n isoform X10	XM_017029329 .1	865	XP_016884818. 1	866
Dystrophi n isoform XI2	XM_011545469 .1	867	XP_011543771. 1	868

The murine dystrophin protein has the following amino acid sequence (Uniprot Accession No. PI 1531, SEQ. ID. NO: 869):

MWWVDCYRDV KKTTKWNASK GKHDNSDDGK RDGTGKKKGS

TRVHANNVNK ARVKNNVDVN

GSTDVDGNHK TGWNHWVKNV MKTMAGTNSK SWVRSTRNYV

NVNTSSWSDG ANAHSHRDDW

121 NSVVSHSATR HANAKCGKDD VATTYDKKSM YTSVVSAVMR TSSKVTRHHH MHYSTVSAGY

181 TSSSKRKSYA TAAYVATSDS TSYSHARDKS DSSMTVNDSY TAVSWSADTR

AGSNDVVKHA

241 HGMMDTSHGV GNVGSVGKGK SDAVMNNSRW CRVASMKSKH KVMDNKKDDW TKTRTKKMGD

301 DKCVHKVDVR VNSTHMVVVV DSSGDHATAA KVGDRWANCR

WTDRWVDKWH TCSTWSKDAM

361 KNTSGKDNMM SSHKSTKDKK KTMKSSNDSA KNKSVTKMWM

NARWDNTKKS SASAVTTTST

421 TTVMTVTMVT TRMVKHAKKR TVDSRKRDVD THSWTRSAVS

SAVYRKGNSD KVNAARKAKR

481 KDASRSAAVM ANGVNASRAS NSRWTCSRVN WYTNTYNMTT

TANKTSTTST AKSKCKDVNR

541 SAKSKKGGMD ADVATNHNHD GVRAKKTDTM RYTMSSRTWS

SKSVYSVTYM RGKASSKNGN h ³³

601 YSDTVKMAKK ASCKYSGHWK KSSVSCKHMN KRKNHKTKWM AVDVKWAGDA KKKCRVGDTS

661 NSVNGGKKSA ASRTRNTWDH CRVYTRKAKA GDKTVSKDSM HWMTAYRDYK TDTAVMKRAK

721 AKTKVKTTVN SVAHASAAKK TTTNYWCTRN GKCKTVWACW

HSYKANKWNV KKTMNVAGTV

781 SNMHHSNNRA TTDGGVMDNT NSRWRHAVRK KSSAKSHSDK AAYTDKVDAA MAKSDTSHSM

841 KKHNGKDANR VSDVAKKDVS MKRKANRSKM DVKMHATKSV

VSSHCVNYKS SVKSVMVKTG

901 RVKKTNKDRV TAKHYNGAKV TRKKCKSRKM RKMNVTWAAT DTTKRSAVGM SNDSVAWGKA

961 TKKKAHKSVT GSKMVGKKTV DKSNSNWAVT SRVWNYKHMT DNTKWHADDS KKKKDKRKAM

1021 NDMRKVDSTR DAAKMANRGD HCRKVVSNRR AASHRKTGKA

SKNSDKAGVN KDNKDMSDNG

1081 TVNRGDNRTD RKRKKTKHNA KDRSRRKKAS HWYYKRADDK CDKKASRDRK KDRKKKNAVR

1141 RAGSNGAAMA VTSKRWRSNA RRNAHTHTMV VTTDMDVSYV

STYTSHASVD HNTCAKDDKS

1201 KNKDNSGRDH KKKTAASATS MKVKVAVAMD GKHRMYKRGR

DRSVKWRHHY DMKWWNVKK

1261 TNNWHAKYKW YKDGGRAVVR TNATGSSKTD VNKGSSRWHD CKARRKRKNV SRDNVWADNA

1321 TGDKVKARGK NTGGAVVSAR DKKKKTNWKV SRAKGVHKDR

DHWSRNYNSA GDKVTVHGKA

1381 DVRSKGHYKK STVKRKDRSW AVNHRRTKDR AGSTTGASAS TVTVTSVVTK TVSKMSSVAA

F ³⁴

1441 DNRAWTTDWS DRVKSRVMVG DDNMKKATDR RTAANKNKTS

NARTTDRRWD VNRRNMK.DST

1501 WAKAVGVRGK DSWKGHTVDA KKTTKAKDRR SVDVANDAKR DYSADDTRKV HMTNNTSWGN

1561 HKRVSAATHR DKS WTATTAN VDASRKKDSR G VRMK WDGTH

TDYHNDNGKR SGSDARRDNM

1621 NKWSKKSNRS HASSDWKRHS VWKDDSRAGG DAVKNDHRAK

RKTKVMSTTV RTGKYRRANV

1681 TRRKAVNAWD KNRSADWRKD ARAADDKRAV KGSWVGDDSD

HKVKARGAKN VNRVNDAHTT

1741 GSYNSTDNTR WRVAVDRVRH AHRDGASHST SVGWRASNKV

YYNHTTTCWD HKMTYSADNN

1801 VRSAYRTAMK RRKACDSSAA CDADHNKNDM DNCTTYDRHN NVNVCVDMCN WNVYDTGRTG

1861 RRVSKTGSCK AHDKYRYKVA SSTGCDRRGH DSRGVASGGS

NSVRSCANNK AADWMRSMVW

1921 VHRVAAATAK HAKCNCKCGR YRSKHNYDCS CSGRVAKGHK

MHYMVYCTTT SGDVRDAKVK

1981 NKRTKRYAKH RMGYVTVGDN MTVTNWVDSA ASSSHDDTHS

RHYASRAMNS NGSYNDSSNS

2041 DDHHYCSNDS SRSASSRGRA DNRNAYDRKH HKGSSMMTSS

RDAAAKRHKG RARMDHNKSH

2101 RRAAKVNGTT VSSSTSRSDS SMRVVGSTSS MGDSDTSTGV MNNSSSRGRN AGKMRDTM

Dystrophin is an important component within muscle tissue that provides structural stability to the dystroglycan complex (DGC) of the cell membrane. While both sexes can carry the mutation, females are rarely affected with the skeletal muscle form of the disease.

Mutations vary in nature and frequency. Large genetic délétions are found in about 6070% of cases, large duplications are found in about 10% of cases, and point mutants or other small changes account for about 15-30% of cases. Bladen et al. (2015), who examined some 7000 mutations, catalogued a total of 5,682 large mutations (80% of total mutations), of which 4,894 (86%) were délétions (1 exon or larger) and 784 (14%) were duplications (I exon or larger). There were 1,445 small mutations (smaller than 1 exon, 20% of ail mutations), of which 358 (25%) were small délétions and 132 (9%) small insertions, while 199 (14%) affected the splice sites. Point mutations totaled 756 (52% of small mutations) with 726 (50%) nonsense mutations and 30 (2%) missense mutations. Finally, 22 (0.3%) mid-intronic mutations were observed. In addition, mutations were identified within the database that would potentially benefit from novel genetic thérapies for DMD including stop codon read-through thérapies (10% of total mutations) and exon skipping therapy (80% of délétions and 55% of total mutations).

B. Symptoms

Symptoms usually appear in boys between the âges of2 and 3 and may be visible in early infancy. Even though symptoms do not appear until early infancy, laboratory testing can identify children who carry the active mutation at birth. Progressive proximal muscle weakness of the legs and pelvis associated with loss of muscle mass is observed first. Eventually this weakness spreads to the arms, neck, and other areas. Early signs may include pseudohypertrophy (enlargement of calf and deltoid muscles), low endurance, and difficulties in standing unaided or inability to ascend staircases. As the condition progresses, muscle tissue expériences wasting and is eventually replaced by fat and fibrotic tissue (fibrosis). By âge 10, braces may be required to aid in walking but most patients are wheelchair dépendent by âge 12. Later symptoms may include abnormal bone development that lead to skeletal deformities, including curvature of the spine. Due to progressive détérioration of muscle, loss of movement occurs, eventually leading to paralysis. Intellectual impairment may or may not be présent but if présent, does not progressively worsen as the child âges. The average life expectancy for males afflicted with DMD is around 25.

The main symptom of Duchenne muscular dystrophy, a progressive neuromuscular disorder, is muscle weakness associated with muscle wasting with the voluntary muscles being First affected, especially those of the hips, pelvic area, thighs, shoulders, and calves. Muscle weakness also occurs later, in the arms, neck, and other areas. Calves are ofiten enlarged.

Symptoms usually appear before âge 6 and may appear in early infancy. Other physical symptoms are:

1. Awkward manner of walking, stepping, or running - (patients tend to walk on their forefeet, because of an increased calf muscle tone. Also, toe walking is a compensatory adaptation to knee extensor weakness.)

2. Frequent falls.

3. Fatigue.

4. Difficulty with motor skills (running, hopping, jumping).

5. Lumbar hyperlordosis, possibly ieading to shortening of the hip-flexor muscles. This has an effect on overall posture and a manner of walking, stepping, or running.

6. Muscle contractures of Achilles tendon and hamstrings impair functionality because the muscle fibers shorten and fibrose in connective tissue.

7. Progressive difficulty walking.

8. Muscle fiber deformities.

9. Pseudohypertrophy (enlarging) of tongue and calf muscles. The muscle tissue is eventually replaced by fat and connective tissue, hence the term pseudohypertrophy.

10. Higher risk of neurobehavioral disorders (e.g., ADHD), learning disorders (dyslexia), and non-progressive weaknesses in spécifie cognitive skills (in particular short-term verbal memory), which are believed to be the resuit of absent or dysfunctional dystrophin in the brain.

11. Eventual loss of ability to walk (usually by the âge of 12).

12. Skeletal deformities (including scoliosis in some cases).

13. Trouble getting up from lying or sitting position.

The condition can often be observed clinically from the moment the patient takes his first steps, and the ability to walk usually completely disintegrates between the time the boy is 9 to 12 years of âge. Most men affected with DMD become essentially “paralyzed from the neck down” by the âge of 21. Muscle wasting begins in the legs and pelvis, then progresses to the muscles of the shoulders and neck, followed by loss of arm muscles and respiratory muscles. Calf muscle enlargement (pseudohypertrophy) is quite obvious. Cardiomyopathy particularly (dilated cardiomyopathy) is common, but the development of congestive heart failure or arrhythmia (irregular heartbeat) is only occasional.

A positive Gowers' sign reflects the more severe impairment of the lower extremities muscles. The child helps himself to get up with upper extremities: first by rising to stand on his arms and knees, and then walking his hands up his legs to stand upright. Affected children usually tire more easily and hâve less overall strength than their peers. Creatine kinase (CPKMM) levels in the bloodstream are extremely high. An electromyography (EMG) shows that weakness is caused by destruction of muscle tissue rather than by damage to nerves. Genetic testing can reveal genetic errors in the Xp21 gene. A muscle biopsy (immunohistochemistry or immunoblotting) or genetic test (blood test) confirms the absence of dystrophin, although improvements in genetic testing often make this unnecessary.

DMD patients may suffer from:

1. Abnormal heart muscle (cardiomyopathy).

2. Congestive heart failure or irregular heart rhythm (arrhythmia).

3. Deformities of the chest and back (scoliosis).

4. Enlarged muscles of the calves, buttocks, and shoulders (around âge 4 or 5). These muscles are eventually replaced by fat and connective tissue (pseudohypertrophy).

5. Loss of muscle mass (atrophy).

6. Muscle contractures in the heels, legs.

7. Muscle deformities.

8. Respiratory disorders, including pneumonia and swallowing with food or fluid passing into the lungs (in late stages of the disease).

C. Causes

Duchenne muscular dystrophy (DMD) is caused by a mutation of the dystrophin gene at locus Xp21, located on the short arm of the X chromosome. Dystrophin is responsible for connecting the cytoskeleton of each muscle fiber to the underlying basal lamina (extracellular matrix), through a protein complex containing many subunits. The absence of dystrophin permits excess calcium to penetrate the sarcolemma (the cell membrane). Alterations in calcium and signaling pathways cause water to enter into the mitochondria, which then burst.

In skeletai muscle dystrophy, mitochondrial dysfunction gives rise to an amplification of stress-induced cytosolic calcium signais and an amplification of stress-induced reactiveoxygen species (ROS) production. In a complex cascading process that involves several pathways and is not clearly understood, increased oxidative stress within the cell damages the sarcolemma and eventually results in the death of the cell. Muscle fibers undergo necrosis and are ultimately replaced with adipose and connective tissue.

DMD is inherited in an X-linked récessive pattern. Females will typically be carriers for the disease while males will be affected. Typically, a female carrier will be unaware they carry a mutation until they hâve an affected son. The son of a carrier mother has a 50% chance of inheriting the defective gene from his mother. The daughter of a carrier mother has a 50% chance of being a carrier and a 50% chance of having two normal copies of the gene. In ail cases, an unaffected father will either pass a normal Y to his son or a normal X to his daughter. Female carriers of an X-linked récessive condition, such as DMD, can show symptoms depending on their pattern of X-inactivation.

Exon délétions preceding exon 51 of the human DMD gene, which disrupt the open reading frame (ORF) by juxtaposing out of frame exons, represent the most common type of human DMD mutation. Skipping of exon 51 can, in principle, restore the DMD ORF in 13% of DMD patients with exon délétions.

Duchenne muscular dystrophy has an incidence of 1 in 5000 male infants. Mutations within the dystrophin gene can either be inherited or occur spontaneously during germline transmission. A table of exemplary but non-limiting mutations and corresponding models are set forth below:

Table 2: Dystrophin mutations and corresponding mouse models

Délétion, small insertion and nonsense mutations	Name of Mouse Model
Exon 44	ΔΕχ44
Exon 52	ΔΕχ52
Exon 43	ΔΕχ43

D. Diagnosis

Genetic counseling is advised for people with a family history of the disorder. Duchenne muscular dystrophy can be detected with about 95% accuracy by genetic studies performed during pregnancy.

DNA test. The muscle-specific isoform of the dystrophin gene is composed of 79 exons, and DNA testing and analysis can usually identify the spécifie type of mutation of the exon or exons that are affected. DNA testing confirms the diagnosis in most cases.

Muscle biopsy. If DNA testing fails to find the mutation, a muscle biopsy test may be performed. A small sample of muscle tissue is extracted (usually with a scalpel instead of a needle) and a dye is applied that reveals the presence of dystrophin. Complété absence of the protein indicates the condition.

Over the past several years DNA tests hâve been developed that detect more of the many mutations that cause the condition, and muscle biopsy is not required as often to confirm the presence of Duchenne's.

Prénatal tests. DMD is carried by an X-linked récessive gene. Males hâve only one X chromosome, so one copy ofthe mutated gene will cause DMD. Fathers cannot pass X-linked traits on to their sons, so the mutation is transmitted by the mother.

If the mother is a carrier, and therefore one of her two X chromosomes has a DMD mutation, there is a 50% chance that a female child will inherit that mutation as one of her two X chromosomes, and be a carrier. There is a 50% chance that a male child will inherit that mutation as his one X chromosome, and therefore hâve DMD.

Prénatal tests can tell whether their unborn child has the most common mutations. There are many mutations responsible for DMD, and some hâve not been identified, so genetic testing only works when family members with DMD hâve a mutation that has been identified.

Prior to invasive testing, détermination of the fêtai sex is important; while males are sometimes affected by this X-linked disease, female DMD is extremely rare. This can be achieved by ultrasound scan at 16 weeks or more recently by free fêtai DNA testing. Chorion villus sampling (CVS) can be done at 11-14 weeks, and has a 1% risk of miscarriage. Amniocentesis can be done after 15 weeks, and has a 0.5% risk of miscarriage. Fêtai blood sampling can be done at about 18 weeks. Another option in the case of unclear genetic test results is fêtai muscle biopsy.

E. Treatment

There is no current cure for DMD, and an ongoing medical need has been recognized by regulatory authorities. Phase l-2a trials with exon skipping treatment for certain mutations hâve halted décliné and produced small clinical improvements in walking. Treatment is generally aimed at controlling the onset of symptoms to maximize the quality of life, and may include the foilowing:

1. Corticosteroids such as prednisolone and deflazacort increase energy and strength and defer severity of some symptoms.

2. Randomized control trials hâve shown that beta-2-agonists increase muscle strength but do not modify disease progression. Follow-up time for most RCTs on beta2-agonists is 5 only around 12 months and hence results cannot be extrapolated beyond that time frame.

3. Mild, non-jarring physical activity such as swimming is encouraged. Inactivity (such as bed rest) can worsen the muscle disease.

4. Physical therapy is helpful to maintain muscle strength, flexibility, and function.

5. Orthopédie appliances (such as braces and wheelchairs) may improve mobility and the ability for self-care. Form-fitting removable leg braces that hold the ankle in place during sleep can defer the onset of contractures.

6. Appropriate respiratory support as the disease progresses is important.

Comprehensive multi-disciplinary care standards/guidelines for DMD hâve been developed by 15 the Centers for Disease Control and Prévention (CDC), and are available at www.treatnmd.eu/dmd/care/diagnosis-management-DMD.

DMD generally progresses through five stages, as outlined in Bushby et al., Lancet Neurol., 9(1): 77-93 (2010) and Bushby et al., Lancet Neurol., 9(2): 177-198 (2010), incorporated by reference in their entireties. During the presymptomatic stage, patients 20 typically show developmental delay, but no gait disturbance. During the early ambulatory stage, patients typically show the Gowers’ sign, waddling gait, and toe walking. During the late ambulatory stage, patients typically exhibit an increasingly labored gait and begin to lose the ability to climb stairs and rise from the floor. During the early non-ambulatory stage, patients are typically able to self-propel for some time, are able to maintain posture, and may 25 develop scoliosis. During the late non-ambulatory stage, upper limb function and postural maintenance is increasingly limited.

In some embodiments, treatment is initiated in the presymptomatic stage ofthe disease. In some embodiments, treatment is initiated in the early ambulatory stage. In some embodiments, treatment is initiated in the late ambulatory stage. In embodiments, treatment is 30 initiated during the early non-ambulatory stage. In embodiments, treatment is initiated during the late non-ambulatory stage.

1. Physical Therapy

Physical therapists are concerned with enabling patients to reach their maximum physical potential. Their aim is to:

1. minimize the development of contractures and deformity by developing a programme of stretches and exercises where appropriate,

2. anticipate and minimize other secondary complications of a physical nature by recommending bracing and durable medical equipment, and

3. monitor respiratory function and advise on techniques to assist with breathing exercises and methods of clearing sécrétions.

2. Respiration Assistance

Modem volume ventilators/respirators, which deliver an adjustable volume (amount) of air to the person with each breath, are valuable in the treatment of people with muscular dystrophy related respiratory problems. The ventilator may require an invasive endotrachéal or tracheotomy tube through which air is directly delivered, but, for some people non-invasive delivery through a face mask or mouthpiece is sufficient. Positive airway pressure machines, particularly bi-level ones, are sometimes used in this latter way. The respiratory equipment may easily fit on a ventilator tray on the bottom or back of a power wheelchair with an external battery for portability.

Ventilator treatment may start in the mid to late teens when the respiratory muscles can begin to collapse. If the vital capacity has dropped below 40% of normal, a volume ventilator/respirator may be used during sleeping hours, a time when the person is most likely to be under ventilating (“hypoventilating”). Hypoventilation during sleep is determined by a thorough history of sleep disorder with an oximetry study and a capillary blood gas (See Pulmonary Function Testing). A cough assist device can help with excess mucus in lungs by hyperinflation of the lungs with positive air pressure, then négative pressure to get the mucus up. If the vital capacity continues to décliné to less than 30 percent of normal, a volume ventilator/respirator may also be needed during the day for more assistance. The person gradually will increase the amount of time using the ventilator/respirator during the day as needed.

F. Prognosis

Duchenne muscular dystrophy is a progressive disease which eventually affects ail voluntary muscles and involves the heart and breathing muscles in later stages. The life 5 expectancy is currently estimated to be around 25, but this varies from patient to patient. Recent advancements in medicine are extending the lives of those afflicted. The Muscular Dystrophy Campaign, which is a leading UK charity focusing on ail muscle disease, States that “with high standards of medical care young men with Duchenne muscular dystrophy are often living well into their 30s.”

In rare cases, persons with DMD hâve been seen to survive into the forties or early fifties, with the use of proper positioning in wheelchairs and beds, ventilator support (via tracheostomy or mouthpiece), airway clearance, and heart médications, if required. Early planning of the required supports for later-life care has shown greater longevity in people living with DMD.

Curiously, in the mdx mouse model of Duchenne muscular dystrophy, the lack of dystrophin is associated with increased calcium levels and skeletal muscle myonecrosis. The intrinsic laryngeal muscles (ILM) are protected and do not undergo myonecrosis. ILM hâve a calcium régulation System profile suggestive of a better ability to handle calcium changes in comparison to other muscles, and this may provide a mechanistic insight for their unique pathophysiological properties. The ILM may facilitate the development of novel strategies for the prévention and treatment of muscle wasting in a variety of clinical scénarios.

IL Other Diseases Benefiting from Genome Editing

In addition to finding application in the treatment of DMD, the inventors’ multipleguide approach can be advantageously applied to other disease States where such intervention 25 could resuit in correction of the underlying causal genetic defects. Some of these are described briefly below.

Limb Girdle Muscular Dystrophy. Limb-girdle muscular dystrophy (LGMD) or Erb's muscular dystrophy is a genetically and clinically heterogeneous group of rare muscular dystrophies. It is characterized by progressive muscle wasting which affects predominantly hip 30 and shoulder muscles. LGMD has an autosomal pattern of inheritance and currently has no known cure. The symptoms of an individual with Limb-girdle muscular dystrophy (LGMD) generally has difficulty walking, going both up and down stairs and raising from a chair. Difficulty bending over and falling on a regular basis are also common. Difficulty lifting certain objects is also a common présentation of LGMD as well as difficulty holding your arms out or above your head. Eventually the ability to walk/run détériorâtes.

The disease inevitably gets worse overtime, although progression is more rapid in some patients than others. Eventually the disease can affect other muscles such as the ones located in the face. The disease commonly leads to dependence on a wheelchair within years of symptom onset, but there is high inter-patient variability, with some patients maintaining mobility.

The muscle weakness is generally symmetric, proximal, and slowly progressive. In most cases, pain is not présent with LGMD, and mental function is not affected. LGMD can begin in childhood, adolescence, young adulthood or even later, the âge of onset is usually between 10 and 30. Both genders are affected equally, when limb-girdle muscular dystrophy begins in childhood the progression appears to be faster and the disease more disabling. When the disorder begins in adolescence or adulthood the disease is generally not as severe and progresses more slowly. There is no sensory neuropathy or autonomie or viscéral dysfunction at présentation.

In terms of the genetics of LGMD is an inherited disorder, though it may be inherited as a dominant or récessive genetic defect. The resuit of the defect is that the muscles cannot properly form certain proteins needed for normal muscle function. Several different proteins can be affected, and the spécifie protein that is absent or defective identifies the spécifie type of muscular dystrophy. Among the proteins affected in LGMD are α, β, γ and δ sarcoglycans. The sarcoglycanopathies could be possibly amenable to gene therapy.

Dysferlinopathy. Dysferlinopathy is an autosomal récessive neuromuscular disorder caused by a deficiency of functional dysferlin protein due to mutations in the dysferlin gene. Dysferlinopathy is characterized by progressive muscle wasting and is most often clinically diagnosed as Limb-girdle muscular dystrophy type 2B (LGMD2B) or Miyoshi muscular dystrophy 1 (MMD1; a type of distal muscular dystrophy), depending on the initial pattern of muscle involvement at diagnosis. Dysferlinopathy is a rare disease, the exact incidence of which has not yet been determined.

The symptoms of dysferlinopathy usually manifest in early adulthood between the âges of 16 and 25 and primarily affect the skeletal muscle of the limbs and the limb girdles (hips and shoulders), leaving critical muscles such as the heart and diaphragm largely unaffected. The majority of dysferlinopathy patients become non-ambulant within 10-20 years of diagnosis, but life expectancy is normal. There is a large amount of variability in the âge of onset and progression of the disease.

Although LGMD2B and MMD1 are both caused by dysferlin deficiency, a diagnosis of LGMD2B is given when weakness initially présents in the proximal muscles (thighs and upper arms) while a diagnosis of MMD1 is given when weakness initially présents in the distal muscles (calves and lower arms). In both cases, weakness eventually progresses to include both distal and proximal muscles. Both LGMD2B and MMD1 are very difficult to diagnose, and patients are often misdiagnosed many times before they are successfully diagnosed with dysferlinopathy.

While dysferlinopathy simply refers to the absence of the dysferlin protein, LGMD2B and Miyoshi myopathy 1 are the confirmed diagnosis only when mutations in the dysferlin gene can be detected. It is essential that patients receive a genetic diagnosis for participation in clinical studies and trials spécifie to dysferlinopathy. The Jain Foundation is a non-profit family organization (www.jain-foundation.org) that helps patients with limb girdle muscular dystrophy to achieve a genetic diagnosis by organizing testing and covering the cost.

Titin Myopathy. Titin, also known as connectin, is a protein that, in humans, is encoded by the TTN gene. Titin is a giant protein, greater than 1 pm in length, that functions as a molecular spring which is responsible for the passive elasticity of muscle. It is composed of 244 individually folded protein domains connected by unstructured peptide sequences. These domains unfold when the protein is stretched and refold when the tension is removed.

Titin is important in the contraction of striated muscle tissues. It connects the Z line to the M line in the sarcomere. The protein contributes to force transmission at the Z line and resting tension in the I band région. It limits the range of motion of the sarcomere in tension, thus contributing to the passive stiffness of muscle. Variations in the sequence of titin between different types of muscle (e.g., cardiac or skeletal) hâve been correlated with différences in the mechanical properties of these muscles.

Titin is a large abundant protein of striated muscle. Titin's primary functions are to stabilize the thick filament, center it between the thin filaments, prevent overstretching of the sarcomere, and to recoil the sarcomere like a spring after it is stretched. An N-terminal Z-disc région and a C-terminal M-line région bind to the Z-line and M-line of the sarcomere, respectively, so that a single titin molécule spans half the length of a sarcomere. Titin also contains binding sites for muscle-associated proteins so it serves as an adhesion template for the assembly of contractile machinery in muscle cells. It has also been identified as a structural protein for chromosomes. Considérable variability exists in the I-band, the M-line and the Zdisc régions of titin. Variability in the I-band région contributes to the différences in elasticity of different titin isoforms and, therefore, to the différences in elasticity of different muscle types. Of the many titin variants identified, five are described with complété transcript information available.

Mutations anywhere within the unusually long sequence of this gene can cause prématuré stop codons or other defects. Titin mutations are associated with hereditary myopathy with early respiratory failure, early-onset myopathy with fatal cardiomyopathy, core myopathy with heart disease, centronuclear myopathy, Limb-girdle muscular dystrophy type 2J, familial dilated cardiomyopathy 9, hypertrophie cardiomyopathy and tibial muscular dystrophy. Further research also suggests that no genetically linked form of any dystrophy or myopathy can be safely excluded from being caused by a mutation on the TTN gene. Truncating mutations in dilated cardiomyopathy patients are most commonly found in the A région; although truncations in the upstream I région might be expected to prevent translation of the A région entirely, alternative splicing créâtes some transcripts that do not encounter the prématuré stop codon, ameliorating its effect. Autoantibodies to titin are produced in patients with the autoimmune disease scleroderma.

Congénital myopathy “central core disease” caused by mutation in ryanodine receptor (mutation with pseudoexons). Central core disease (CCD), also known as central core myopathy, is an autosomal dominant congénital myopathy (inborn muscle disorder). It was first described by Shy and Magee in 1956. It is characterized by the appearance of the myofibril under the microscope.

The symptoms of CCD are variable, but usually involve hypotonia (decreased muscle tone) at birth, mild delay in child development (highly variable between cases), weakness of the facial muscles, and skeletal malformations such as scoliosis and hip dislocation.

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Symptoms may be présent at birth or may appear at any stage of life. There appears to be a growing number of people who do not become symptomatic until adulthood to middle âge. While generally not progressive, again there appears to be a growing number of people who do expérience a slow clinically significant progression of symptomatology. These cases 5 may hypothetically be due to the large number of gene mutations of ryanodine receptor malfunction, and with continued research may in fact be found to be clinical variants.

The diagnosis is made on the combination of typical symptoms and the appearance on biopsy (tissue sample) from muscle. The name dérivés from the typical appearance of the biopsy on light microscopy, where the muscle cells hâve cores that are devoid of mitochondria 10 and spécifie enzymes. Respiratory insufficiency develops in a small proportion of cases.

Creatine kinase and electromyography (EMG) tend to be normal.

Central core disease is inherited in an autosomal dominant fashion. Most cases hâve demonstrable mutations in the ryanodine receptor type 1 (RYR1) gene, which are often de novo (newly developed). People with CCD are at risk for malignant hyperthermia (MH) when 15 receiving general anesthésia.

There is no spécifie treatment but triggering anesthetics are avoided and relatives are screened for RYR1 mutations as these may make them susceptible to MH.

Myotonie Dystrophy. Myotonie dystrophy is a long term genetic disorder that affects muscle function. Symptoms include gradually worsening muscle loss and weakness. Muscles 20 often contract and are unable to relax. Other symptoms may include cataracts, intellectual disability, and heart conduction problems. In men there may be early balding and an inability to hâve children.

Myotonie dystrophy is an autosomal-dominant disorder which is typically inherited from a person's parents. There are two main types: type 1 (DM1 ) due to mutations in the DMPK 25 gene and type 2 (DM2) due to mutations in the CNBP gene. The disorder generally worsens in each génération. A type of DM1 may be apparent at birth. DM2 is generally milder. They are types of muscular dystrophy. Diagnosis is confirmed by genetic testing.

There is no cure. Treatments may include braces or wheelchairs, pacemakers, and noninvasive positive pressure ventilation. The médications mexiletine or carbamazepine are 30 occasionally helpful. Pain if it occurs may be treated with tricyclic antidepressants and nonsteroidal anti-inflammatory drugs (NSAIDs).

Myotonie dystrophy affects more than 1 in 8,000 people worldwide. While myotonie dystrophy can occur at any âge, onset is typically in the 20s and 30s. It is the most common form of muscular dystrophy that begins in adulthood. It was first described in 1909 with the underlying cause of type 1 determined in 1992.

Présentation of symptoms and signs varies considerably by form (DM1/DM2), severity and even unusual DM2 phenotypes. DM 1 symptoms for DM2 include problems with executive function (e.g., organization, concentration, word-finding) and hypersomnia. Conduction abnormalities are more common in DM1 than DM2, but ail people are advised to hâve an annual ECG. Both types are also associated with insulin résistance. Myotonie dystrophy may hâve a cortical cataract with a blue dot appearance, or a posterior subcapsular cataract.

DM2 is generally milder than DM1, with generally fewer DM2 people requiring assistive devices than DM1 people. In addition, the severe congénital form that affects babies in DM1 has not been found in DM2 and the early onset of symptoms is rarely noted to appear in younger people in the medical literature.

Symptoms may appear at any time from infancy to adulthood. DM causes general weakness, usually beginning in the muscles of the hands, feet, neck, or face. It slowly progresses to involve other muscle groups, including the heart. DM affects a wide variety of other organ Systems as well.

Myotonie dystrophy is a genetic condition which is inherited in an autosomal dominant pattern and thus will be passed along to 50% of a carrier's offspring, on average. Myotonie dystrophy is one of several known trinucleotide repeat disorders. Certain areas of DNA hâve repeated sequences of two or three nucléotides.

Myotonie dystrophy (DM) is an inherited disease. A severe form of DM, congénital myotonie dystrophy, may appear in newborns of mothers who hâve DM. Congénital myotonie dystrophy can also be inherited via the paternal gene, although it is said to be relatively rare. Congénital means that the condition is présent from birth.

In DM1, the affected gene is called DMPK, which codes for myotonie dystrophy protein kinase, a protein expressed predominantly in skeletal muscle. The gene is located on the long arm of chromosome 19.

In DM1, there is an expansion of the cytosine-thymine-guanine (CTG) triplet repeat in the DMPK gene. Between 5 and 37 repeats is considered normal, while individuals with b 48 between 38 and 49 repeats are considered to hâve a pre-mutation and are at risk of having children with further expanded repeats and, therefore, symptomatic disease. Individuals with greaterthan 50 repeats are almost invariably symptomatic, with some noted exceptions. Longer repeats are usually associated with earlier onset and more severe disease.

DMPK alleles with greater than 37 repeats are unstable and additional trinucleotide repeats may be inserted during cell division in mitosis and meiosis. Consequently, the children of individuals with premutations or mutations inherit DMPK alleles which are longer than their parents and therefore are more likely to be affected or display an earlier onset and greater severity of the condition, a phenomenon known as anticipation. Interestingly, paternal 10 transmission of the condition is very uncommon, possibly due to sélection pressures against sperm with expanded repeats, but anticipation tends to be less severe than in cases of maternai inheritance.

The RNA from the expanded trinucleotide repeat région forms intranucleoplasmic hairpin loops due to the extensive hydrogen bonding between C-G base pairs, and it has been 15 demonstrated that these sequester the splicing regulator MBNL1 to form distinctive foci by labelling it with GFP and a probe oligonucleotide with the red-fluorescent dye Cyanine5 (Cy5).

DM2 is caused by a defect of the ZNF9 gene on chromosome 3. The spécifie defect is a repeat of the cytosine-cytosine-thymine-guanosine (CCTG) tetranucleotide in the ZNF9 gene. As it involves the repeat of four nucléotides, it is not a trinucleotide repeat disorder, but rather 20 a tetranucleotide repeat disorder.

The repeat expansion for DM2 is much larger than for DM1, ranging from 75 to over 11,000 repeats. Unlike in DM1, the size of the repeated DNA expansion in DM2 does not appear to make a différence in the âge of onset or disease severity. Anticipation appears to be less significant in DM2 and most current reviews only report mild anticipation as a feature of 25 DM2.

Friedreich’s ataxia. Friedreich's ataxia is an autosomal récessive inherited disease that causes progressive damage to the nervous System. It manifests in initial symptoms of poor coordination such as gait disturbance; it can also lead to scoliosis, heart disease and diabètes, but does not affect cognitive function. The disease progresses until a wheelchair is required for 30 mobility. Its incidence in the general population is roughly 1 in 50,000.

The particular genetic mutation (expansion of an intronic GAA triplet repeat in the FXN gene) leads to reduced expression of the mitochondrial protein frataxin. Over time this deficiency causes the aforementioned damage, as well as frequent fatigue due to effects on cellular metabolism.

The ataxia of Friedreich's ataxia results from the degeneration of nervous tissue in the spinal cord, in particular sensory neurons essential (through connections with the cerebellum) for directing muscle movement of the arms and legs. The spinal cord becomes thinner and nerve cells lose some of their myelin sheath (the insulating covering on some nerve cells that helps conduct nerve impulses).

Symptoms typically begin sometime between the âges of 5 to 15 years, but in Late Onset FA may occur in the 20s or 30s. Symptoms include any combination, but not necessarily ail, of muscle weakness in the arms and legs, loss of coordination, vision impairment, hearing impairment, slurred speech, curvature of the spine, high plantar arches (pes cavus deformity of the foot), diabètes (about 20% of people with Friedreich's ataxia develop carbohydrate intolérance and 10% develop diabètes mellitus) and heart disorders (e.g., atrial fibrillation, and résultant tachycardia (fast heart rate) and hypertrophie cardiomyopathy).

It présents before 22 years of âge with progressive staggering or stumbling gait and frequent falling. Lower extremities are more severely involved. The symptoms are slow and progressive. Long-term observation shows that many patients reach a plateau in symptoms in the patient's early adulthood. On average, after 10-15 years with the disease, patients are usually wheelchair bound and require assistance with ail activities of daily living.

The following physical signs may be detected on physical examination: cerebellar signs such as nystagmus, fast saccadic eye movements, truncal ataxia, dysarthria, dysmetria, lower motor neuron lésions, such as absent deep tendon reflexes; pyramidal signs such as extensor plantar responses, and distal weakness are commonly found; dorsal column aspects, such as loss of vibratory and proprioceptive sensation occurs. Cardiac involvement occurs in 91% of patients, including cardiomegaly (up to dilated cardiomyopathy), symmetrical hypertrophy, heart murmurs, and conduction defects. Médian âge of death is j5 years, while females hâve better prognosis with a 20-year survival of 100% as compared to 63% in men. Twenty percent of cases are found in association with diabètes mellitus.

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Friedreich's ataxia is an autosomal récessive disorder that occurs when the FXN gene contains amplified intronic GAA repeats (an example ot trinucleotide repeat expansion). The FXN gene encodes the protein frataxin. GAA repeat expansion causes frataxin levels to be reduced. Frataxin is an iron-binding protein responsible for forming iron-sulphur clusters. One 5 resuit of frataxin deficiency is mitochondrial iron overload which can cause damage to many proteins. The exact rôle of frataxin in normal physiology remains unclear. The gene is located on chromosome 9.

The mutant gene contains expanded GAA triplet repeats in the First intron; in a few pedigrees, point mutations hâve been detected. Because the defect is located in an intron (which 10 is removed from the mRNA transcript between transcription and translation), this mutation does not resuit in the production of abnormal frataxin proteins. Instead, the mutation causes gene silencing (i.e·, the mutation decreases the transcription of the gene) through induction of a heterochromatin structure in a manner similar to position-effect variegation.

Besides reducing expression of frataxin, long tracts of GAA repeats induce 15 chromosome breaks in in vivo yeast studies.

Huntington's disease. Huntington's disease (HD), also known as Huntington's chorea, is an inherited disorder that results in death of brain cells. The earliest symptoms are often subtle problems with mood or mental abilities. A general lack of coordination and an unsteady gait often follow. As the disease advances, uncoordinated, jerky body movements become more 20 apparent. Physical abilities gradually worsen until coordinated movement becomes difficult and the person is unable to talk. Mental abilities generally décliné into dementia. The spécifie symptoms vary somewhat between people. Symptoms usually begin between 30 and 50 years of âge, but can start at any âge. The disease may develop earlier in life in each successive génération. About 8% of cases start before the âge of 20 years and typically présent with 25 symptoms more similar to Parkinson's disease. People with HD often underestimate the degree of their problems.

HD is typically inherited from a person's parents with 10% of cases due to a new mutation. The disease is caused by an autosomal dominant mutation in either of an individual's two copies of a gene called Huntingtin. This means a child of an affected person typically has 30 a 50% chance of inheriting the disease. The Huntingtin gene provides the genetic information for a protein that is also called “huntingtin.” Expansion of CAG (cytosine-adenine-guanine) triplet repeats in the gene coding for the Huntingtin protein results in an abnormal protein, which gradually damages cells in the brain, through mechanisms that are not fully understood. Diagnosis is by genetic testing, which can occur at any point in time regardless of whether or not symptoms are présent. This fact raises several ethical debates: the âge at which an individual is considered mature enough to choose testing; whether parents hâve the right to hâve their children tested; and managing confidentiality and disclosure oftest results.

There is no cure for HD. Full-time care is required in the later stages of the disease. Treatments can relieve some symptoms and in some improve quality of life. The best evidence for treatment of the movement problems is with tetrabenazine. HD affects about 4 to 15 in 100,000 people of European descent. It is rare among Japanese and occurs at an unknown rate in Africa. The disease affects men and women equally. Complications such as pneumonia, heart disease, and physical injury from faits reduce life expectancy. Suicide is the cause of death in about 9% of cases. Death typically occurs fifteen to twenty years from when the disease was first detected.

Symptoms of Huntington's disease most commonly become noticeable between the âges of 35 and 44 years, but they can begin at any âge from infancy to old âge. In the early stages, there are subtle changes in personality, cognition, and physical skills. The physical symptoms are usually the first to be noticed, as cognitive and behavioral symptoms are generally not severe enough to be recognized on their own at the earlier stages. Almost everyone with Huntington's disease eventually exhibits similar physical symptoms, but the onset, progression and extent of cognitive and behavioral symptoms vary significantly between individuals.

The most characteristic initial physical symptoms are jerky, random, and uncontrollable movements called chorea. Chorea may be initially exhibited as general restlessness, small unintentionally initiated or uncompleted motions, lack of coordination, or slowed saccadic eye movements. These minor motor abnormalities usually précédé more obvious signs of motor dysfunction by at least three years. The clear appearance of symptoms such as rigidity, writhing motions or abnormal posturing appear as the disorder progresses. These are signs that the System in the brain that is responsible for movement has been affected. Psychomotor functions become increasingly impaired, such that any action that requires muscle control is affected. Common conséquences are physical instability, abnormal facial expression, and difficulties chewing, swallowing, and speaking. Eating difficulties commonly cause weight loss and may lead to malnutrition. Sleep disturbances are also associated symptoms. Juvénile HD differs from these symptoms in that it generally progresses faster and chorea is exhibited briefly, if at ail, with rigidity being the dominant symptom. Seizures are also a common symptom of this form of HD.

Cognitive abilities are progressively impaired. Especially affected are executive functions which include planning, cognitive flexibility, abstract thinking, rule acquisition, initiation of appropriate actions, and inhibition of inappropriate actions. As the disease progresses, memory déficits tend to appear. Reported impairments range from short-term memory déficits to long-term memory difficulties, including déficits in episodic (memory of one's life), procédural (memory of the body of how to perform an activity) and working memory. Cognitive problems tend to worsen over time, ultimately leading to dementia. This pattern of déficits has been called a subcortical dementia syndrome to distinguish it from the typical effects of cortical dementias, e.g., Alzheimer's disease.

Reported neuropsychiatrie manifestations are anxiety, dépréssion, a reduced display of émotions (blunted affect), egocentrism, aggression, and compulsive behavior, the latter of which can cause or worsen addictions, including alcoholism, gambling, and hypersexuality. Difficulties in recognizing other people's négative expressions hâve also been observed. The prevalence of these symptoms is highly variable between studies, with estimated rates for lifetime prevalence of psychiatrie disorders between 33% and 76%. For many sufferers and their families, these symptoms are among the most distressing aspects of the disease, often affecting daily functioning and constituting reason for institutionalization. Suicidai thoughts and suicide attempts are more common than in the general population. Often individuals hâve reduced awareness of chorea, cognitive and emotional impairments.

Mutant Huntingtin is expressed throughout the body and associated with abnormalities in peripheral tissues that are directly caused by such expression outside the brain. These abnormalities include muscle atrophy, cardiac failure, impaired glucose tolérance, weight loss, osteoporosis, and testicular atrophy.

Ail humans hâve two copies of the Huntingtin gene (HTT), which codes for the protein Huntingtin (HTT). The gene is also called HD and IT15, which stands for ’interesting transcript 15'. Part of this gene is a repeated section called a trinucleotide repeat, which varies in length between individuals and may change length between générations. If the repeat is présent in a healthy gene, a dynamic mutation may increase the repeat count and resuit in a defective gene. When the length of this repeated section reaches a certain threshold, it produces an altered form b ⁵³ of the protein, called mutant Huntingtin protein (mHTT). The differing functions of these proteins are the cause of pathological changes which in tum cause the disease symptoms. The Huntington's disease mutation is genetically dominant and almost fully pénétrant: mutation of either of a person's /fTTalleles causes the disease. It is not inherited according to sex, but the 5 length of the repeated section of the gene and hence its severity can be influenced by the sex of the affected parent.

HD is one of several trinucleotide repeat disorders which are caused by the length of a repeated section of a gene exceeding a normal range. The HTT gene is located on the short arm of chromosome 4 at 4p 16.3. HTTcontains a sequence ofthree DNA bases—cytosine-adenine10 guanine (CAG)—repeated multiple times (i.e., ... CAGCAGCAG ...), known as a trinucleotide repeat. CAG is the 3-letter genetic code (codon) for the amino acid glutamine, so a sériés of them results in the production of a chain of glutamine known as a polyglutamine tract (or polyQ tract), and the repeated part of the gene, the PolyQ région.

Generally, people hâve fewer than 36 repeated glutamines in the polyQ région which 15 results in production of the cytoplasmic protein Huntingtin. However, a sequence of 36 or more glutamines results in the production of a protein which has different characteristics. This altered form, called mutant huntingtin (mHTT), increases the decay rate of certain types of neurons. Régions of the brain hâve differing amounts and reliance on these types of neurons, and are affected accordingly. Generally, the number of CAG repeats is related to how much this process 20 is affected, and accounts for about 60% of the variation of the âge of the onset of symptoms.

The remaining variation is attributed to environment and other genes that modify the mechanism of HD. 36-39 repeats resuit in a reduced-penetrance form of the disease, with a much later onset and slower progression of symptoms. In some cases the onset may be so late that symptoms are never noticed. With very large repeat counts, HD has full penetrance and 25 can occur under the âge of 20, when it is then referred to as juvénile HD, akinetic-rigid, or Westphal variant HD. This accounts for about 7% of HD carriers.

Ataxia telangiectasia. Ataxia-telangiectasia (AT or A-T), also referred to as ataxiatelangiectasia syndrome or Louis-Bar syndrome, is a rare, neurodegenerative, autosomal récessive disease causing severe disability. Ataxia refers to poor coordination and 30 telangiectasia to small dilated blood vessels, both of which are hallmarks of the disease.

A-T affects many parts of the body. It impairs certain areas of the brain including the cerebellum, causing difficulty with movement and coordination. It weakens the immune

System, causing a prédisposition to infection. It prevents repair of broken DNA, increasmg the risk of cancer.

Symptoms most often first appear in early childhood (the toddler stage) when children begin to walk. Though they usually start walking at a normal âge, they wobble or sway when walking, standing still or sitting, and may appear almost as if they are drunk. In late pre-school and early school âge, they develop difficulty moving their eyes in a natural manner from one place to the next (oculomotor apraxia). They develop slurred or distorted speech, and swallowing problems. Some hâve an increased number of respiratory tract infections (ear infections, sinusitis, bronchitis, and pneumonia). Because not ali children develop in the same manner or at the same rate, it may be some years before A-T is properly diagnosed. Most children with A-T hâve stable neurologie symptoms for the first 4-5 years of life, but begin to show increasing problems in early school years.

A-T is caused by a defect in the ATM gene, which is responsible for managing the cell’s response to multiple forms of stress including double-strand breaks in DNA. In simple terms, the protein produced by the ATM gene recognizes that there is a break in DNA, recruits other proteins to fix the break, and stops the cell from making new DNA until the repair is complété.

There is substantial variability in the severity of features of A-T among affected individuals, and at different âges. The foilowing symptoms or problems are either common or important features of A-T:

Ataxia (difficulty with control of movement) that is apparent early but worsens in school to pre-teen years.

Oculomotor apraxia (difficulty with coordination of head and eye movement when shifting gaze from one place to the next).

Involuntary movements.

Telangiectasia (dilated blood vessels) over the white (sciera) of the eyes, making them appear bloodshot. These are not apparent in infancy and may first appear at âge 5-8 years. Telangiectasia may also appear on sun-exposed areas of skin.

Problems with infections, especially of the ears, sinuses and lungs.

Increased incidence of cancer (primarily, but not exclusively, lymphomas and leukemias).

Delayed onset or incomplète pubertal development, and very early ménopausé.

Slowed rate of growth (weight and/or height).

Drooling particularly in young children when they are tired or concentrating on activities.

Dysarthria (slurred, slow, or distorted speech sounds).

Diabètes in adolescence or later.

Prématuré changes in haïr and skin.

Many children are initially misdiagnosed as having ataxie cérébral palsy. The diagnosis of AT may not be made until the preschool years when the neurologie symptoms of impaired gait, hand coordination, speech and eye movement appear or worsen, and the telangiectasia first appear. Because A-T is so rare, doctors may not be familiar with the symptoms, or methods of making a diagnosis. The late appearance of telangiectasia may be a barrier to the diagnosis. It may take some time before doctors consider A-T as a possibility because of the early stability of symptoms and signs.

A-T is caused by mutations in the ATM (ATM serine/threonine kinase or ataxiatelangiectasia mutated) gene, which was cloned in 1995. ATM is located on human chromosome 11(1 lq22.3) and is made up of 69 exons spread across 150kb of genomic DNA.

The mode of inheritance for A-T is autosomal récessive. Each parent is a carrier, meaning that they hâve one normal copy of the A-T gene (ATM) and one copy which is mutated. A-T occurs if a child inherits the mutated A-T gene from each parent, so in a family with two carrier parents, there is 1 chance in 4 that a child born to the parents will hâve the disorder. Prénatal diagnosis (and carrier détection) can be carried out in families if the errors (mutation) in an affected child’s two ATM genes hâve been identified. The process of getting this done can be complicated and, as it requires time, should be arranged before conception.

Looking for mutations in the ATM gene of an unrelated person (for example, the spouse of a known A-T carrier) présents significant challenges. Genes often hâve variant spellings (polymorphisms) which do not affect function. In a gene as large as ATM, such variant ⁵⁶ spellings are likely to occur and doctors cannot always predict whether a spécifie variant will or will not cause disease. Genetic counseling can help family members of an A-T patient understand what can or cannot be tested, and how the test results should be interpreted.

Carriers of A-T, such as the parents of a person with A-T, hâve one mutated copy of 5 the ATM gene and one normal copy. They are generally healthy, but there is an increased risk of breast cancer in women. This finding has been confirmed in a variety of different ways, and is the subject of current research. Standard surveillance (including monthly breast self-exams and mammography at the usual schedule for âge) is recommended, unless additional tests are indicated because the individual has other risk factors (e.g, family history of breast cancer).

Neurofibromatosis type 1 and 2. Neurotibromatosis (NF) is a group of three conditions in which tumors grow in the nervous System. The three types are neurofibromatosis type 1 (NF1), neurofibromatosis type 2 (NF2), and schwannomatosis. In NF1 symptoms include light brown spots on the skin, freckles in the armpit and groin, small bumps within nerves, and scoliosis. In NF2 there may be hearing loss, cataracts at a young âge, balance problems, flesh colored skin flaps, and muscle wasting. The tumors are generally noncancerous.

The cause is a genetic mutation in certain genes. In half of cases these are inherited from a person's parents while in the rest they occur during early development. The tumors involve supporting cells in the nervous System rather than the neurons. In NF1 the tumors are 20 neurofibromas (tumors of the peripheral nerves) while in NF2 and schwannomatosis tumors of Schwann cells are more common. Diagnosis is typically based on the signs and symptoms and occasionally supported by genetic testing.

There is no known prévention or cure. Surgery may be done to remove tumors that are causing problems or hâve become cancerous. Radiation and chemotherapy may also be used if 25 cancer occurs. A cochlear implant or auditory brainstem implant may help some who hâve hearing loss.

In the United States about 1 in 3,500 people hâve NF 1 and 1 in 25,000 hâve NF2. Males and females are affected equally frequently. In NF1 symptom are often présent at birth and otherwise develop before 10 years of âge. While the condition typically worsens with time 30 most people with NF1 hâve a normal life expectancy. In NF2 symptoms may not become apparent until early adulthood. NF2 increases the risk of early death. Descriptions of the condition occur as far back as the 1 st century.

Neurofibromatosis (NF1) in early life may cause learning and behavior problems about 60% of children who hâve NF1 hâve a mild form of difficulty in school. In terms of signs the individual might hâve are the following: six or more light brown dermatological spots (“café-au-lait spots), at least two neurofibromas, at least two growths on the eye's iris, and abnormal growth of the spine (scoliosis).

Neurofibromatosis is an autosomal dominant disorder, which means only one copy of the affected gene is needed for the disorder to develop. Therefore, if only one parent has neurofibromatosis, his or her children hâve a 50% chance of developing the condition as well. The affected child could hâve mild NF1 even though inherited from a parent with a severe form of the disorder. There two types of neurofibromatosis. Neurofibromatosis type I is characterized by nerve tissue growth into tumors (neurofibromas) that may be benign and may cause serious damage by compressing nerves and other tissues. Neurofibromatosis type II exhibits bilateral acoustic neuromas (tumors of the vestibulocochlear nerve or cranial nerve 8 (CN VIII) also known as schwannoma), often leading to hearing loss.

Schwannomatosis, in which painful schwannomas develop on spinal and peripheral nerves.

CEP290 mutation in Leber congénital amaurosis. Centrosomal protein of 290 kDa is a protein that in humans is encoded by the CEP290 gene. CEP290 is located on the Q arm of chromosome 12. The gene CEP290 is a centrosomal protein that plays an important rôle in centrosome and cilia development. This gene is vital in the formation of the primary cilium, a small antenna-like projections of the cell membrane that plays an important rôle in the photoreceptors at the back of the retina (which detect light and color) and in the kidney, brain, and many other organs of the body. Knocking down levels of the CEP290 gene transcript resulted in dramatic suppression of ciliogenesis in retinal pigment épithélial cells in culture, proving just how important CEP290 is to cilia formation.

On a molecular level, CEP290 has been shown to play a critical regulatory and structural rôle in primary cilium formation. Recent studies hâve implicated CEP290 as a microtubule and membrane binding protein that might serve as a structural link between the microtubule core of the cilium and the overlying ciliary membrane. Disruption of CEP290's microtubule binding domain in the rd!6 mouse mode! of CEP290 disease has been shown to resuit in rapid and dramatic retinal degeneration, demonstrating the importance of CEP290 microtubule binding in disease. The rôle of CEP290 in promoting ciliogensis is inhibited both by auto-regulatory domains found at either end of the CEP290 protein and through CEP290's interaction with the inhibitory protein CPI 10.

The discovery of the CEP290 gene has led researchers to find another gene critical in retinal function, LCA5. Clinical trials involving gene replacement of these two genes hâve started in Philadelphia, where researchers are hopeful that Leber Congénital Amaurosis will one day be cured.

This gene encodes a protein with 13 putative coiled-coil domains, a région with homology to SMC chromosome ségrégation ATPases, six KID motifs, three tropomyosin homology domains and an ATP/GTP binding site motif A. The protein is localized to the centrosome and cilia and has sites for N-glycosylation, tyrosine sulfation, phosphorylation, Nmyristoylation, and amidation.

Mutations in this gene hâve been associated with Joubert syndrome and nephronophthisîs, and recently with a frequent form of Leber's Congénital Amaurosis, called LC A10. The presence of antibodies against this protein is associated with several forms of cancer.

A mutation in this gene leads to infant and child blindness, a disease known as Leber Congénital Amaurosis. As of today, 35 different mutations in CEP290 are responsible for causing LCA. Other mutations in CEP290 hâve also been identified in causing Meckel Syndrome and Joubert Syndrome, a few among many syndromes. A defective CEP290 gene is usually the cause of these disorders due to abnormal cilia. It is unknown how one mutation in a gene can cause so many different types of syndromes, particularly many of which affect the Central Nervous System.

III. CRISPR Systems

A. CRISPRs

CRISPRs (clustered regularly interspaced short palindromie repeats) are DNA loci containing short répétitions of base sequences. Each répétition is followed by short segments of “spacer DNA” from previous exposures to a virus. CRISPRs are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea. CRISPRs are often associated with Cas genes that code for proteins related to CRISPRs. The CRISPR/Cas System is a prokaryotic immune System that confers résistance to foreign genetic éléments such as plasmids and phages and provides a form of acquired immunity. CRISPR spacers recognize and silence these exogenous genetic éléments like RNAi in eukaryotic organisms.

CRISPR repeats range in size from 24 to 48 base pairs. They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromie. Repeats are separated by spacers of similar length. Some CRISPR spacer sequences exactly match sequences from plasmids and phages, although some spacers match the prokaryote’s genome (self-targeting spacers). New spacers can be added rapidly in response to phage infection.

B. Cas Nucleases

CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays. As of 2013, more than forty different Cas protein families had been described. Of these protein families, Casl appears to be ubiquitous among different CRISPR/Cas Systems. Particular combinations of cas genes and repeat structures hâve been used to define 8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with an additionai gene module encoding repeat-associated mysterious proteins (RAMPs). More than one CRISPR subtype may occur in a single genome. The sporadic distribution of the CRISPR/Cas subtypes suggests that the System is subject to horizontal gene transfer during microbial évolution.

Exogenous DNA is apparently processed by proteins encoded by Cas genes into small éléments (~30 base pairs in length), which are then somehow inserted into the CRISPR locus near the leader sequence. RNAs from the CRISPR loci are constitutively expressed and are processed by Cas proteins to small RNAs composed of individual, exogenously-derived sequence éléments with a flanking repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic éléments at the RNA or DNA level. Evidence suggests functional diversity among CRISPR subtypes. The Cse (Cas subtype Ecoli) proteins (called CasA-E in E. coli) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacerrepeat units that Cascade retains. In other prokaryotes, Cas6 processes the CRISPR transcripts. Interestingly, CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Casl and Cas2. The Cmr (Cas RAMP module) proteins found in Pyrococcusfuriosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs. RNA-guided CRISPR enzymes are classified as type V restriction enzymes.

Cas9 is a nuclease, an enzyme specialized for cutting DNA, with two active cutting sites, one for each strand of the double hélix. The team demonstrated that they could disable one or both sites while preserving Cas9’s ability to locate its target DNA. Jinek et al. (2012) combined tracrRNA and spacer RNA into a single-guide RNA molécule that, mixed with Cas9, can find and eut the correct DNA targets and such synthetic guide RNAs are used for gene editing.

Cas9 proteins are highly enriched in pathogenic and commensal bacteria. CRISPR/Casmediated gene régulation may contribute to the régulation of endogenous bacterial genes, particularly during bacterial interaction with eukaryotic hosts. For example, Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein that is critical for F. novicida to dampen host response and promote virulence. Wang et al. (2013) showed that coinjection of Cas9 mRNA and sgRNAs into the germline (zygotes) generated mice with mutations. Dehvery of Cas9 DNA sequences also is contemplated.

The Systems CRISPR/Cas are separated into three classes. Class 1 uses several Cas proteins together with the CRISPR RNAs (crRNA) to build a functional endonuclease. Class 2 CRISPR Systems use a single Cas protein with a crRNA. Cpfl has been recently identified as a Class H, Type V CRISPR/Cas Systems containing a 1,300 amino acid protein. See also U.S. Patent Publication 2014/0068797, which is incorporated by référencé in its entirety.

In some embodiments, the compositions ofthe disclosure include a small version of a Cas9 from the bacterium Staphylococcus aureus (UniProt Accession No. J7RUA5). The small version of the Cas9 provides advantages over wildtype or full length Cas9. In some embodiments the Cas9 is a spCas9 (AddGene).

C. Cpfl Nucleases

Clustered Regularly Interspaced Short Palindromie Repeats from Prevotella and Francisella 1 or CRISPR/Cpfl is a DNA-editing technology which shares some similarities with the CRISPR/Cas9 System. Cpfl is an RNA-guided endonuclease ofa class II CRISPR/Cas System. This acquired immune mechanism is found in Prevotella and Francisella bacteria. It prevents genetic damage from viruses. Cpfl genes are associated with the CRISPR locus, b ⁶¹ coding for an endonuclease that use a guide RNA to find and cleave viral DNA. Cpfl is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 System limitations.

Cpfl appears in many bacterial species. The ultimate Cpfl endonuclease that was developed into a tool for genome editing was taken from one of the first 16 species known to harbor it.

In embodiments, the Cpfl is a Cpfl enzyme from Acidaminococcus (species BV3L6, UniProt Accession No. U2UMQ6; SEQ ID NO: 870), having the sequence set forth below:

MTQFEGFTNL YQVSKTLRFE L1PQGKTLKH IQEQGFIEED

KARNDHYKEL KPI1DRIYKT

YADQCLQLVQ LDWENLSAAI DSYRKEKTEE TRNALIEEQA TYRNAIHDYF IGRTDNLTDA

121 INKRHAEIYK GLFKAELFNG KVLKQLGTVT TTEHENALLR SFDKFTTYFS GFYENRKNVF

181 SAEDISTAIP HRIVQDNFPK FKENCH1FTR LITAVPSLRE HFENVKKAIG

IFVSTSIEEV

241 FSFPFYNQLL TQTQIDLYNQ LLGGISREAG TEKIKGLNEV LNLAIQKNDE TAHIIASLPH

301 RFIPLFKQIL SDRNTLSFIL EEFKSDEEVI QSFCKYKTLL RNENVLETAE

ALFNELNSID

361 LTHIFISHKK LETISSALCD HWDTLRNALY ERRISELTGK ITKSAKEKVQ RSLKHED1NL

421 QEIISAAGKE LSEAFKQKTS EILSHAHAAL DQPLPTTLKK QEEKEILKSQ LDSLLGLYHL

481 LDWFAVDESN EVDPEFSARL TGIKLEMEPS LSFYNKARNY ATKKPYSVEK

FKLNFQMPTL

541 ASGWDVNKEK NNGAILFVKN GLYYLGIMPK QKGRYKALSF EPTEKTSEGF DKMYYDYFPD

601 AAKMIPKCST QLKAVTAHFQ THTTPILLSN NFIEPLE1TK EIYDLNNPEK

EPKKFQTAYA

661 KKTGDQKGYR EALCKWIDFT RDFLSKYTKT TSIDLSSLRP SSQYKDLGEY

YAELNPLLYH

721 ISFQRIAEKE IMDAVETGKL YLFQIYNKDF AKGHHGKPNL HTLYWTGLFS

PENLAKTSIK

781 LNGQAELFYR PKSRMKRMAH RLGEKMLNKK LKDQKTPIPD TLYQELYDYV NHRLSHDLSD

841 EARALLPNVI TKEVSHEIIK DRRFTSDKFF FHVPITLNYQ AANSPSKFNQ RVNAYLKEHP

901 ETPIIGIDRG ERNLIY1TV1 DSTGKILEQR SLNTIQQFDY QKKLDNREKE RVAARQAWSV

961 VGTIKDLKQG YLSQVIF1E1V DLMIHYQAVV VLENLNFGFK SKRTGIAEKA VYQQFEKMLI

1021 DKLNCLVLKD YPAEKVGGVL NPYQLTDQFT SFAKMGTQSG FLFYVPAPYT SKIDPLTGFV

1081 DPFVWKTIKN HESRKHFLEG FDFLHYDVKT GDFILHFKMN RNLSFQRGLP GFMPAWDIVF

141 EKNETQFDAK GTPFIAGKRI VPVIENHRFT GRYRDLYPAN ELIALLEEKG IVFRDGSNIL

1201 PKLLENDDSH AIDTMVALIR SVLQMRNSNA ATGEDYINSP VRDLNGVCFD SRFQNPEWPM

1261 DADANGAYHI ALKGQLLLNH LKESKDLKLQ NGISNQDWLA YIQELRN

In some embodiments, the Cpfl is a Cpfl enzyme from Lachnospiraceae (species ND2006, UniProt Accession No. A0A182DWE3; SEQ ID NO: 871), having the sequence set forth below:

AASKLEKFTN CYSLSKTLRF KAIPVGKTQE NIDNKRLLVE DEKRAEDYKG VKKLLDRYYL

SFINDVLHSI KLKNLNNYIS LFRKKTRTEK ENKELENLEI NLRKEIAKAF KGAAGYKSLF

121 KKDIIETILP EAADDKDEIA LVNSFNGFTT AFTGFFDNRE NMFSEEAKST SIAFRCINEN

181 LTRYISNMDI FEKVDAIFDK HEVQEIKEKI LNSDYDVEDF FEGEFFNFVL TQEGIDVYNA

241 IIGGFVTESG EKIKGLNEYI NLYNAKTKQA LPKFKPLYKQ VLSDRESLSF YGEGYTSDEE b ⁶³

301 VLEVFRNTLN KNSEIFSSIK KLEKLFKNFD EYSSAGIFVK NGPAISTISK DIFGEWNLIR

361 DKWNAEYDDI HLKKKAVVTE KYEDDRRKSF KKIGSFSLEQ LQEYADADLS VVEKLKEIII

421 QKVDEIYKVY GSSEKLFDAD FVLEKSLKKN DAVVAIMKDL LDSVKSFENY

IKAFFGEGKE

481 TNRDESFYGD FVLAYDILLK VDHIYDAIRN YVTQKPYSKD KFKLYFQNPQ FMGGWDKDKE

541 TDYRATILRY GSKYYLAIMD KKYAKCLQKI DKDDVNGNYE KINYKLLPGP

NKMLPKVFFS

601 KKWMAYYNPS EDIQKIYKNG TFKKGDMFNL NDCHKLIDFF KDSISRYPKW SNAYDFNFSE

661 TEKYKDIAGF YREVEEQGYK VSFESASKKE VDKLVEEGKL YMFQIYNKDF SDKSHGTPNL

721 HTMYFKLLFD ENNHGQIRLS GGAELFMRRA SLKKEELVVH PANSPIANKN

PDNPKKTTTL

781 SYDVYKDKRF SEDQYELHIP IA1NKCPKNI FKINTEVRVL LKHDDNPYVI GIDRGERNLL

841 YIVVVDGKGN IVEQYSLNEIINNFNGIRIK TDYHSLLDKK EKERFEARQN

WTSIENIKEL

901 KAGYISQVVH KICELVEKYD AVIALEDLNS GFKNSRVKVE KQVYQKFEKM LIDKLNYMVD

961 KKSNPCATGG ALKGYQITNK FESFKSMSTQ NGFIFYIPAW LTSKIDPSTG FVNLLKTKYT

1021 SIADSKKFIS SFDRIMYVPE EDLFEFALDY KNFSRTDADY IKKWKLYSYG

NRIR1FAAAK

1081 KNNVFAWEEV CLTSAYKELF NKYGINYQQG DIRALLCEQS DKAFYSSFMA LMSLMLQMRN

1141 SITGRTDVDF LISPVKNSDG IFYDSRNYEA QENA1LPKNA DANGAYNIAR KVLWAIGQFK

1201 KAEDEKLDKV K1AISNKEWL EYAQTSVK

In some embodiments, the Cpfl is codon optimized for expression in mammalian cells.

In some embodiments, the Cpfl is codon optimized for expression in human cells or mouse cells.

The Cpfl locus contains a mixed alpha/beta domain, a RuvC-I followed by a helical région, a RuvC-II and a zinc finger-like domain. The Cpfl protein has a RuvC-like 10 endonuclease domain that is similar to the RuvC domain of Cas9. Furthermore, Cpfl does not hâve a HNH endonuclease domain, and the N-terminal of Cpfl does not hâve the alpha-helical récognition lobe of Cas9.

Cpfl CRISPR-Cas domain architecture shows that Cpfl is functionally unique, being classified as Class 2, type V CRISPR System. The Cpfl loci encode Casl, Cas2 and Cas4 15 proteins more similar to types I and III than from type II Systems. Database searches suggest the abundance of Cpfl-family proteins in many bacterial species.

Functional Cpfl does not require a tracrRNA, therefore, only crRNA is required. This benefits genome editing because Cpfl is not only smaller than Cas9, but also it has a smaller sgRNA molécule (proximately half as many nucléotides as Cas9).

2o The Cpfl-crRNA complex cleaves target DNA or RNA by identification of a protospacer adjacent motif 5'-YTN-3' (where Y is a pyrimidine and N is any nucleobase) or 5'-TTN-3', in contrast to the G-rich PAM targeted by Cas9. After identification of PAM, Cpfl introduces a sticky-end-like DNA double- stranded break of 4 or 5 nucléotides overhang.

The CRISPR/Cpfl System consist of a Cpfl enzyme and a guide RNA that finds and 25 positions the complex at the correct spot on the double hélix to cleave target DNA. CRISPR/Cpfl Systems activity has three stages:

Adaptation, during which Casl and Cas2 proteins facilitate the adaptation of small fragments of DNA into the CRISPR array;

Formation of crRNAs: processing of pre-cr-RNAs producing of mature crRNAs to 30 guide the Cas protein; and

F 65

Interférence, in which the Cpfl is bound to a crRNA to form a binary complex to identify and cleave a target DNA sequence.

D. gRNA

As an RNA guided protein, Cas9 requires a short RNA to direct the récognition of DNA targets. Though Cas9 preferentially interrogates DNA sequences containing a PAM sequence NGG it can bind here without a protospacer target. However, the Cas9-gRNA complex requires a close match to the gRNA to create a double strand break. CRISPR sequences in bacteria are expressed in multiple RNAs and then processed to create guide strands for RNA. Because 10 Eukaryotic Systems lack some of the proteins required to process CRISPR RNAs the synthetic construct gRNA was created to combine the essential pièces of RNA for Cas9 targeting into a single RNA expressed with the RNA polymerase type III promoter U6. Synthetic gRNAs are slightly over lOObp at the minimum length and contain a portion which is targets the 20 protospacer nucléotides immediately preceding the PAM sequence NGG; gRNAs do not 15 contain a PAM sequence.

In some embodiments, the gRNA targets a site within a wildtype dystrophin gene. In some embodiments, the gRNA targets a site within a mutant dystrophin gene. In some embodiments, the gRNA targets a dystrophin intron. In some embodiments, the gRNA targets a dystrophin exon. In some embodiments, the gRNA targets a site in a dystrophin exon that is 20 expressed and is présent in one or more of the dystrophin isoforms shown in Table 1. In embodiments, the gRNA targets a dystrophin splice site. In some embodiments, the gRNA targets a splice donor site on the dystrophin gene. In embodiments, the gRNA targets a splice acceptor site on the dystrophin gene.

In embodiments, the guide RNA targets a mutant DMD exon. In some embodiments, 25 the mutant exon is exon 23 or 51. In some embodiments, the guide RNA targets at least one of exons 1, 23, 41, 44, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 of the dystrophin gene. In embodiments, the guide RNA targets at least one of introns 44, 45, 50, 51, 52, 53, 54, or 55 of the dystrophin gene. In preferred embodiments, the guide RNAs are designed to induce skipping of exon 51 or exon 23. In embodiments, the gRNA is targeted to a splice acceptor 30 site of exon 51 or exon 23.

Suitable gRNAs for use in various compositions and methods disclosed herein are provided as SEQ ID NOs. 383-705, 709-71 1,715-717, 790-862, 864. (Tables 7, 9, 11, 13, and 15). In preferred embodiments, the gRNA is selected from any one of SEQ ID No: 790 to SEQ ID No: 862.

In some embodiments, gRNAs of the disclosure comprise a sequence that is complementary to a target sequence within a coding sequence or a non-coding sequence corresponding to the DMD gene, and, therefore, hybridize to the target sequence. In some embodiments, gRNAs for Cpfl comprise a single crRNA containing a direct repeat scaffold sequence followed by 24 nucléotides of guide sequence. In some embodiments, a “guide” sequence of the crRNA comprises a sequence of the gRNA that is complementary to a target sequence. In some embodiments, crRNA ofthe disclosure comprises a sequence ofthe gRNA that is not complementary to a target sequence. “Scaffold” sequences ofthe disclosure link the gRNA to the Cpfl polypeptide. “Scaffold” sequences of the disclosure are not équivalent to a tracrRNA sequence of a gRNA-Cas9 construct.

In some embodiments, a nucleic acid may comprise one or more sequences encoding a gRNA. In some embodiments, a nucleic acid may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 sequences encoding a gRNA. In some embodiments, ail ofthe sequences encode the same gRNA. In some embodiments, ail of the sequences encode different gRNAs. In some embodiments, at least 2 of the sequences encode the same gRNA, for example 20 at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the sequences encode the same gRNA.

E. Cas9 versus Cpfl

Cas9 requires two RNA molécules to eut DNA while Cpfl needs one. The proteins also eut DNA at different places, offering researchers more options when selecting an edfting site.

Cas9 cuts both strands in a DNA molécule at the same position, leaving behind blunt ends. Cpfl leaves one strand longer than the other, creating 'sticky' ends that are easier to work with. Cpfl appears to be more able to insert new sequences at the eut site, compared to Cas9. Although the CRISPR/Cas9 System can efficiently disable genes, it is challenging to insert genes or generate a knock-in. Cpfl lacks tracrRNA, utilizes a T-rich PAM and cleaves DNA 30 via a staggered DNA DSB.

In summary, important différences between Cpfl and Cas9 Systems are that Cpfl recognizes different PAMs, enabling newtargeting possibilities, créâtes 4-5 nt long sticky ends, instead of blunt ends produced by Cas9, enhancing the efficiency of genetic insertions and specificity during NHEJ or HDR, and cuts target DNA further away from PAM, further away from the Cas9 cutting site, enabling new possibilities for cleaving the DNA.

Table 3: Différences between Cas9 and Cpfl

Feature	Cas9	Cpfl
Structure	Two RNA required (Or 1 fusion transcript (crRNA+tracrRNA=gRNA))	One RNA required
Cutting mechanism	Blunt end cuts	Staggered end cuts
Cutting site	Proximal to récognition site	Distal from récognition site
Target sites	G-rich PAM	T-rich PAM

F. CRISPR -mediated gene editing

The first step in editing the DMD gene using CRISPR/Cpfl or CRISPR/Cas9 is to identify the genomic target sequence. The genomic target for the gRNAs of the disclosure can be any ~24 nucléotide DNA sequence, provided that the sequence is unique compared to the rest of the genome. In some embodiments, the genomic target sequence corresponds to a sequence within exon 51, exon 45, exon 44, exon 53, exon 46, exon 52, exon 50, exon 43, exon 6, exon 7, exon 8, and/or exon 55 of the human dystrophin gene. In some embodiments, the genomic target sequence is a 5’ or 3’ splice site of exon 51, exon 45, exon 44, exon 53, exon 46, exon 52, exon 50, exon 43, exon 6, exon 7, exon 8, and/or exon 55 of the human dystrophin gene. In some embodiments, the genomic target sequence corresponds to a sequence within an intron immediately upstream or downstream of exon 51, exon 45, exon 44, exon 53, exon 46, exon 52, exon 50, exon 43, exon 6, exon 7, exon 8, and/or exon 55 of the human dystrophin gene. Exemplary genomic target sequences can be found in Tables 6, 8, 10, 12, and 14.

The next step in editing the DMD gene is to identify ail Protospacer Adjacent Motif (PAM) sequences within the genetic région to be targeted. The target sequence must be immediately upstream of a PAM. Once ail possible PAM sequences and putative target sites hâve been identified, the next step is to choose which site is likely to resuit in the most efficient on-target cleavage. The gRNA targeting sequence needs to match the target sequence, and the gRNA targeting sequence must not match additional sites within the genome. In preferred embodiments, the gRNA targeting sequence has perfect homology to the target with no homology elsewhere in the genome. In some embodiments, a given gRNA targeting sequence will hâve additional sites throughout the genome where partial homology exists. These sites are called “off-targets” and should be considered when designing a gRNA. In general, offtarget sites are not cleaved as efficiently when mismatches occur near the PAM sequence, so gRNAs with no homology or those with mismatches close to the PAM sequence will hâve the highest specificity. In addition to “off-target activity”, factors that maximize cleavage of the desired target sequence (“on-target activity”) must be considered. It is known to those of skill in the art that two gRNA targeting sequences, each having 100% homology to the target DNA may not resuit in équivalent cleavage efficiency. In fact, cleavage efficiency may increase or decrease depending upon the spécifie nucléotides within the selected target sequence. Close examination of predicted on-target and off-target activity of each potential gRNA targeting sequence is necessary to design the best gRNA. Several gRNA design programs hâve been developed that are capable of locating potential PAM and target sequences and ranking the associated gRNAs based on their predicted on-target and off-target activity (e.g. CRISPRdirect, available at www.crispr.dbcls.jp).

The next step is to synthesize and clone desired gRNAs. Targeting oligos can be synthesized, annealed, and inserted into plasmids containing the gRNA scaffold using standard restriction-ligation cloning. However, the exact cloning strategy will dépend on the gRNA vector that is chosen. The gRNAs for Cpfl are notably simpler than the gRNAs for Cas9, and only consist of a single crRNA containing direct repeat scaffold sequence followed by ~24 25 nucléotides of guide sequence.

Each gRNA should then be validated in one or more target cell lines. For example, after the Cas9 or Cpfl and the gRNA are delivered to the cell, the genomic target région may be amplified using PCR and sequenced according to methods known to those of skill in the art.

In some embodiments, gene editing may be performed in vitro or ex vivo. In some embodiments, cells are contacted in vitro or ex vivo with a Cas9 or a Cpfl and a gRNA that targets a dystrophin splice site. In some embodiments, the cells are contacted with one or more nucleic acids encoding the Cas9 or Cpfl and the guide RNA. In some embodiments, the one or more nucleic acids are introduced into the cells using, for example, lipofection or electroporation. Gene editing may also be performed in zygotes. In embodiments, zygotes may be injected with one or more nucleic acids encoding Cas9 or Cpfl and a gRNA that targets a dystrophin splice site. The zygotes may subsequently be injected into a host.

In embodiments, the Cas9 or Cpfl is provided on a vector. In embodiments, the vector contains a Cas9 derived from S. pyogenes (SpCas9, SEQ 1D NO. 872). In embodiments, the vector contains a Cas9 derived from S. aureus (SaCas9, SEQ 1D NO. 873). In embodiments, the vector contains a Cpfl sequence derived from a Lachnospiraceae bacterium. See, for example, Uniprot Accession No. A0A182DWE3; SEQ ID NO. 871. In embodiments, the vector contains a Cpfl sequence derived from an Acidaminococcus bacterium. See, for example, Uniprot Accession No. U2UMQ6; SEQ ID NO. 870. In some embodiments, the Cas9 or Cpfl sequence is codon optimized for expression in human cells or mouse cells. In some embodiments, the vector further contains a sequence encoding a fluorescent protein, such as GFP, which allows Cas 9 or Cpfl-expressing cells to be sorted using fluorescence activated cell sorting (FACS). In some embodiments, the vector is a viral vector such as an adenoassociated viral vector.

In embodiments, the gRNA is provided on a vector. In some embodiments, the vector is a viral vector such as an adeno-associated viral vector. In embodiments, the Cas9 or Cpfl and the guide RNA are provided on the same vector. In embodiments, the Cas9 or Cpfl and the guide RNA are provided on different vectors.

In some embodiments, the cells are additionally contacted with a single-stranded DMD oligonucleotide to effect homology directed repair. In some embodiments, small INDELs restore the protein reading frame of dystrophin (“reframing” strategy). When the reframing strategy is used, the cells may be contacted with a single gRNA. In embodiments, a splice donor or splice accepter site is disrupted, which results in exon skipping and restoration of the protein reading frame (“exon skipping” strategy). When the exon skipping strategy is used, the cells may be contacted with two or more gRNAs.

Efflciency of in vitro or ex vivo Cas9 or Cpfl-mediated DNA cleavage may be assessed using techniques known to those of skill in the art, such as the T7 El assay. Restoration of DMD expression may be confirmed using techniques known to those of skill in the art, such as RT-PCR, western blotting, and immunocytochemistry.

⁷⁰

In some embodiments, in vitro or ex vivo gene editing is performed in a muscle or satellite cell. In some embodiments, gene editing is performed in iPSC or iCM cells. In embodiments, the iPSC cells are differentiated after gene editing. For example, the iPSC cells may be differentiated into a muscle cell or a satellite cell after editing. In embodiments, the 5 iPSC cells are differentiated into cardiac muscle cells, skeletal muscle cells, or smooth muscle cells. In embodiments, the iPSC cells are differentiated into cardiomyocytes. iPSC cells may be induced to differentiate according to methods known to those of skill in the art.

In some embodiments, contacting the cell with the Cas9 or the Cpfl and the gRNA restores dystrophin expression. In embodiments, cells which hâve been edited in vitro or ex 10 vivo, or cells derived therefrom, show levels of dystrophin protein that is comparable to wildtype cells. In embodiments, the edited cells, or cells derived therefrom, express dystrophin at a level that is 50%, 60%, 70%, 80%, 90%, 95% or any percentage in between of wildtype dystrophin expression levels. In embodiments, the cells which hâve been edited in vitro or ex vivo, or cells derived therefrom, hâve a mitochondrial number that is comparable to that of 15 wildtype cells. In embodiments the edited cells, or cells derived therefrom, hâve 50%, 60%, 70%, 80%, 90%, 95% or any percentage in between as many mitochondria as wildtype cells. In embodiments, the edited cells, or cells derived therefrom, show an increase in oxygen consumption rate (OCR) compared to non-edited cells at baseline.

G. RNA Pol III and Pol III Promoters

In eukaryotes, RNA polymerase III (also called Pol III) transcribes DNA to synthesize ribosomal 5S rRNA, tRNA and other small RNAs. The genes transcribed by RNA Pol III fall in the category of “housekeeping” genes whose expression is required in ail cell types and most environmental conditions. Therefore, the régulation of Pol III transcription is primarily tied to the régulation of cell growth and the cell cycle, thus requiring fewer regulatory proteins than 25 RNA polymerase IL Under stress conditions however, the protein Mafl represses Pol III activity.

In the process of transcription (by any polymerase) there are three main stages: (i) initiation, requiring construction of the RNA polymerase complex on the gene's promoter; (ii) élongation, the synthesis ofthe RNA transcript; and (iii) termination, the fmishing of RNA 30 transcription and disassembly of the RNA polymerase complex.

Promoters under the control of RNA Pol III include those for ribosomal 5S rRNA, tRNA and few other small RNAs such as U6 spliceosomal RNA, RNase P and RNase MRP RNA, 7SL RNA (the RNA component of the signal récognition particles), Vault RNAs, Y RNA, SINEs (short interspersed répétitive éléments), 7SK RNA, two microRNAs, several 5 small nucleolar RNAs and several few regulatory antisense RNAs

IV. Nucleic Acid Delivery

As discussed above, in certain embodiments, expression cassettes are employed to express a transcription factor product, either for subséquent purification and delivery to a cell/subject, or for use directly in a genetic-based delivery approach. Provided herein are 10 expression vectors which contain one or more nucleic acids encoding Cas9 or Cpfl and at least one DMD guide RNA that targets a dystrophin splice site. In some embodiments, a nucleic acid encoding Cas9 or Cpfl and a nucleic acid encoding at least one guide RNA are provided on the same vector. In further embodiments, a nucleic acid encoding Cas9 or Cpfl and a nucleic acid encoding least one guide RNA are provided on separate vectors.

Expression requires that appropriate signais be provided in the vectors, and include various regulatory éléments such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes ot interest in cells. Eléments designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug sélection markers for establishing permanent, stable cell 20 clones expressing the products are also provided, as is an element that links expression of the drug sélection markers to expression of the polypeptide.

A. Regulatory Eléments

Throughout this application, the term “expression cassette” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or ail of 25 the nucleic acid encoding sequence is capable of being transcribed and translated, i.e., is under the control of a promoter. A “promoter’ refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the spécifie transcription of a gene. The phrase “under transcriptional control means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase 30 initiation and expression of the gene. An “expression vector” is meant to include expression cassettes comprised in a genetic construct that is capable of réplication, and thus including one or more of origins of réplication, transcription termination signais, poly-A régions, selectable markers, and multipurpose cloning sites.

The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized dérivés from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, hâve shown that promoters are composed of discrète functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more récognition sites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucléotidyl transferase gene and the promoter for the SV40 laie genes, a discrète element overlying the start site itself helps to fix the place of initiation.

In some embodiments, the Cas9 or Cpfl constructs of the disclosure are expressed by a muscle-cell spécifie promoter. This muscle-cell spécifie promoter may be constitutively active or may be an inducible promoter.

Additional promoter éléments regulate the frequency of transcriptional initiation. Typically, these are located in the région 30-110 bp upstream of the start site, although a number of promoters hâve recently been shown to contain functional éléments downstream of the start site as well. The spacing between promoter éléments frequently is flexible, so that promoter function is preserved when éléments are inverted or moved relative to one another. In the tk promoter, the spacing between promoter éléments can be increased to 50 bp apart before activity begins to décliné. Depending on the promoter, it appears that individual éléments can function either co-operatively or independently to activate transcription.

In certain embodiments, viral promûtes such as the human cytomégalovirus (CMV) immédiate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraIdehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose. By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, sélection of a promoter that is regulated in response to spécifie physiologie signais can permit inducible expression of the gene product.

Enhancers are genetic éléments that increase transcription from a promoter located at a distant position on the same molécule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual éléments, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer région as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter région or its component éléments. On the other hand, a promoter must hâve one or more éléments that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to hâve a very similar modular organization.

Below is a list of promoters/enhancers and inducible promoters/enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct. Additionally, any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

The promoter and/or enhancer may be, for example, immunoglobulin light chain, immunoglobulin heavy chain, T-cell receptor, HLA DQ a and/or DQ β, β-interferon, interleukin-2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-Dra, β-Actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagénase, albumin, α-fetoprotein, t-globin, β-globin, c-fos, c-HA-ras, insulin, neural cell adhesion molécule (NCAM), αι-antitrypain, H2B (TH2B) histone, mouse and/or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human sérum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), duchenne muscular dystrophy, SV40, polyoma, retroviruses, papilloma virus, hepatitis B virus, human immunodeficiency virus, cytomégalovirus (CMV), and gibbon ape leukemia virus.

In some embodiments, inducible éléments may be used. In some embodiments, the inducible element is, for example, MTII, MMTV (mouse mammary tumor virus), β-interferon, adenovirus 5 E2, collagénase, stromelysin, SV40, murine MX gene, GRP78 gene, a-2macroglobulin, vimentin, MHC class I gene H-2Kb, HSP70, proliferin, tumor necrosis factor, and/or thyroid stimulating hormone a gene. In some embodiments, the inducer is phorbol ester (TFA), heavy metals, glucocorticoids, poly(rl)x, poly(rc), E1A, phorbol ester (TPA), interferon, Newcastle Disease Virus, A23187, IL-6, sérum, interferon, SV40 large T antigen, PMA, and/or thyroid hormone. Any of the inducible éléments described herein may be used with any of the inducers described herein.

Of particular interest are muscle spécifie promoters. These include the myosin light chain-2 promoter, the α-actin promoter, the troponin 1 promoter; the Na⁺/Ca²⁺ exchanger promoter, the dystrophin promoter, the a7 integrin promoter, the brain natriuretic peptide promoter and the αΒ-crystallin/small heat shock protein promoter, α-myosin heavy chain promoter and the ANF promoter. In some embodiments, the muscle spécifie promoter is the CK8 promoter. The CK8 promoter has the following sequence (SEQ ID NO. 874):

CTAGACTAGC ATGCTGCCCA TGTAAGGAGG CAAGGCCTGG

GGACACCCGA GATGCCTGGT

TATAATTAAC CCAGACATGT GGCTGCCCCC CCCCCCCCAA

CACCTGCTGC CTCTAAAAAT

121 AACCCTGCAT GCCATGTTCC CGGCGAAGGG CCAGCTGTCC

CCCGCCAGCT AGACTCAGCA

181 CTTAGTTTAG GAACCAGTGA GCAAGTCAGC CCTTGGGGCA GCCCATACAA GGCCATGGGG

241 CTGGGCAAGC TGCACGCCTG GGTCCGGGGT GGGCACGGTG CCCGGGCAAC GAGCTGAAAG

301 CTCATCTGCT CTCAGGGGCC CCTCCCTGGG GACAGCCCCT CCTGGCTAGT CACACCCTGT

361 AGGCTCCTCT ATATAACCCA GGGGCACAGG GGCTGCCCTC ATTCTACCAC CACCTCCACA

421 GCACAGACAG ACACTCAGGA GCCAGCCAGC

In some embodiments, the muscle-cell cell spécifie promoter is a variant of the CK8 promoter, called CK8e. The CK8e promoter has the following sequence (SEQ ID NO. 875):

TGCCCATGTA AGGAGGCAAG GCCTGGGGAC ACCCGAGATG

CCTGGTTATA ATTAACCCAG

ACATGTGGCT GCCCCCCCCC CCCCAACACC TGCTGCCTCT

AAAAATAACC CTGCATGCCA

121 TGTTCCCGGC GAAGGGCCAG CTGTCCCCCG CCAGCTAGAC

TCAGCACTTA GTTTAGGAAC

181 CAGTGAGCAA GTCAGCCCTT GGGGCAGCCC ATACAAGGCC

ATGGGGCTGG GCAAGCTGCA

241 CGCCTGGGTC CGGGGTGGGC ACGGTGCCCG GGCAACGAGC

TGAAAGCTCA TCTGCTCTCA

301 GGGGCCCCTC CCTGGGGACA GCCCCTCCTG GCTAGTCACA

CCCTGTAGGC TCCTCTATAT

361 AACCCAGGGG CACAGGGGCT GCCCTCATTC TACCACCACC TCCACAGCAC AGACAGACAC

421 TCAGGAGCCA GCCAGC

Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. Any polyadenylation sequence may be employed such as human growth hormone and SV40 polyadenylation signais. Also contemplated as an element of the expression cassette is a terminator. These éléments can serve to enhance message levels and to minimize read through from the cassette into other sequences.

B. 2A Peptide

In some embodiments, a 2A-like self-cleaving domain from the insect virus Thosea asigna (TaV 2A peptide)(SEQ ID NO. 876, EGRGSLLTCGDVEENPGP) is used. These 2Alike domains hâve been shown to function across eukaryotes and cause cleavage of amino acids to occur co-translationally within the 2A-like peptide domain. Therefore, inclusion of TaV 2A peptide allows the expression of multiple proteins from a single mRNA transcript. Importantly, the domain of TaV when tested in eukaryotic Systems has shown greater than 99% cleavage activity. Other acceptable 2A-like peptides include, but are not limited to, equine rhinitis A virus (ERAV) 2A peptide (SEQ ID NO. 877; QCTNYALLKLAGDVESNPGP), porcine teschovirus-1 (PTV1) 2A peptide (SEQ ID NO. 878; ATNFSLLKQAGDVEENPGP) and foot and mouth disease virus (FMDV) 2A peptide (SEQ ID NO. 879; PVKQLLNFDLLKLAGDVESNPGP) or modified versions thereof.

In some embodiments, the 2A peptide is used to express a reporter and a Cas9 or a Cpfl simultaneously. The reporter may be, for example, GFP.

Other self-cleaving peptides that may be used include, but are not limited to nuclear inclusion protein a (Nia) protease, a PI protease, a 3C protease, a L protease, a 3C-like protease, or modified versions thereof.

C. Delivery of Expression Vectors

There are a number of ways in which expression vectors may be introduced into cells. In certain embodiments, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently hâve made them attractive candidates for the transfer of foreign genes into mammalian cells . These hâve a relatively low capacity for foreign DNA sequences and hâve a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animais.

One of the preferred methods for in vivo delivery involves the use of an adenovirus expression vector. “Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein. In this context, expression does not require that the gene product be synthesized.

The expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kB, linear, double-stranded DNA virus, allows substitution of large pièces of adénoviral DNA with foreign sequences up to 7 kB. In contrast to retrovirus, the adénoviral infection of host cells does not resuit in chromosomal intégration because adénoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually ail épithélial cells regardless of their cell cycle stage. So far, adénoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vector because of its midsized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis éléments necessary for viral DNA réplication and packaging. The early (E) and late (L) régions of the genome contain different transcription units that are divided by the onset ot viral DNA réplication. The El région (E1A and E1B) encodes proteins responsible for the régulation of transcription of the viral genome and a few cellular genes. The expression of the E2 région (E2A and E2B) results in the synthesis of the proteins for viral DNA réplication. These proteins are involved in DNA réplication, late gene expression and host cell shut-off. The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and ail the mRNAs issued from this promoter possess a 5D-tripartite leader (TPL) sequence which makes them preferred mRNAs for translation.In one System, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.

Génération and propagation of the current adenovirus vectors, which are réplication déficient, dépend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins. Since the E3 région is dispensable from the adenovirus genome, the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both régions. In nature, adenovirus can package approximately 105% ofthe wild-type genome, providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the El and E3 régions, the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vectorborne cytotoxicity. Also, the réplication deficiency ofthe El-deleted virus is incomplète.

Helper cell Unes may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or épithélial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or épithélial cells. As stated above, the preferred helper cell line is 293.

Improved methods for culturing 293 cells and propagating adenovirus are known in the art. In one format, natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for I to 4 h. The medium is then replaced with 50 ml of fresh medium and shaking initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.

The adenoviruses of the disclosure are réplication defective, or at least conditionally réplication defective. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the présent disclosure.

As stated above, the typical vector according to the présent disclosure is réplication defective and will not hâve an adenovirus El région. Thus, it will be most convenient to introduce the polynucleotide encoding the gene of interest at the position from which the Elcoding sequences hâve been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical. The polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 région in E3 replacement vectors, or in the E4 région where a helper cell line or helper virus compléments the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10⁹-10¹² plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require intégration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, hâve low genotoxicity to host cells. No side effects hâve been reported in studies of vaccination with wild-type adenovirus, demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.

Adenovirus vectors hâve been used in eukaryotic gene expression and vaccine development. Animal studies suggested that recombinant adenovirus could be used for gene therapy. Studies in administering recombinant adenovirus to different tissues include trachea instillation, muscle injection, peripheral intravenous injections and stereotactic inoculation into the brain.

The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reversetranscription. The resulting DNA then stably intégrâtes into cellular chromosomes as a provirus and directs synthesis of viral proteins. The intégration results in the rétention of the viral gene sequences in the récipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are présent at the 5’ and 3’ ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for intégration in the host cell genome.

In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate précipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, intégration and stable expression require the division of host cells.

A novel approach designed to allow spécifie targeting of retrovirus vectors was recently developed based on the Chemical modification of a retrovirus by the Chemical addition of lactose residues to the viral envelope. This modification could permit the spécifie infection of hépatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses may be used, in which biotinylated antibodies against a retroviral envelope protein and against a spécifie cell receptor are used. The antibodies are coupled via the biotin components by using streptavidin. Using antibodies against major histocompatibility complex class I and class II antigens, it has been demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al., 1989).

There are certain limitations to the use of retrovirus vectors in ail aspects of the présent disclosure. For example, retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfère with the function of flanking genes. Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This can resuit from recombination events in which the intact- sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome. However, new packaging cell Unes are now available that should greatly decrease the likelihood of recombination (see, for example, Markowitz et al., 1988; Hersdorffer et al., 1990).

Other viral vectors may be employed as expression constructs in the présent disclosure. Vectors derived from viruses such as vaccinia virus adeno-associated virus (AAV) and herpesviruses may be employed. They offer several attractive features for various mammalian cells.

In embodiments, the AAV vector is replication-defective or conditionally réplication defective. In embodiments, the AAV vector is a recombinant AAV vector. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVIO, AAV11 or any combination thereof.

In some embodiments, a single viral vector is used to deliver a nucleic acid encoding a Cas9 or a Cpfl and at least one gRNA to a cell. In some embodiments, Cas9 or Cpfl is provided to a cell using a first viral vector and at least one gRNA is provided to the cell using a second viral vector.

ln some embodiments, a single viral vector is used to deliver a nucleic acid encoding Cas9 or Cpfl and at least one gRNA to a cell. In some embodiments, Cas9 or Cpfl is provided to a cell using a first viral vector and at least one gRNA is provided to the cell using a second viral vector. In order to effect expression of sense or antisense gene constructs, the expression construct must be delivered into a cell. The cell may be a muscle cell, a satellite cell, a mesangioblast, a bone marrow derived cell, a stromal cell or a mesenchymal stem cell. In embodiments, the cell is a cardiac muscle cell, a skeletal muscle cell, or a smooth muscle cell. In embodiments, the cell is a cell in the tibialis anterior, quadriceps, soleus, diaphragm or heart. In some embodiments, the cell is an induced pluripotent stem cell (iPSC) or inner cell mass cell (iCM). In further embodiments, the cell is a human iPSC or a human iCM. In some embodiments, human iPSCs or human iCMs of the disclosure may be derived from a cultured stem cell line, an adult stem cell, a placental stem cell, or from another source of adult or embryonic stem cells that does not require the destruction of a human embryo. Delivery to a cell may be accomplished in vitro, as in laboratory procedures for transforming cells Unes, or in vivo or ex vivo, as in the treatment of certain disease States. One mechanism for delivery is via viral infection where the expression construct is encapsidated in an infections viral particle.

Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the présent disclosure. These include calcium phosphate précipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection. Some of these techniques may be successfully adapted for in vivo or ex vivo use.

Once the expression construct has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This intégration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and réplication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dépendent on the type of expression construct employed.

In yet another embodiment, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well.

Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate précipitâtes into liver and spleen of adult and newborn mice demonstrating active viral réplication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. DNA encoding a gene of interest may also be transferred in a similar 10 manner in vivo and express the gene product.

In still another embodiment for transferring a naked DNA expression construct into cells may involve particle bombardment. This method dépends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them. Several devices for accelerating small particles hâve been 15 developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force. The microprojectiles used hâve consisted of biologically inert substances such as tungsten or gold beads.

In some embodiments, the expression construct is delivered directly to the liver, skin, and/or muscle tissue of a subject. This may require surgical exposure of the tissue or cells, to 20 eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment.

Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the présent disclosure.

In a further embodiment, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an 25 inner aqueous medium. Multilamellar liposomes hâve multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutés between the lipid bilayers. Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitto has been very successful. A reagent known as Lipofectamine 2000™ is widely used and commercially available.

In certain embodiments, the liposome may be complexed with a hemagglutinating virus (HVJ) to facilitate fusion with the cell membrane and promote cell entry of liposomeencapsulated DNA. In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression constructs hâve been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the présent disclosure. Where a bacterial promoter is employed in the DNA construct, it also will be désirable to include within the liposome an appropriate bacterial polymerase.

Other expression constructs which can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the sélective uptake of macromolecules by receptor-mediated endocytosis in almost ail eukaryotic cells. Because ofthe cell type-specific distribution ofvarious receptors, the delivery can be highly spécifie.

Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Severai ligands hâve been used tor receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) and transferrin. A synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells.

D. AAV-Cas9 vectors

In some embodiments, a Cas9 may be packaged into an AAV vector. In some embodiments, the AAV vector is a wildtype AAV vector. In some embodiments, the AAV vector contains one or more mutations. In some embodiments, the AAV vector is isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11 or any combination thereof.

Exemplary AAV-Cas9 vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence région comprising the Cas9 sequence. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof. In some embodiments, the ITRs comprise or consist of full-length and/or wildtype sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of truncated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of elongated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of sequences comprising a sequence variation compared to a wildtype sequence for the same AAV serotype. In some embodiments, the sequence variation comprises one or more of a substitution, délétion, insertion, inversion, or transposition. In some embodiments, the ITRs comprise or consist of at least 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs comprise or consist of 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs hâve a length of 110 ± 10 base pairs. In some embodiments, the ITRs hâve a length of 120 ± 10 base pairs. In some embodiments, the ITRs hâve a length of 130 ± 10 base pairs. In some embodiments, the ITRs hâve a length of140 ± 10 base pairs. In some embodiments, the ITRs hâve a length of 150 ± 10 base pairs. In some embodiments, the ITRs hâve a length of 115, 145, or 141 base pairs. In some embodiments, the ITRs hâve a sequence selected from SEQ. ID. NO: 880, SEQ 1D NO: 881, SEQ ID NO; 882, SEQ 1D NO: 883 and SEQ ID. NO: 946.

In some embodiments, the AAV-Cas9 vector may contain one or more nuclear localization signais (NLS). In some embodiments, the AAV-Cas9 vector contains 1,2, 3, 4, or 5 nuclear localization signais. Exemplary NLS include the c-myc NLS (SEQ ID NO: 884), the SV40 NLS (SEQ ID NO: 885), the hnRNPAI M9 NLS (SEQ ID NO: 886), the nucleoplasmin NLS (SEQ ID NO: 887), the sequence

RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 888) of the IBB domain from importin-alpha, the sequences VSRKRPRP (SEQ ID NO: 889) and PPKKARED (SEQ ID NO: 890) of the myoma T protein, the sequence PQPKKKPL (SEQ ID NO: 891) of human p53, the sequence SALIKK.KK.KMAP (SEQ ID NO: 892) of mouse c-abl IV, the sequences DRLRR (SEQ ID NO: 893) and PKQKKRK (SEQ ID NO: 894) of the influenza virus NSI, the sequence RKLKKKIKKL (SEQ ID NO: 895) of the Hepatitis virus delta antigen and the sequence REKKKFLKRR (SEQ ID NO: 896) of the mouse Mxl protein. Further acceptable nuclear localization signais include bipartite nuclear localization sequences such as the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 897) of the human poly(ADP-ribose) polymerase or the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 898) of the steroid hormone receptors (human) glucocorticoid.

In some embodiments, the AAV-Cas9 vector may comprise additional éléments to facilitate packaging of the vector and expression of the Cas9. In some embodiments, the AAVCas9 vector may comprise a polyA sequence. In some embodiments, the polyA sequence may be a mini-polyA sequence. In some embodiments, the AAV-CAs9 vector may comprise a transposable element. In some embodiments, the AAV-Cas9 vector may comprise a regulator element. In some embodiments, the regulator element is an activator or a repressor.

In some embodiments, the AAV-Cas9 may contain one or more promoters. In some embodiments, the one or more promoters drive expression of the Cas9. In some embodiments, the one or more promoters are muscle-specific promoters. Exemplary muscle-specific promoters include myosin light chain-2 promoter, the α-actin promoter, the troponin 1 promoter, the Na+/Ca2+ exchanger promoter, the dystrophin promoter, the a7 integrin promoter, the brain natriuretic peptide promoter, the αΒ-crystallin/small heat shock protein promoter, α-myosin heavy chain promoter, the ANF promoter, the CK8 promoter and the CK8e promoter.

In some embodiments, the AAV-Cas9 vector may be optimized for production in yeast, bacteria, insect cells, or mammalian cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in human cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in a bacculovirus expression System.

In some embodiments, the AAV-Cas9 vector comprises a sequence selected from SEQ IDNO: 899, SEQ ID NO: 900, SEQ IDNO: 901, or SEQ ID NO: 902, as shown in Table 4.

Table 4: Exemplary gene editing constructs (from ITR to ITR for delivery via AAV vector)

SE Q ID NO:	Sequence
899	GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCA AAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGC GAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTA GTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGAT

TAATTAATGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCC TGGTTATAATTAACCCAGACATGTGGCTGCCCCCCCCCCCCCAACACCTG CTGCCTCTAAAAATAACCCTGCATGCCATGTTCCCGGCGAAGGGCCAGC TGTCCCCCGCCAGCTAGACTCAGCACTTAGTTTAGGAACCAGTGAGCAA GTCAGCCCTTGGGGCAGCCCATACAAGGCCATGGGGCTGGGCAAGCTGC ACGCCTGGGTCCGGGGTGGGCACGGTGCCCGGGCAACGAGCTGAAAGC TCATCTGCTCTCAGGGGCCCCTCCCTGGGGACAGCCCCTCCTGGCTAGTC ACACCCTGTAGGCTCCTCTATATAACCCAGGGGCACAGGGGCTGCCCTC ATTCTACCACCACCTCCACAGCACAGACAGACACTCAGGAGCCAGCCAG CGCTAGCGCCACCATGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTAT CCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATC GGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGC CCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCA AGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCG AGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGA AGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAA GGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAA GAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGAC GAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGA AACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGC CCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGAC CTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGC AGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGT GGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTG GAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCG GCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAA CTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTAC GACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCG ACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGA CATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCT ATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAA GCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCG ACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCC AGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGG CACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAA GCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGA GAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGA AGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTA CTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACC AGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTG GACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCG ATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTA CGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACC GAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCC ATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGC TG A AAG AGG ACTACTTC AAGA A A ATCG AGTGCTTCG ACTCCGTGG A A AT CTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGAT CTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACG AGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAG AGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGAC

AAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGG CTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAG ACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCA TGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAA AGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAAT CTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGG TGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACA TCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGA AGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGC TGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGC AGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTA CGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGAC CATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGG TGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCT CCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGA acgccaagctgattacccagagaaagttcgacaatctgaccaaggccga GAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACA GCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGA CTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGA AGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAG GATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCC ACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTA CCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGAC GTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACC GCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGAT TACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAAC GGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACC GTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACC GAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGG AACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAG TACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGC CAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCT GCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATC GACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATC ATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGA GAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCT GCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAG CTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAAC AGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTC CAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCC TACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATC ATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTA CTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTG CTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACAC GGATCGACCTGTCTCAGCTGGGAGGCGACAAGCGTCCTGCTGCTACTAA GAAAGCTGGTCAAGCTAAGAAAAAGAAATGAATCGATTAGCAATAAAG GATCGTTTATTTTCATTGGAAGCGTGTGTTGGTTTTTTGATCAGGCGCGG GTACCAGGTCGCGGCCGCTCTAGAGCATGGCTACGTAGATAAGTAGCAT GGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCA CTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGT

CGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCG CGCAGAGAGGGA

900

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAA GCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGA GCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGTGGCC AACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTA CTTATCTACGTAGCCATGCTCTAGATTAATTAATGCCCATGTAAGGAGGC AAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTAACCCAGACATGT GGCTGCCCCCCCCCCCCCAACACCTGCTGCCTCTAAAAATAACCCTGCAT GCCATGTTCCCGGCGAAGGGCCAGCTGTCCCCCGCCAGCTAGACTCAGC ACTTAGTTTAGGAACCAGTGAGCAAGTCAGCCCTTGGGGCAGCCCATAC AAGGCCATGGGGCTGGGCAAGCTGCACGCCTGGGTCCGGGGTGGGCAC GGTGCCCGGGCAACGAGCTGAAAGCTCATCTGCTCTCAGGGGCCCCTCC CTGGGGACAGCCCCTCCTGGCTAGTCACACCCTGTAGGCTCCTCTATATA ACCCAGGGGCACAGGGGCTGCCCTCATTCTACCACCACCTCCACAGCAC AGACAGACACTCAGGAGCCAGCCAGCGCTAGCGCCACCATGATGGCCCC AAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAA GAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCC GTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGG GCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCT GTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGC CAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGA GATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGA CTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCAC CCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACC CCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGG CCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGG GGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGG ACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGA AAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCC AGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCC GGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGG GCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAA ACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTG GCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACC TGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGAT CACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCAC CACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTG AGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGG CTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAG CCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGA ACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCA TCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCA GGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAG ATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAA ACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCC CCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTT

CATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTG CTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGC TGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCT GAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAA CCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAAT CGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAAC GCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGG ACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCT GACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAA AACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGG CGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGC ATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCG ACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCT GACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGAT AGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGA AGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGA TGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGA ACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGC GGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAAC ACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTA CCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAAC CGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAA GGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCG GGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAA GAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAG TTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGAT AAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACA AAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACG AGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCA AGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGA GATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTG GGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGT ACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCG AGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACAT CATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGG AAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGG GATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCC AAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCA AAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAA AGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGT GGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAG AAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGA agcagcttcgagaagaatcccatcgactttctggaagccaagggctaca AAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTT CGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACT GCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTG TACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATG AGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGAT CATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCT A ATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCA

TCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCT GGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAG AGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGA GCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGG CGACAAGCGTCCTGCTGCTACTAAGAAAGCTGGTCAAGCTAAGAAAAAG AAATGAATCGATTAGCAATAAAGGATCGTTTATTTTCATTGGAAGCGTG TGTTGGTTTTTTGATCAGGCGCGGGTACCAGGTCGCGGCCGCTCTAGAGC ATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGA ACCCCTAGTGATGGAGTTAGGAACCCCTAGTGATGGAGTTGGCCACTCC CTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCC CGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCA GCTGCC'GCAGG

901

AAGATGACGGTTTGTCACATGGAGTTGGCAGGATGTTTGATTAAAAACA TAACAGGAAGAAAAATGCCCCGCTGTGGGCGGACAAAATAGTTGGGAA CTGGGAGGGGTGGAAATGGAGTTTTTAAGGATTATTTAGGGAAGAGTGA CAAAATAGATGGGAACTGGGTGTAGCGTCGTAAGCTAATACGAAAATTA AAAATGACAAAATAGTTTGGAACTAGATTTCACTTATCTGGTTCGGATCT CCTAGGCGATATCAGTGATCAcggatctcgaccaattgacattattgaagcaACTAGTATCG ATtttatcagggttattgtctcagaCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGA GGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCC TCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACT AGGGGTTCCTGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGA TTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGATTAATTAAT GCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATA ATTAACCCAGACATGTGGCTGCCCCCCCCCCCCCAACACCTGCTGCCTCT AAAAATAACCCTGCATGCCATGTTCCCGGCGAAGGGCCAGCTGTCCCCC GCCAGCTAGACTCAGCACTTAGTTTAGGAACCAGTGAGCAAGTCAGCCC TTGGGGCAGCCCATACAAGGCCATGGGGCTGGGCAAGCTGCACGCCTGG GTCCGGGGTGGGCACGGTGCCCGGGCAACGAGCTGAAAGCTCATCTGCT CTCAGGGGCCCCTCCCTGGGGACAGCCCCTCCTGGCTAGTCACACCCTGT AGGCTCCTCTATATAACCCAGGGGCACAGGGGCTGCCCTCATTCTACCA CCACCTCCACAGCACAGACAGACACTCAGGAGCCAGCCAGCGCTAGCGC CACCATGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGT CCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAA CTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAG AAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAAC CTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCC GGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGA TCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGA CAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAG AAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCT ACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGA CAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCAC ATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCG ACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAA CCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAG GCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGA

TCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGAT TGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTG GCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGAC CTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTC TGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAG AGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAG AGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGC GGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAA GAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTT CTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAA CTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCT TCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGC CATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGG GAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCC CTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCG AGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCG CTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCT GCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTC ACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATG AGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGAC CTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAG GACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCG TGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAA AATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACAT TCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATG ATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGA TGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCC GGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCC TGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCT GATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAG GTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCG GCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGG ACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGA TCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACA GCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCA GCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACG AGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGA CCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATC GTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGA CCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAG AGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCA AGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAG GCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGT GGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCG GATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAA AGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTC CAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACG CCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAA GCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGG AAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAG

TACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCT GGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGA AACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGG AAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTG CAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGC GATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGC GGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAG TGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGG GGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTT TCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAA GCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATG CTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCT CCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAA GGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCAC AAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGA GAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAA CAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCA CCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTG ACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGG ACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGAT CGACCTGTCTCAGCTGGGAGGCGACAAGCGTCCTGCTGCTACTAAGAAA GCTGGTCAAGCTAAGAAAAAGAAATGAATCGATTAGCAATAAAGGATC GTTTATTTTCATTGGAAGCGTGTGTTGGTTTTTTGATCAGGCGCGGGTAC CAGGTCGCGGCCGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCG GGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTaggaacccctagtga tggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggct ttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggCATGCAAGCTGTAGCCAA CCACTAGAACTATAGCTAGAGTCCTGGGCGAACAAACGATGCTCGCCTT CCAGAAAACCGAGGATGCGAACCACTTCATCCGGGGTCAGCACCACCGG CAAGCGCCGCGACGGCCGAGGTCTTCCGATCTCCTGAAGCCAGGGCAGA TCCGTGCACAGCACCTTGCCGTAGAAGAACAGCAAGGCCGCCAATGCCT GACGATGCGTGGAGACCGAAACCTTGCGCTCGTTCGCCAGCCAGGACAG AAATGCCTCGACTTCGCTGCTGCCCAAGGTTGCCGGGTGACGCACACCG TGGAAACGGATGAAGGCACGAACCCAGTTGACATAAGCCTGTTCGGTTC GTAAACTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAAC CTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCT tgttatgactgtttttttgtacagtctatGcctcgggcatccaagcagca AGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGAT GTTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACA AAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCC CTGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAG TTCGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACC TCGGGAACTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGAC CAAGAAGCGGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAAGTTTGA GCAGCCGCGTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAG CACCGGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATG AGGCCAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGG TGACGATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAA GTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAACAATTCGTTCA AGCCGAGATCGGCTTCCCGGCCGCGGAGTTGTTCGGTAAATTGTCACAA

CGCCGCGAATATAGTCTTTACCATGCCCTTGGCCACGCCCCTCTTTAATA CGACGGGCAATTTGCACTTCAGAAAATGAAGAGTTTGCTTTAGCCATAA CAAAAGTCCAGTATGCTTTTTCACAGCATAACTGGACTGATTTCAGTTTA CAACTATTCTGTCTAGTTTAAGACTTTATTGTCATAGTTTAGATCTATTTT GTTCAGTTTAAGACTTTATTGTCCGCCCACACCCGCTTACGC

902

AAGATGACGGTTTGTCACATGGAGTTGGCAGGATGTTTGATTAAAAACA TAACAGGAAGAAAAATGCCCCGCTGTGGGCGGACAAAATAGTTGGGAA CTGGGAGGGGTGGAAATGGAGTTTTTAAGGATTATTTAGGGAAGAGTGA CAAAATAGATGGGAACTGGGTGTAGCGTCGTAAGCTAATACGAAAATTA AAAATGACAAAATAGTTTGGAACTAGATTTCACTTATCTGGTTCGGATCT CCTAGGCGATATCAGTGATCAcggatctcgaccaattgacattattgaagcaACTAGTATCG ATtttatcagggttattgtctcagaGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTG AGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGC CTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCAC TAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGT AGCCATGCTCTAGATTAATTAATGCCCATGTAAGGAGGCAAGGCCTGGG GACACCCGAGATGCCTGGTTATAATTAACCCAGACATGTGGCTGCCCCC CCCCCCCCAACACCTGCTGCCTCTAAAAATAACCCTGCATGCCATGTTCC CGGCGAAGGGCCAGCTGTCCCCCGCCAGCTAGACTCAGCACTTAGTTTA GGAACCAGTGAGCAAGTCAGCCCTTGGGGCAGCCCATACAAGGCCATG GGGCTGGGCAAGCTGCACGCCTGGGTCCGGGGTGGGCACGGTGCCCGG GCAACGAGCTGAAAGCTCATCTGCTCTCAGGGGCCCCTCCCTGGGGACA GCCCCTCCTGGCTAGTCACACCCTGTAGGCTCCTCTATATAACCCAGGGG CACAGGGGCTGCCCTCATTCTACCACCACCTCCACAGCACAGACAGACA CTCAGGAGCCAGCCAGCGCTAGCGCCACCATGATGGCCCCAAAGAAGA AGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACA GCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCAC CGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACAC CGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGAC AGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGA AGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCA GCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAG AGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTT CGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATC TACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTG CGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTT CCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCT GTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCC ATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGA GCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGA AGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGAC CCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAG CTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGA TCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGA CGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAG GCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGG ACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTA

CAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATT GACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCC TGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAG AGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCA CCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGAT TTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGA CCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAG ATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAAC TTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGC GGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAA GCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAA GTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGC GAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAA GTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCT TCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCT GGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTG GACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGA CACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGC CCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATA CACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGA CAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTC GCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTA AAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGC ACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCAT CCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCG GCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGAC CACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGA AGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGT GGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAG AATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGT CCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGA CTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAA GAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTA CTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGAC AATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCC GGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCAC GTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATG ACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGT GTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAAC AACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCG CCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGA AATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAAC TTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGC CTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGG GCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAA TATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTC TATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGA CTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTAT TCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTG

AAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGC TTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAG TGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCT GGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAA GGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTG GCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAG AAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCG AGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCT GGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGA GAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAG CCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTA CACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATC ACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACA AGCGTCCTGCTGCTACTAAGAAAGCTGGTCAAGCTAAGAAAAAGAAATG AATCGATTAGCAATAAAGGATCGTTTATTTTCATTGGAAGCGTGTGTTGG _{ttttttgatcaggcgcgggtaccaggtcgcggccgctctagagcatggc} TACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCC TAGTGATGGAGTTGGCCAC TCCCTCTCTGCGCGCTCGCTCGCTCACTGAG GCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCT CAGTGAGCGAGCGAGCGCGCAGAGAGGGACATGCAAGCTGTAGCCAAC CACTAGAACTATAGCTAGAGTCCTGGGCGAACAAACGATGCTCGCCTTC CAGAAAACCGAGGATGCGAACCACTTCATCCGGGGTCAGCACCACCGGC AAGCGCCGCGACGGCCGAGGTCTTCCGATCTCCTGAAGCCAGGGCAGAT CCGTGCACAGCACCTTGCCGTAGAAGAACAGCAAGGCCGCCAATGCCTG ACGATGCGTGGAGACCGAAACCTTGCGCTCGTTCGCCAGCCAGGACAGA AATGCCTCGACTTCGCTGCTGCCCAAGGTTGCCGGGTGACGCACACCGT GGAAACGGATGAAGGCACGAACCCAGTTGACATAAGCCTGTTCGGTTCG TAAACTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACC TTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTT GTTATGACTGTTTTTTTGTACAGTCTATGCCTCGGGCATCCAAGCAGCAA GCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATG TTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAA AGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCC TGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGT TCGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCT CGGGAACTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACC AAGAAGCGGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAAGTTTGAG CAGCCGCGTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAGC ACCGGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGA GGCCAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGT GACGATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAG TGATGCACTTTGATATCGACCCAAGTACCGCCACCTAACAATTCGTTCAA GCCGAGATCGGCTTCCCGGCCGCGGAGTTGTTCGGTAAATTGTCACAAC GCCGCGAATATAGTCTTTACCATGCCCTTGGCCACGCCCCTCTTTAATAC GACGGGCAATTTGCACTTCAGAAAATGAAGAGTTTGCTTTAGCCATAAC AAAAGTCCAGTATGCTTTTTCACAGCATAACTGGACTGATTTCAGTTTAC AACTATTCTGTCTAGTTTAAGACTTTATTGTCATAGTTTAGATCTATTTTG TTCAGTTTAAGACTTTATTGTCCGCCCACACCCGCTTACGC

In some embodiments of the gene editing constructs of the disclosure, including those embodiments encompassing SEQ ID NOs: 899-902, the construct comprises or consists of a promoter and a nuclease. In some embodiments, the construct comprises or consists of an CK8e promoter and a Cas9 nuclease. In some embodiments, the construct comprises or consists of an CK8e promoter and a Cas9 nuclease isolated or derived from Staphylococcus pyogenes (“SpCas9”). In some embodiments, the CK8e promoter comprises or consists of a nucléotide sequence of

TGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTA ACCCAGACATGTGGCTGCCCCCCCCCCCCCAACACCTGCTGCCTCTAAAAATAAC CCTGCATGCCATGTTCCCGGCGAAGGGCCAGCTGTCCCCCGCCAGCTAGACTCAG CACTTAGTTTAGGAACCAGTGAGCAAGTCAGCCCTTGGGGCAGCCCATACAAGG CCATGGGGCTGGGCAAGCTGCACGCCTGGGTCCGGGGTGGGCACGGTGCCCGGG CAACGAGCTGAAAGCTCATCTGCTCTCAGGGGCCCCTCCCTGGGGACAGCCCCTC CTGGCTAGTCACACCCTGTAGGCTCCTCTATATAACCCAGGGGCACAGGGGCTGC CCTCATTCTACCACCACCTCCACAGCACAGACAGACACTCAGGAGCCAGCCAGC (SEQ ID NO: 875). In some embodiments, the SpCas9 nuclease comprises or consists of a nucléotide sequence of

GACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCC GTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAAC ACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGC GGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACAC CAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGC CAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGA GGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGC CTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAG CACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAG TTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTG GACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAAC CCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGC AAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAAT GGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCÇCAACTTCAAGA GCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACG ACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGT

TTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGT GAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGA CGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCC

TGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTA CATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCT GGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACC TGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACC TGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAA GGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGT GGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGA GGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGC CCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAA GGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTG ACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGC GAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACC GTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTG GAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATC TGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACA TTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGA GGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCT GAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACG GCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACG GCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAA AGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCA CATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGT GAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACAT CGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACA GCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAG ATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTAC CTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATC

AACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGG ACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAG AGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGG

CAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAG GCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAG CTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGG ATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATC ACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAG TGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCG TGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACG GCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAA ATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCA AGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGA CAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCG TGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGC AGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGC TGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCC CCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCA AGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGC AGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTG AAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAAC GGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTG GCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGC TGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACA AGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGA TCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGG ATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCA ATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAG GTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCAC CGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGAC (SEQ ID NO:872). In some embodiments, the construct comprising a promoter and a nuclease further comprises ai least two inverted terminal repeat (ITR) sequences. In some embodiments, the construct comprising a promoter and a nuclease further comprises at least two ITR sequences from isolated or derived from an AAV of serotype 2 (AAV2). In some embodiments, the construct comprising a promoter and a nuclease further comprises at least two ITR sequences each comprising or consisting of a nucléotide sequence of

GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC GCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGA GAGGGA (SEQ ID NO:880). In some embodiments, the construct comprising a promoter and a nuclease further comprises at least two ITR sequences, wherein the first ITR sequence comprises or consists of a nucléotide sequence of CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCG GGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAG AGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO: 881) and the second ITR sequence comprises or consist of a nucléotide sequence of AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCAC TGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTC AGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG (SEQ ID NO: 882). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding an CK8e promoter, a sequence encoding a SpCas9 nuclease and a second AAV2 ITR. In some embodiments, the construct comprising or consisting of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease and a second ITR, further comprises a poly A sequence. In some embodiments, the polyA sequence comprises or consists of a minipolyA sequence. Exemplary minipolyA sequences of the disclosure comprise or consist of a nucléotide sequence of

TAGCAATAAAGGATCGTTTATTTTCATTGGAAGCGTGTGTTGGTTTTTTGATCAGG CGCG (SEQ ID NO: 903). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease, a minipoly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding an CK8e promoter, a sequence encoding a SpCas9 nuclease, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprising, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR, further comprises at least one nuclear localization signal. In some embodiments, the construct comprising, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a

100 nuclease, a poly A sequence and a second ITR, further comprises at least two nuclear localization signais. Exemplary nuclear localization signais of the disclosure comprise or consist of a nucléotide sequence of

AAGCGTCCTGCTGCTACTAAGAAAGCTGGTCAAGCTAAGAAAAAGAAA (SEQ ID

NO. 887), or a nucléotide sequence of

ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCC (SEQ ID NO. 885). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 885, a sequence encoding a SpCas9 nuclease, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding a CK8e promoter, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO. 885, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprising, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a poly A sequence and a second ITR, further comprises a stop codon. The stop codon may hâve a sequence of TAG (SEQ ID NO. 904), TAA (SEQ ID NO. 905), or TGA (SEQ ID NO. 906). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5' to 3’ a first AAV2 ITR, a sequence encoding a CK8e promoter, a nuclear localization signal having a sequence of

101

SEQ ID NO: 885, a sequence encoding a SpCas9 nuclease, a stop codon, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding an CK8e promoter, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, a stop codon, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding an CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO. 885, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, a stop codon a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprising or consisting of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence and a second ITR, further comprises transposable element inverted repeats. Exemplary transposable element inverted repeats of the disclosure comprise or consist of a nucléotide sequence of

TGTGGGCGGACAAAATAGTTGGGAACTGGGAGGGGTGGAAATGGAGTTTTTAAG GATTATTTAGGGAAGAGTGACAAAATAGATGGGAACTGGGTGTAGCGTCGTAAG CTAATACGAAAATTAAAAATGACAAAATAGTTTGGAACTAGATTTCACTTATCTG GTT (SEQ ID NO. 907) and/or a nucléotide sequence of GAATATAGTCTTTACCATGCCCTTGGCCACGCCCCTCTTTAATACGACGGGCAAT TTGCACTTCAGAAAATGAAGAGTTTGCTTTAGCCATAACAAAAGTCCAGTATGCT TTTTCACAGCATAACTGGACTGATTTCAGTTTACAACTATTCTGTCTAGTTTAAGA CTTTATTGTCATAGTTTAGATCTATTTTGTTCAGTTTAAGACTTTATTGTCCGCCCA CA (SEQ ID NO. 908). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat having a sequence of SEQ ID NO. 907, a first AAV2 ITR, a sequence encoding a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO. 885, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, a stop codon a minipoly A sequence, a second AAV2 ITR, and a second transposable

102 element inverted repeat having a sequence of SEQ ID NO. 908. In some embodiments, the construct comprisingorconsisting of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence, a second ITR, and a second transposable element inverted repeat, further comprises a regulatory sequence. Exemplary regulatory sequences of the disclosure comprise or consist of a nucléotide sequence of CATGCAAGCTGTAGCCAACCACTAGAACTATAGCTAGAGTCCTGGGCGAACAAA CGATGCTCGCCTTCCAGAAAACCGAGGATGCGAACCACTTCATCCGGGGTCAGC ACCACCGGCAAGCGCCGCGACGGCCGAGGTCTTCCGATCTCCTGAAGCCAGGGC AGATCCGTGCACAGCACCTTGCCGTAGAAGAACAGCAAGGCCGCCAATGCCTGA CGATGCGTGGAGACCGAAACCTTGCGCTCGTTCGCCAGCCAGGACAGAAATGCC TCGACTTCGCTGCTGCCCAAGGTTGCCGGGTGACGCACACCGTGGAAACGGATG AAGGCACGAACCCAGTTGACATAAGCCTGTTCGGTTCGTAAACTGTAATGCAAGT AGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTA

ACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATG CCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTAT GGAGCAGCAACGATGTTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGC CCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCG GCCCTGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTT CGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAA CTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTT GTTGGCGCTCTCGCGGCTTACGTTCTGCCCAAGTTTGAGCAGCCGCGTAGTGAGA TCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCAC CGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATC TACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAAGTTGG GCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAAC AATTCGTTCAAGCCGAGATCGGCTTCCCGGCCGCGGAGTTGTTCGGTAAATTGTC ACAACGCCG (SEQ 1D NO. 909). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence, a second ITR, a regulatory sequence and a second transposable element inverted

103 repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat having a sequence of SEQ IDNO. 907, a first AAV2 1TR, a sequence encoding a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO. 885, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, a stop codon a minipoly A sequence, a second AAV2 1TR, a regulatory sequence having a sequence of SEQ ID NO. 909, and a second transposable element inverted repeat having a sequence of SEQ ID NO. 908. In some embodiments, the construct may further comprise one or more spacer sequences. Exemplary spacer sequences of the disclosure hâve length from 1-1500 nucléotides, inclusive ofall ranges therebetween. In some embodiments, the spacer sequences may be located either 5’ to or 3’ to an ITR, a promoter, a nuclear localization sequence, a nuclease, a stop codon, a polyA sequence, a transposable element inverted repeat, and/or a regulator element. In some embodiments, the construct may hâve a sequence comprising or consisting of SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, or SEQ ID NO: 902.

E. AAV-sgRNA vectors

In some embodiments, at least a first sequence encoding a gRNA and a second sequence encoding a gRNA may be packaged into an AAV vector. In some embodiments, at least a first sequence encoding a gRNA, a second sequence encoding a gRNA, and a third sequence encoding a gRNA may be packaged into an AAV vector. In some embodiments, at least a first sequence encoding a gRNA, a second sequence encoding a gRNA, a third sequence encoding a gRNA, and a fourth sequence encoding a gRNA may be packaged into an AAV vector. In some embodiments, at least a first sequence encoding a gRNA, a second sequence encoding a gRNA, a third sequence encoding a gRNA, a fourth sequence encoding a gRNA, and a fifth sequence encoding a gRNA may be packaged into an AAV vector. In some embodiments, a plurality of sequences encoding a gRNA are packaged into an AAV vector. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 sequences encoding a gRNA may be packaged into an AAV vector. In some embodiments, each sequence encoding a gRNA is different. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ofthe sequences encoding a gRNA are the same. In some embodiments, ail of the sequence encoding a gRNA are the same.

In some embodiments, the AAV vector is a wildtype AAV vector. In some embodiments, the AAV vector contains one or more mutations. In some embodiments, the

104

AAV vector is isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof.

Exemplary AAV-sgRNA vectors contain two 1TR (inverted terminal repeat) sequences which flank a central sequence région comprising the sgRNA sequences. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof. In some embodiments, the ITRs are isolated or derived from an AAV vector of a first serotype and a sequence encoding a capsid protein of the AAV-sgRNA vector is isolated or derived from an AAV vector of a second serotype. In some embodiments, the first serotype and the second serotype are the same. In some embodiments, the first serotype and the second serotype are not the same. In some embodiments, the first serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, or AAV11. In some embodiments, the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, or AAV11. In some embodiments, the first serotype is AAV2 and the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the first serotype is AAV2 and the second serotype is AAV9.

Exemplary AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence région comprising the gRNA sequences. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11 or any combination thereof. In some embodiments, a first ITR is isolated or derived from an AAV vector of a first serotype, a second ITR is isolated or derived from an AAV vector of a second serotype and a sequence encoding a capsid protein ofthe AAV-sgRNA vector is isolated or derived from an AAV vector of a third serotype. In some embodiments, the first serotype and the second serotype are the same. In some embodiments, the first serotype and the second serotype are not the same. In some embodiments, the first serotype, the second serotype, and the third serotype are the same. In some embodiments, the first serotype, the second serotype, and the third serotype are not the same. In some embodiments, the first serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, or AAV 11. In some embodiments, the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, or AAV1 1. In some embodiments, the third serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, or AAV11. In some embodiments, the first serotype is AAV2, the second serotype is AAV4 and the third serotype is AAV1, AAV2, AAV3, AAV4, AAV5,

105

AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the first serotype is AAV2, the second serotype is AAV4 and the third serotype is AAV9. Exemplary AAVsgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence région comprising the sgRNA sequences. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AA VIO, AAV11 or any combination thereof. In some embodiments, the ITRs comprise or consist of full-length and/or wildtype sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of truncated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of elongated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of sequences comprising a sequence variation compared to a wildtype sequence for the same AAV serotype. In some embodiments, the sequence variation comprises one or more of a substitution, délétion, insertion, inversion, or transposition. In some embodiments, the ITRs comprise or consist of at least 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,

136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs comprise or consist of 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,

128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,

147, 148, 149 or 150 base pairs. In some embodiments, the ITRs hâve a length of 110 ± 10 base pairs. In some embodiments, the ITRs hâve a length of 120 ± 10 base pairs. In some embodiments, the ITRs hâve a length of130 ± 10 base pairs. In some embodiments, the ITRs hâve a length of 140 ± 10 base pairs. In some embodiments, the ITRs hâve a length of 150 ± 10 base pairs. In some embodiments, the ITRs hâve a length of 115, 145, or 141 base pairs. In some embodiments, the ITRs hâve a sequence selected from SEQ ID NO: 880, SEQ ID NO: 881, SEQ ID NO: 882, or SEQ ID NO: 883.

In some embodiments, the AAV-sgRNA vector may comprise additional éléments to facilitate packaging of the vector and expression of the sgRNA. In some embodiments, the AAV-sgRNA vector may comprise a transposable element. In some embodiments, the AAVsgRNA vector may comprise a regulatory element. In some embodiments, the regulatory element comprises an activator or a repressor. In some embodiments, the AAV-sgRNA sequence may comprise a non-functional or “stuffer” sequence. Exemplary stuffer sequences of the disclosure may hâve some (a non-zero percentage of) identity or homology to a genomic

106 sequence of a mammal (including a human). Alternatively, exemplary stuffer sequences of the disclosure may hâve no identify or homology to a genomic sequence of a mammal (including a human). Exemplary stuffer sequences of the disclosure may comprise or consist of naturally occurring non-coding sequences or sequences that are neither transcribed nor translated following administration ofthe AAV vector to a subject.

In some embodiments, the AAV-sgRNA vector may be optimized for production in yeast, bacteria, insect cells, or mammalian cells. In some embodiments, the AAV-sgRNA vector may be optimized for expression in human cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in a bacculovirus expression System.

In some embodiments, the AAV-sgRNA vector comprises at least one promoter. In some embodiments, the AAV-sgRNA vector comprises at least two promoters. In some embodiments, the AAV-sgRNA vector comprises at least three promoters. In some embodiments, the AAV-sgRNA vector comprises at least four promoters. In some embodiments, the AAV-sgRNA vector comprises at least five promoters. Exemplary promoters include, for example, immunoglobulin light chain, immunoglobulin heavy chain, T-cell receptor, HLA DQ a and/or DQ β, β-interferon, interleukin-2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-Dra, β-Actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagénase, albumin, α-fetoprotein, tglobin, β-globin, c-fos, c-HA-ras, insulin, neural cell adhesion molécule (NCAM), aiantitrypain, H2B (TH2B) histone, mouse and/or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human sérum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), duchenne muscular dystrophy, SV40, polyoma, retroviruses, papilloma virus, hepatitis B virus, human immunodeficiency virus, cytomégalovirus (CMV), and gibbon ape leukemia virus. Further exemplary promoters include the U6 promoter, the H1 promoter, and the 7SK promoter.

In some embodiments, the sequence encoding the gRNA comprises a sequence selected from SEQ ID Nos: 383-705, 709-71 1,715-717, 790-862, and 864.

In some embodiments, the AAV vector comprises a first sequence encoding a gRNA and a second sequence encoding a gRNA, a first promoter drives expression of the first sequence encoding a gRNA and a second promoter drives expression of the second sequence encoding a gRNA. In some embodiments, the first and second promoters are the same. In some embodiments, the first and second promoters are different. In some embodiments, the first and second promoters are selected from the Fil promoter, the U6 promoter, and the 7SK

107 promoter. In some embodiments, the first sequence encoding a gRNA and the second sequence encoding a gRNA are identical. In some embodiments, the first sequence encoding a gRNA and the second sequence encoding a gRNA are not identical.

In some embodiments, the AAV vector comprises a first sequence encoding a gRNA, a second sequence encoding a gRNA, and a third sequence encoding a gRNA, a first promoter drives expression of the first sequence encoding a gRNA, a second promoter drives expression of the second sequence encoding a gRNA, and a third promoter drives expression of a third sequence encoding a gRNA. In some embodiments, at least two of the first, second, and third promoters are the same. In some embodiments, each of the first, second, and third promoters are different. In some embodiments, the first, second, and third promoters are selected from the H1 promoter, the U6 promoter, and the 7SK promoter. In some embodiments, the first promoter is the U6 promoter. In some embodiments, the second promoter is the H1 promoter. In some embodiments, the third promoter is the 7SK promoter. In some embodiments, the first promoter is the U6 promoter, the second promoter is the H1 promoter, and the third promoter is the 7SK promoter. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, and the third sequence encoding a gRNA are identical. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, and the third sequence encoding a gRNA are not identical.

In some embodiments, the AAV vector comprises a first sequence encoding a gRNA, a second sequence encoding a gRNA, a third sequence encoding a gRNA, and a fourth sequence encoding a gRNA, a first promoter drives expression of the first sequence encoding a gRNA, a second promoter drives expression of the second sequence encoding a gRNA, a third promoter drives expression of the third sequence encoding a gRNA, and a fourth promoter drives expression of the fourth sequence encoding a gRNA. In some embodiments, at least two of the first, second, third, and fourth promoters are the same. In some embodiments, each ofthe first, second, third, and fourth promoters are different. In some embodiments, each of the first, second, third and fourth promoters are selected from the H1 promoter, the U6 promoter, and the 7SK promoter. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, the third sequence encoding a gRNA, and the fourth sequence encoding a gRNA are identical. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, the third sequence encoding a gRNA, and the fourth sequence encoding a gRNA are not identical.

108

In some embodiments, the AAV vector comprises a first sequence encoding a gRNA, a second sequence encoding a gRNA, a third sequence encoding a gRNA, a fourth sequence encoding a gRNA, and a fifth sequence encoding a gRNA, a first promoter drives expression of the first sequence encoding a gRNA, a second promoter drives expression of the second sequence encoding a gRNA, a third promoter drives expression of the third sequence encoding a gRNA, a fourth promoter drives expression of the fourth sequence encoding a gRNA, and a fifth promoter drives expression of the fifth sequence encoding a gRNA. In some embodiments, at least two of the first, second, third, fourth, and fifth promoters are the same. In some embodiments, each of the first, second, third, fourth, and fifth promoters are different. In some embodiments, each of the first, second, third, and fourth promoters are different. In some embodiments, each of the first, second, third, fourth and fifth promoters are selected from the H1 promoter, the U6 promoter, and the 7SK promoter. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, the third sequence encoding a gRNA, the fourth sequence encoding a gRNA, and the fifth sequence encoding a gRNA are identical. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, the third sequence encoding a gRNA, the fourth sequence encoding a gRNA, and the fifth sequence encoding a gRNA are not identical.

In some embodiments, the AAV-sgRNA vector comprises a sequence selected from SEQ ID NO: 910, SEQ ID NO: 911, SEQ ID NO: 912, or SEQ ID NO: 913. In some embodiments, the AAV-sgRNA vector comprises a sequence selected from SEQ ID NO: 914, SEQ ID NO: 915, SEQ ID NO: 916, or SEQ ID NO: 917. In some embodiments, the AAVsgRNA vector comprises a sequence selected from SEQ ID NO: 918, SEQ ID NO: 919, SEQ ID NO: 920, or SEQ ID NO: 921. Exemplary AAV-sgRNA vectors are provided in Table 5.

Table 5. Exemplary AAV-sgRNA vectors.

SE Q ID NO	Sequence
910	CCACTCCCTCTATGCGCGCTCGCTCACTCACTCGGCCCTGGAGACCAAAG GTCTCCAGACTGCCGGCCTCTGGCCGGCAGGGCCGAGTGAGTGAGCGAG CGCGCATAGAGGGAGTGGGTACCTCCATCATCTAGGTTTGCCAGATCTGA TATCGGCGCGCCCCTGGGCGCGCCCGAGTCCAACACCCGTGGGAATCCC ATGGGCACCATGGCCCCTCGCTCCAAAAATGCTTTCGCGTCGCGCAGACA

109

CTGCTCGGTAGTTTCGGGGATCAGCGTTTGAGTAAGAGCCCGCGTCTGAA CCCTCCGCGCCGCCCCGGCCCCAGTGGAAAGACGCGCAGGCAAAACGCA CCACGTGACGGAGCGTGACCGCGCGCCGAGCGCGCGCCAAGGTCGGGCA GGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACA AGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGAT ATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTG CAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTT GAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAAC ACCGCACTAGAGTAACAGTCTGACGTTTAAGAGCTATGCTGGAAACAGC ATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCA CCGAGTCGGTGCTTTTTTTGCGGCCGCGCTCGGCGCGCCCATATTTGCAT GTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATT TGGGAATCTTATAAGTTCTGTATGAGACCACGGTACACCGCACTAGAGTA ACAGTCTGACGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAA TAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTT TTTTGCGGCCGCTGACGGCGCGCCCTGCAGTATTTAGCATGCCCCACCCA TCTGCAAGGCATTCTGGATAGTGTCAAAACAGCCGGAAATCAAGTCCGTT TATCTCAAACTTTAGCATTTTGGGAATAAATGATATTTGCTATGCTGGTTA AATTAGATTTTAGTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGTAAC TTGACCTAAGTGTAAAGTTGAGATTTCCTTCAGGTTTATATAGCTTGTGC GCCGCCTGGGTACACCGCACTAGAGTAACAGTCTGACGTTTAAGAGCTAT GCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGGCCGCGCTTGCGGCCGC CTCGAGTGATCAAAAAAACCAACACACGCTTCCAATGAAAATAAACGAT CCTTTATTGCTAGCCTTTACTTGTACAGCTCGTCCATGCCGAGAGTGATCC CGGCGGCGGTCACGAACTCCAGCAGGACCATGTGATCGCGCTTCTCGTTG GGGTCTTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTCGGGCAG CAACACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGTGGTCGGCG AGCTGCACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCACCTT GATGCCGTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTA GTTGTACTCCAGCTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGA TGCCCTTCAGCTCGATGCGGTTCACCAGGGTGTCGCCCTCGAACTTCACC TCGGCGCGGGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTC CTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCA TGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGT CACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAAC TTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCCTCGCCGGACAC GCTGAACTTGTGGCCGTTTACGTCGCCGTCCAGCTCGACCAGGATGGGCA CCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACCATGGTGGCGACCGGT GGATCCCGGGCCCGCGGGGTGGCTTTACCAACAGTACCCGGAATGCCAA GCTTACTTAGATCGCAGTCTCGACGCTGGCTGGCTCCTGAGTGTCTGTCT GTGCTGTGGAGGTGGTGGTAGAATGAGGGCAGCCCCTGTGCCCCTGGGT TATATAGAGGAGCCTACAGGGTGTGACTAGCCAGGAGGGGCTGTCCCCA GGGAGGGGCCCCTGAGAGCAGATGAGCTTTCAGCTCGTTGCCCGGGCAC CGTGCCCACCCCGGACCCAGGCGTGCAGCTTGCCCAGCCCCATGGCCTTG TATGGGCTGCCCCAAGGGCTGACTTGCTCACTGGTTCCTAAACTAAGTGC TGAGTCTAGCTGGCGGGGGACAGCTGGCCCTTCGCCGGGAACATGGCAT GCAGGGTTATTTTTAGAGGCAGCAGGTGTTGGGGGGGGGGGGGCAGCCA CATGTCTGGGTTAATTATAACCAGGCATCTCGGGTGTCCCCAGGCCTTGC CTCCTTACATGGGCACGTCGACGATATCAGATCTAGGAACCCCTAGTGAT

110

GGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCC GGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAG CGAGCGAGCGCGCAGAGAGGGAGTGG

918

CCACTCCCTCTATGCGCGCTCGCTCACTCACTCGGCCCTGGAGACCAAAG GTCTCCAGACTGCCGGCCTCTGGCCGGCAGGGCCGAGTGAGTGAGCGAG CGCGCATAGAGGGAGTGGGTACCTCCATCATCTAGGTTTGCCAGATCTGA TATCGGCGCGCCCCTGGGCGCGCCCGAGTCCAACACCCGTGGGAATCCC ATGGGCACCATGGCCCCTCGCTCCAAAAATGCTTTCGCGTCGCGCAGACA CTGCTCGGTAGTTTCGGGGATCAGCGTTTGAGTAAGAGCCCGCGTCTGAA CCCTCCGCGCCGCCCCGGCCCCAGTGGAAAGACGCGCAGGCAAAACGCA CCACGTGACGGAGCGTGACCGCGCGCCGAGCGCGCGCCAAGGTCGGGCA GGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACA AGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGAT ATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTG CAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTT GAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAAC ACCGCACCAGAGTAACAGTCTGACGTTTAAGAGCTATGCTGGAAACAGC ATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCA CCGAGTCGGTGCTTTTTTTGCGGCCGCGCTCGGCGCGCCCATATTTGCAT GTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATT TGGGAATCTTATAAGTTCTGTATGAGACCACGGTACACCGCACCAGAGTA ACAGTCTGACGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAA TAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTT TTTTGCGGCCGCTGACGGCGCGCCCTGCAGTATTTAGCATGCCCCACCCA TCTGCAAGGCATTCTGGATAGTGTCAAAACAGCCGGAAATCAAGTCCGTT TATCTCAAACTTTAGCATTTTGGGAATAAATGATATTTGCTATGCTGGTTA AATTAGATTTTAGTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGTAAC TTGACCTAAGTGTAAAGTTGAGATTTCCTTCAGGTTTATATAGCTTGTGC GCCGCCTGGGTACACCGCACCAGAGTAACAGTCTGACGTTTAAGAGCTA TGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGGCCGCGCTGTTTAAAC CAACAAAATCAGCAGCTAATGAAGGCAAGTCAGCAGGTCACTCATCATT TTCCACTTCGGCAATGCAGTGGGATTATTCCAACAGAGGTTTTTCACAGC ATTCCTTCAGTTAACTGGAGATCGAATCTTGATTTTCACAGATATACTTG GCAAGGTCCGCCCTGTCATCAGCACATTCAAGCAGATCTCCATGGCAGCA TTCCGTGTGGACTTAGGTAAGATCTGTCACTAACTTGGAAACTTCTGCAA ACTCAGCTTAGGGAAATCTCTGGCTCAGGCGAGCTACTGCCTCAGCTTAG AAAGCTCTTTCTCCAAATTATTGGAGACTGGCACACTTAAGTCCCTGTTA GGCAGACGAAGCCTTCCCTTCATCCCGAAGTTCATCGAGCTTTGGCAACA GGCAGGCAGCTTTATCAGCAGCTTGGCAACATTCTGTAAAAGCAGCTTTA TACCTTTAAGCAAAGAAAAGGAGTTCCGGGGCATAAAAGTAAGGATGTC TTCTGGCAATTTATAATAAGTATTTTTTCAAAAATGTCTCTTCATTGTCAT GAAAAGCAGTGCATCACACATCAACCTCTGGTCTCACCAATCGGGGGAG GTTTGGGTTGTTTACTTAGTGTTGCAAGAATTATTTTATTCTCTCAGGTTC TTGTTTTGCACAGCAGTCAGCTCATTCACCATAGGTTTCACGAAGAGTTG CTGCGGCCGCCTCGATAATACGACTCACTATAGGGTCGACGATATCAGAT CTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCT

111

CGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGG TCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGG

914

CCACTCCCTCTATGCGCGCTCGCTCACTCACTCGGCCCTGGAGACCAAAG GTCTCCAGACTGCCGGCCTCTGGCCGGCAGGGCCGAGTGAGTGAGCGAG CGCGCATAGAGGGAGTGGGTACCTCCATCATCTAGGTTTGCCAGATCTGA TATCGGCGCGCCCCTGGGCGCGCCCGAGTCCAACACCCGTGGGAATCCC ATGGGCACCATGGCCCCTCGCTCCAAAAATGCTTTCGCGTCGCGCAGACA CTGCTCGGTAGTTTCGGGGATCAGCGTTTGAGTAAGAGCCCGCGTCTGAA CCCTCCGCGCCGCCCCGGCCCCAGTGGAAAGACGCGCAGGCAAAACGCA CCACGTGACGGAGCGTGACCGCGCGCCGAGCGCGCGCCAAGGTCGGGCA GGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACA AGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGAT ATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTG CAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTT GAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAAC ACCGCACCAGAGTAACAGTCTGAGGTTTAAGAGCTATGCTGGAAACAGC ATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCA CCGAGTCGGTGCTTTTTTTGCGGCCGCGCTCGGCGCGCCCATATTTGCAT GTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATT TGGGAATCTTATAAGTTCTGTATGAGACCACGGTACACCGCACCAGAGTA ACAGTCTGAGGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAA TAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTT TTTTGCGGCCGCTGACGGCGCGCCCTGCAGTATTTAGCATGCCCCACCCA TCTGCAAGGCATTCTGGATAGTGTCAAAACAGCCGGAAATCAAGTCCGTT TATCTCAAACTTTAGCATTTTGGGAATAAATGATATTTGCTATGCTGGTTA AATTAGATTTTAGTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGTAAC TTGACCTAAGTGTAAAGTTGAGATTTCCTTCAGGTTTATATAGCTTGTGC GCCGCCTGGGTACACCGCACCAGAGTAACAGTCTGAGGTTTAAGAGCTA TGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGGCCGCGCTGTTTAAAC CAACAAAATCAGCAGCTAATGAAGGCAAGTCAGCAGGTCACTCATCATT TTCCACTTCGGCAATGCAGTGGGATTATTCCAACAGAGGTTTTTCACAGC ATTCCTTCAGTTAACTGGAGATCGAATCTTGATTTTCACAGATATACTTG GCAAGGTCCGCCCTGTCATCAGCACATTCAAGCAGATCTCCATGGCAGCA TTCCGTGTGGACTTAGGTAAGATCTGTCACTAACTTGGAAACTTCTGCAA ACTCAGCTTAGGGAAATCTCTGGCTCAGGCGAGCTACTGCCTCAGCTTAG AAAGCTCTTTCTCCAAATTATTGGAGACTGGCACACTTAAGTCCCTGTTA GGCAGACGAAGCCTTCCCTTCATCCCGAAGTTCATCGAGCTTTGGCAACA GGCAGGCAGCTTTATCAGCAGCTTGGCAACATTCTGTAAAAGCAGCTTTA TACCTTTAAGCAAAGAAAAGGAGTTCCGGGGCATAAAAGTAAGGATGTC TTCTGGCAATTTATAATAAGTATTTTTTCAAAAATGTCTCTTCATTGTCAT GAAAAGCAGTGCATCACACATCAACCTCTGGTCTCACCAATCGGGGGAG GTTTGGGTTGTTTACTTAGTGTTGCAAGAATTATTTTATTCTCTCAGGTTC TTGTTTTGCACAGCAGTCAGCTCATTCACCATAGGTTTCACGAAGAGTTG CTGCGGCCGCCTCGATAATACGACTCACTATAGGGTCGACGATATCAGAT CTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCT

112

CGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGG TCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGG

911

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAA GCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGA GCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTAGATCT GATATCGGCGCGCCCCTGGGCGCGCCCGAGTCCAACACCCGTGGGAATC CCATGGGCACCATGGCCCCTCGCTCCAAAAATGCTTTCGCGTCGCGCAGA CACTGCTCGGTAGTTTCGGGGATCAGCGTTTGAGTAAGAGCCCGCGTCTG AACCCTCCGCGCCGCCCCGGCCCCAGTGGAAAGACGCGCAGGCAAAACG CACCACGTGACGGAGCGTGACCGCGCGCCGAGCGCGCGCCAAGGTCGGG CAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATA CAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAG ATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTT TGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAA CTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGA AACACCGCACTAGAGTAACAGTCTGACGTTTAAGAGCTATGCTGGAAAC AGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTG GCACCGAGTCGGTGCTTTTTTTGCGGCCGCGCTCGGCGCGCCCATATTTG CATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGG ATTTGGGAATCTTATAAGTTCTGTATGAGACCACGGTACACCGCACTAGA GTAACAGTCTGACGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTT AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTG _{ctttttttgcggccgctgacggcgcgccctgcagtatttagcatgcccca} CCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCCGGAAATCAAGT CCGTTTATCTCAAACTTTAGCATTTTGGGAATAAATGATATTTGCTATGCT GGTTAAATTAGATTTTAGTTAAATTTCCTGCTGAAGCTCTAGTACGATAA GTAACTTGACCTAAGTGTAAAGTTGAGATTTCCTTCAGGTTTATATAGCT TGTGCGCCGCCTGGGTACACCGCACTAGAGTAACAGTCTGACGTTTAAGA GCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCA ACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGGCCGCGCTTGCG GCCGCCTCGAGTGATCAAAAAAACCAACACACGCTTCCAATGAAAATAA ACGATCCTTTATTGCTAGCCTTTACTTGTACAGCTCGTCCATGCCGAGAGT GATCCCGGCGGCGGTCACGAACTCCAGCAGGACCATGTGATCGCGCTTCT CGTTGGGGTCTTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTCG GGCAGCAACACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGTGGT CGGCGAGCTGCACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTC ACCTTGATGCCGTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTG TTGTAGTTGTACTCCAGCTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAG TCGATGCCCTTCAGCTCGATGCGGTTCACCAGGGTGTCGCCCTCGAACTT CACCTCGGCGCGGGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGC GCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGC TTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTCAGGG TGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGAT G AACTTC AGGGTC AGCTTGCCG T AGGTGGC ATCGCCCTCGCCCTCGCCGG ACACGCTGAACTTGTGGCCGTTTACGTCGCCGTCCAGCTCGACCAGGATG GGCACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACCATGGTGGCGAC ÇGGTGGATCCCGGGCCCGCGGGGTGGCTTTACCAACAGTACCCGGAATG

113

CCAAGCTTACTTAGATCGCAGTCTCGACGCTGGCTGGCTCCTGAGTGTCT GTCTGTGCTGTGGAGGTGGTGGTAGAATGAGGGCAGCCCCTGTGCCCCTG GGTTATATAGAGGAGCCTACAGGGTGTGACTAGCCAGGAGGGGCTGTCC CCAGGGAGGGGCCCCTGAGAGCAGATGAGCTTTCAGCTCGTTGCCCGGG CACCGTGCCCACCCCGGACCCAGGCGTGCAGCTTGCCCAGCCCCATGGC CTTGTATGGGCTGCCCCAAGGGCTGACTTGCTCACTGGTTCCTAAACTAA GTGCTGAGTCTAGCTGGCGGGGGACAGCTGGCCCTTCGCCGGGAACATG GCATGCAGGGTTATTTTTAGAGGCAGCAGGTGTTGGGGGGGGGGGGGCA GCCACATGTCTGGGTTAATTATAACCAGGCATCTCGGGTGTCCCCAGGCC TTGCCTCCTTACATGGGCACGTCGACGATATCAGATCTAGGAACCCCTAG TGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCC GGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAG TGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

919

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAA GCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGA GCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTAGATCT GATATCGGCGCGCCCCTGGGCGCGCCCGAGTCCAACACCCGTGGGAATC CCATGGGCACCATGGCCCCTCGCTCCAAAAATGCTTTCGCGTCGCGCAGA CACTGCTCGGTAGTTTCGGGGATCAGCGTTTGAGTAAGAGCCCGCGTCTG AACCCTCCGCGCCGCCCCGGCCCCAGTGGAAAGACGCGCAGGCAAAACG CACCACGTGACGGAGCGTGACCGCGCGCCGAGCGCGCGCCAAGGTCGGG CAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATA CAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAG ATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTT TGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAA CTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGA AACACCGCACCAGAGTAACAGTCTGACGTTTAAGAGCTATGCTGGAAAC AGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTG GCACCGAGTCGGTGCTTTTTTTGCGGCCGCGCTCGGCGCGCCCATATTTG CATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGG ATTTGGGAATCTTATAAGTTCTGTATGAGACCACGGTACACCGCACCAGA GTAACAGTCTGACGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTT AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTG CTTTTTTTGCGGCCGCTGACGGCGCGCCCTGCAGTATTTAGCATGCCCCA CCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCCGGAAATCAAGT CCGTTTATCTCAAACTTTAGCATTTTGGGAATAAATGATATTTGCTATGCT GGTTAAATTAGATTTTAGTTAAATTTCCTGCTGAAGCTCTAGTACGATAA GTAACTTGACCTAAGTGTAAAGTTGAGATTTCCTTCAGGTTTATATAGCT TGTGCGCCGCCTGGGTACACCGCACCAGAGTAACAGTCTGACGTTTAAG AGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATC AACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGGCCGCGCTGTT TAAACCAACAAAATCAGCAGCTAATGAAGGCAAGTCAGCAGGTCACTCA TCATTTTCCACTTCGGCAATGCAGTGGGATTATTCCAACAGAGGTTTTTC ACAGCATTCCTTCAGTTAACTGGAGATCGAATCTTGATTTTCACAGATAT ACTTGGCAAGGTCCGCCCTGTCATCAGCACATTCAAGCAGATCTCCATGG CAGCATTCCGTGTGGACTTAGGTAAGATCTGTCACTAACTTGGAAACTTC TGCAAACTCAGCTTAGGGAAATCTCTGGCTCAGGCGAGCTACTGCCTCAG

114

CTTAGAAAGCTCTTTCTCCAAATTATTGGAGACTGGCACACTTAAGTCCC TGTTAGGCAGACGAAGCCTTCCCTTCATCCCGAAGTTCATCGAGCTTTGG CAACAGGCAGGCAGCTTTATCAGCAGCTTGGCAACATTCTGTAAAAGCA GCTTTATACCTTTAAGCAAAGAAAAGGAGTTCCGGGGCATAAAAGTAAG GATGTCTTCTGGCAATTTATAATAAGTATTTTTTCAAAAATGTCTCTTCAT TGTCATGAAAAGCAGTGCATCACACATCAACCTCTGGTCTCACCAATCGG GGGAGGTTTGGGTTGTTTACTTAGTGTTGCAAGAATTATTTTATTCTCTCA GGTTCTTGTTTTGCACAGCAGTCAGCTCATTCACCATAGGTTTCACGAAG AGTTGCTGCGGCCGCCTCGATAATACGACTCACTATAGGGTCGACGATAT CAGATCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGC TCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGC TTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

915

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAA GCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGA GCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTAGATCT GATATCGGCGCGCCCCTGGGCGCGCCCGAGTCCAACACCCGTGGGAATC CCATGGGCACCATGGCCCCTCGCTCCAAAAATGCTTTCGCGTCGCGCAGA CACTGCTCGGTAGTTTCGGGGATCAGCGTTTGAGTAAGAGCCCGCGTCTG AACCCTCCGCGCCGCCCCGGCCCCAGTGGAAAGACGCGCAGGCAAAACG CACCACGTGACGGAGCGTGACCGCGCGCCGAGCGCGCGCCAAGGTCGGG CAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATA CAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAG ATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTT TGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAA CTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGA AACACCGCACCAGAGTAACAGTCTGAGGTTTAAGAGCTATGCTGGAAAC AGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTG GCACCGAGTCGGTGCTTTTTTTGCGGCCGCGCTCGGCGCGCCCATATTTG CATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGG ATTTGGGAATCTTATAAGTTCTGTATGAGACCACGGTACACCGCACCAGA GTAACAGTCTGAGGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTT AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTG ctttttttgcggccgctgacggcgcgccctgcagtatttagcatgcccca CCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCCGGAAATCAAGT CCGTTTATCTCAAACTTTAGCATTTTGGGAATAAATGATATTTGCTATGCT GGTTAAATTAGATTTTAGTTAAATTTCCTGCTGAAGCTCTAGTACGATAA GTAACTTGACCTAAGTGTAAAGTTGAGATTTCCTTCAGGTTTATATAGCT TGTGCGCCGCCTGGGTACACCGCACCAGAGTAACAGTCTGAGGTTTAAG AGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATC AACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGGCCGCGCTGTT TAAACCAACAAAATCAGCAGCTAATGAAGGCAAGTCAGCAGGTCACTCA TCATTTTCCACTTCGGCAATGCAGTGGGATTATTCCAACAGAGGTTTTTC ACAGCATTCCTTCAGTTAACTGGAGATCGAATCTTGATTTTCACAGATAT ACTTGGCAAGGTCCGCCCTGTCATCAGCACATTCAAGCAGATCTCCATGG CAGCATTCCGTGTGGACTTAGGTAAGATCTGTCACTAACTTGGAAACTTC TGCAAACTCAGCTTAGGGAAATCTCTGGCTCAGGCGAGCTACTGCCTCAG CTTAGAAAGCTCTTTCTCCAAATTATTGGAGACTGGCACACTTAAGTCCC

115

TGTTAGGCAGACGAAGCCTTCCCTTCATCCCGAAGTTCATCGAGCTTTGG CAACAGGCAGGCAGCTTTATCAGCAGCTTGGCAACATTCTGTAAAAGCA GCTTTATACCTTTAAGCAAAGAAAAGGAGTTCCGGGGCATAAAAGTAAG GATGTCTTCTGGCAATTTATAATAAGTATTTTTTCAAAAATGTCTCTTCAT TGTCATGAAAAGCAGTGCATCACACATCAACCTCTGGTCTCACCAATCGG GGGAGGTTTGGGTTGTTTACTTAGTGTTGCAAGAATTATTTTATTCTCTCA GGTTCTTGTTTTGCACAGCAGTCAGCTCATTCACCATAGGTTTCACGAAG AGTTGCTGCGGCCGCCTCGATAATACGACTCACTATAGGGTCGACGATAT CAGATCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGC TCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGC TTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

912

AAGATGACGGTTTGTCACATGGAGTTGGCAGGATGTTTGATTAAAAACAT AACAGGAAGAAAAATGCCCCGCTGTGGGCGGACAAAATAGTTGGGAACT GGGAGGGGTGGAAATGGAGTTTTTAAGGATTATTTAGGGAAGAGTGACA AAATAGATGGGAACTGGGTGTAGCGTCGTAAGCTAATACGAAAATTAAA AATGACAAAATAGTTTGGAACTAGATTTCACTTATCTGGTTCGGATCTCC TAGGCGATATCAGTGATCACGGATCTCGACCAATTGACATTATTGAAGCA ACTAGTATCGATTTTATCAGGGTTATTGTCTCAGACCTGCAGGCAGCTGC GCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGC GACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGA GTGGCCAACTCCATCACTAGGGGTTCCTAGATCTGATATCGGCGCGCCCC TGGGCGCGCCCGAGTCCAACACCCGTGGGAATCCCATGGGCACCATGGC CCCTCGCTCCAAAAATGCTTTCGCGTCGCGCAGACACTGCTCGGTAGTTT CGGGGATCAGCGTTTGAGTAAGAGCCCGCGTCTGAACCCTCCGCGCCGC CCCGGCCCCAGTGGAAAGACGCGCAGGCAAAACGCACCACGTGACGGA GCGTGACCGCGCGCCGAGCGCGCGCCAAGGTCGGGCAGGAAGAGGGCCT ATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAG AGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAA TACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAAT TATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTC GATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCACTAGA GTAACAGTCTGACGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTT AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTG CTTTTTTTGCGGCCGCGCTTCGGCGCGCCCATATTTGCATGTCGCTATGTG TTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTA TAAGTTCTGTATGAGACCACGGTACACCGCACTAGAGTAACAGTCTGAC GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGGCC GCTGACGGCGCGCCCTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGC ATTCTGGATAGTGTCAAAACAGCCGGAAATCAAGTCCGTTTATCTCAAAC TTTAGCATTTTGGGAATAAATGATATTTGCTATGCTGGTTAAATTAGATTT TAGTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGTAACTTGACCTAAG TGTAAAGTTGAGATTTCCTTCAGGTTTATATAGCTTGTGCGCCGCCTGGG TACACCGCACTAGAGTAACAGTCTGACGTTTAAGAGCTATGCTGGAAAC AGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTG GCACCGAGTCGGTGCTTTTTTTGCGGCCGCGCTGCGGCCGCCTCGAGTGA TCAAAAAAACCAACACACGCTTCCAATGAAAATAAACGATCCTTTATTGC

116

TAGCCTTTACTTGTACAGCTCGTCCATGCCGAGAGTGATCCCGGCGGCGG TCACGAACTCCAGCAGGACCATGTGATCGCGCTTCTCGTTGGGGTCTTTG CTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTCGGGCAGCAACACGG GGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGTGGTCGGCGAGCTGCAC GCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCACCTTGATGCCGT TCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGTTGTACT CCAGCTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTC AGCTCGATGCGGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCGCG GGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGACGT AGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCG GGGTAGCGGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGTCACGAGGG TGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGT CAGCTTGCCGTAGGTGGCATCGCCCTCGCCCTCGCCGGACACGCTGAACT TGTGGCCGTTTACGTCGCCGTCCAGCTCGACCAGGATGGGCACCACCCCG GTGAACAGCTCCTCGCCCTTGCTCACCATGGTGGCGACCGGTGGATCCCG GGCCCGCGGGGTGGCTTTACCAACAGTACCCGGAATGCCAAGCTTACTTA GATCGCAGTCTCGACGCTGGCTGGCTCCTGAGTGTCTGTCTGTGCTGTGG AGGTGGTGGTAGAATGAGGGCAGCCCCTGTGCCCCTGGGTTATATAGAG GAGCCTACAGGGTGTGACTAGCCAGGAGGGGCTGTCCCCAGGGAGGGGC CCCTGAGAGCAGATGAGCTTTCAGCTCGTTGCCCGGGCACCGTGCCCACC CCGGACCCAGGCGTGCAGCTTGCCCAGCCCCATGGCCTTGTATGGGCTGC CCCAAGGGCTGACTTGCTCACTGGTTCCTAAACTAAGTGCTGAGTCTAGC TGGCGGGGGACAGCTGGCCCTTCGCCGGGAACATGGCATGCAGGGTTAT TTTTAGAGGCAGCAGGTGTTGGGGGGGGGGGGGCAGCCACATGTCTGGG TTAATTATAACCAGGCATCTCGGGTGTCCCCAGGCCTTGCCTCCTTACAT GGGCACGTCGACGATATCAGATCTAGGAACCCCTAGTGATGGAGTTGGC CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGG TCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGC GCGCAGCTGCCTGCAGGCATGCAAGCTGTAGCCAACCACTAGAACTATA GCTAGAGTCCTGGGCGAACAAACGATGCTCGCCTTCCAGAAAACCGAGG ATGCGAACCACTTCATCCGGGGTCAGCACCACCGGCAAGCGCCGCGACG GCCGAGGTCTTCCGATCTCCTGAAGCCAGGGCAGATCCGTGCACAGCAC CTTGCCGTAGAAGAACAGCAAGGCCGCCAATGCCTGACGATGCGTGGAG ACCGAAACCTTGCGCTCGTTCGCCAGCCAGGACAGAAATGCCTCGACTTC GCTGCTGCCCAAGGTTGCCGGGTGACGCACACCGTGGAAACGGATGAAG GCACGAACCCAGTTGACATAAGCCTGTTCGGTTCGTAAACTGTAATGCAA GTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGC GGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTT GTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGG GTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGCAACG ATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGGTGGCTCAAG TATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCAAGTCAAATCCA TGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAGACGTAGCCACC TACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGCTCCGTAG TAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCG CTCTCGCGGCTTACGTTCTGCCCAAGTTTGAGCAGCCGCGTAGTGAGATC TATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGC CACCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTT ATGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTC TATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTTGATATCGACC

117

CAAGTACCGCCACCTAACAATTCGTTCAAGCCGAGATCGGCTTCCCGGCC GCGGAGTTGTTCGGTAAATTGTCACAACGCCGCGAATATAGTCTTTACCA TGCCCTTGGCCACGCCCCTCTTTAATACGACGGGCAATTTGCACTTCAGA AAATGAAGAGTTTGCTTTAGCCATAACAAAAGTCCAGTATGCTTTTTCAC AGCATAACTGGACTGATTTCAGTTTACAACTATTCTGTCTAGTTTAAGAC TTTATTGTCATAGTTTAGATCTATTTTGTTCAGTTTAAGACTTTATTGTCC GCCCACACCCGCTTACGC

920

AAGATGACGGTTTGTCACATGGAGTTGGCAGGATGTTTGATTAAAAACAT AACAGGAAGAAAAATGCCCCGCTGTGGGCGGACAAAATAGTTGGGAACT GGGAGGGGTGGAAATGGAGTTTTTAAGGATTATTTAGGGAAGAGTGACA AAATAGATGGGAACTGGGTGTAGCGTCGTAAGCTAATACGAAAATTAAA AATGACAAAATAGTTTGGAACTAGATTTCACTTATCTGGTTCGGATCTCC TAGGCGATATCAGTGATCACGGATCTCGACCAATTGACATTATTGAAGCA ACTAGTATCGATTTTATCAGGGTTATTGTCTCAGACCTGCAGGCAGCTGC GCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGC GACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGA GTGGCCAACTCCATCACTAGGGGTTCCTAGATCTGATATCGGCGCGCCCC TGGGCGCGCCCGAGTCCAACACCCGTGGGAATCCCATGGGCACCATGGC CCCTCGCTCCAAAAATGCTTTCGCGTCGCGCAGACACTGCTCGGTAGTTT CGGGGATCAGCGTTTGAGTAAGAGCCCGCGTCTGAACCCTCCGCGCCGC CCCGGCCCCAGTGGAAAGACGCGCAGGCAAAACGCACCACGTGACGGA GCGTGACCGCGCGCCGAGCGCGCGCCAAGGTCGGGCAGGAAGAGGGCCT ATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAG AGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAA TACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAAT TATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTC GATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCACCAGA GTAACAGTCTGACGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTT AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTG _{CTTTTTTTGCGGCCGCGCTCGGCGCGCCCATATTTGCATGTCGCTATGTGT} TCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTAT AAGTTCTGTATGAGACCACGGTACACCGCACCAGAGTAACAGTCTGACG TTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGGCCG CTGACGGCGCGCCCTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCA TTCTGGATAGTGTCAAAACAGCCGGAAATCAAGTCCGTTTATCTCAAACT TTAGCATTTTGGGAATAAATGATATTTGCTATGCTGGTTAAATTAGATTTT AGTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGTAACTTGACCTAAGT GTAAAGTTGAGATTTCCTTCAGGTTTATATAGCTTGTGCGCCGCCTGGGT ACACCGCACCAGAGTAACAGTCTGACGTTTAAGAGCTATGCTGGAAACA GCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG CACCGAGTCGGTGCTTTTTTTGCGGCCGCGCTGTTTAAACCAACAAAATC AGCAGCTAATGAAGGCAAGTCAGCAGGTCACTCATCATTTTCCACTTCGG CAATGCAGTGGGATTATTCCAACAGAGGTTTTTCACAGCATTCCTTCAGT TAACTGGAGATCGAATCTTGATTTTCACAGATATACTTGGCAAGGTCCGC CCTGTCATCAGCACATTCAAGCAGATCTCCATGGCAGCATTCCGTGTGGA CTTAGGTAAGATCTGTCACTAACTTGGAAACTTCTGCAAACTCAGCTTAG

118

GGAAATCTCTGGCTCAGGCGAGCTACTGCCTCAGCTTAGAAAGCTCTTTC TCCAAATTATTGGAGACTGGCACACTTAAGTCCCTGTTAGGCAGACGAAG CCTTCCCTTCATCCCGAAGTTCATCGAGCTTTGGCAACAGGCAGGCAGCT TTATCAGCAGCTTGGCAACATTCTGTAAAAGCAGCTTTATACCTTTAAGC AAAGAAAAGGAGTTCCGGGGCATAAAAGTAAGGATGTCTTCTGGCAATT TATAATAAGTATTTTTTCAAAAATGTCTCTTCATTGTCATGAAAAGCAGT GCATCACACATCAACCTCTGGTCTCACCAATCGGGGGAGGTTTGGGTTGT TTACTTAGTGTTGCAAGAATTATTTTATTCTCTCAGGTTCTTGTTTTGCAC AGCAGTCAGCTCATTCACCATAGGTTTCACGAAGAGTTGCTGCGGCCGCC TCGATAATACGACTCACTATAGGGTCGACGATATCAGATCTAGGAACCCC TAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAG GCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCT CAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGCATGCAAGCTGTAGCC AACCACTAGAACTATAGCTAGAGTCCTGGGCGAACAAACGATGCTCGCC TTCCAGAAAACCGAGGATGCGAACCACTTCATCCGGGGTCAGCACCACC GGCAAGCGCCGCGACGGCCGAGGTCTTCCGATCTCCTGAAGCCAGGGCA GATCCGTGCACAGCACCTTGCCGTAGAAGAACAGCAAGGCCGCCAATGC CTGACGATGCGTGGAGACCGAAACCTTGCGCTCGTTCGCCAGCCAGGAC AGAAATGCCTCGACTTCGCTGCTGCCCAAGGTTGCCGGGTGACGCACACC GTGGAAACGGATGAAGGCACGAACCCAGTTGACATAAGCCTGTTCGGTT CGTAAACTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAA CCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCT TGTTATGACTGTTTTTTTGTACAGTCTATGCCTCGGGCATCCAAGCAGCA AGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATG TTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAA AGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCT GACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTT CGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCG GGAACTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAA GAAGCGGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAAGTTTGAGCA GCCGCGTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACC GGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGAGGC CAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACG ATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGAT GCACTTTGATATCGACCCAAGTACCGCCACCTAACAATTCGTTCAAGCCG AGATCGGCTTCCCGGCCGCGGAGTTG TTCGGTAAATTGTCACAACGCCGC GAATATAGTCTTTACCATGCCCTTGGCCACGCCCCTCTTTAATACGACGG GCAATTTGCACTTCAGAAAATGAAGAGTTTGCTTTAGCCATAACAAAAGT CCAGTATGCTTTTTCACAGCATAACTGGACTGATTTCAGTTTACAACTATT CTGTCTAGTTTAAGACTTTATTGTCATAGTTTAGATCTATTTTGTTCAGTT TAAGACTTTATTGTCCGCCCACACCCGCTTACGC

916

AAGATGACGGTTTGTCACATGGAGTTGGCAGGATGTTTGATTAAAAACAT AACAGGAAGAAAAATGCCCCGCTGTGGGCGGACAAAATAGTTGGGAACT GGGAGGGGTGGAAATGGAGTTTTTAAGGATTATTTAGGGAAGAGTGACA AAATAGATGGGAACTGGGTGTAGCGTCGTAAGCTAATACGAAAATTAAA AATGACAAAATAGTTTGGAACTAGATTTCACTTATCTGGTTCGGATCTCC TAGGCGATATCAGTGATCACGGATCTCGACCAATTGACATTATTGAAGCA

119

ACTAGTATCGATTTTATCAGGGTTATTGTCTCAGACCTGCAGGCAGCTGC GCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGC GACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGA GTGGCCAACTCCATCACTAGGGGTTCCTAGATCTGATATCGGCGCGCCCC TGGGCGCGCCCGAGTCCAACACCCGTGGGAATCCCATGGGCACCATGGC CCCTCGCTCCAAAAATGCTTTCGCGTCGCGCAGACACTGCTCGGTAGTTT CGGGGATCAGCGTTTGAGTAAGAGCCCGCGTCTGAACCCTCCGCGCCGC CCCGGCCCCAGTGGAAAGACGCGCAGGCAAAACGCACCACGTGACGGA GCGTGACCGCGCGCCGAGCGCGCGCCAAGGTCGGGCAGGAAGAGGGCCT ATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAG AGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAA TACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAAT TATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTC GATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCACCAGA GTAACAGTCTGAGGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTT AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTG _{CTTTTTTTGCGGCCGCGCTCGGCGCGCCCATATTTGCATGTCGCTATGTGT} TCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTAT AAGTTCTGTATGAGACCACGGTACACCGCACCAGAGTAACAGTCTGAGG TTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGGCCG CTGACGGCGCGCCCTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCA TTCTGGATAGTGTCAAAACAGCCGGAAATCAAGTCCGTTTATCTCAAACT TTAGCATTTTGGGAATAAATGATATTTGCTATGCTGGTTAAATTAGATTTT AGTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGTAACTTGACCTAAGT GTAAAGTTGAGATTTCCTTCAGGTTTATATAGCTTGTGCGCCGCCTGGGT ACACCGCACCAGAGTAACAGTCTGAGGTTTAAGAGCTATGCTGGAAACA GCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG CACCGAGTCGGTGCTTTTTTTGCGGCCGCGCTGTTTAAACCAACAAAATC AGCAGCTAATGAAGGCAAGTCAGCAGGTCACTCATCATTTTCCACTTCGG CAATGCAGTGGGATTATTCCAACAGAGGTTTTTCACAGCATTCCTTCAGT TAACTGGAGATCGAATCTTGATTTTCACAGATATACTTGGCAAGGTCCGC CCTGTCATCAGCACATTCAAGCAGATCTCCATGGCAGCATTCCGTGTGGA CTTAGGTAAGATCTGTCACTAACTTGGAAACTTCTGCAAACTCAGCTTAG GGAAATCTCTGGCTCAGGCGAGCTACTGCCTCAGCTTAGAAAGCTCTTTC TCCAAATTATTGGAGACTGGCACACTTAAGTCCCTGTTAGGCAGACGAAG CCTTCCCTTCATCCCGAAGTTCATCGAGCTTTGGCAACAGGCAGGCAGCT TTATCAGCAGCTTGGCAACATTCTGTAAAAGCAGCTTTATACCTTTAAGC AAAGAAAAGGAGTTCCGGGGCATAAAAGTAAGGATGTCTTCTGGCAATT TATAATAAGTATTTTTTCAAAAATGTCTCTTCATTGTCATGAAAAGCAGT GCATCACACATCAACCTCTGGTCTCACCAATCGGGGGAGGTTTGGGTTGT TTACTTAGTGTTGCAAGAATTATTTTATTCTCTCAGGTTCTTGTTTTGCAC AGCAGTCAGCTCATTCACCATAGGTTTCACGAAGAGTTGCTGCGGCCGCC TCGATAATACGACTCACTATAGGGTCGACGATATCAGATCTAGGAACCCC TAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAG GCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCT CAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGCATGCAAGCTGTAGCC AACCACTAGAACTATAGCTAGAGTCCTGGGCGAACAAACGATGCTCGCC TTCCAGAAAACCGAGGATGCGAACCACTTCATCCGGGGTCAGCACCACC GGCAAGCGCCGCGACGGCCGAGGTCTTCCGATCTCCTGAAGCCAGGGCA

120

GATCCGTGCACAGCACCTTGCCGTAGAAGAACAGCAAGGCCGCCAATGC CTGACGATGCGTGGAGACCGAAACCTTGCGCTCGTTCGCCAGCCAGGAC AGAAATGCCTCGACTTCGCTGCTGCCCAAGGTTGCCGGGTGACGCACACC GTGGAAACGGATGAAGGCACGAACCCAGTTGACATAAGCCTGTTCGGTT CGTAAACTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAA CCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCT TGTTATGACTGTTTTTTTGTACAGTCTATGCCTCGGGCATCCAAGCAGCA AGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATG TTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAA AGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCT GACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTT CGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCG GGAACTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAA GAAGCGGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAAGTTTGAGCA GCCGCGTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACC GGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGAGGC CAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACG ATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGAT GCACTTTGATATCGACCCAAGTACCGCCACCTAACAATTCGTTCAAGCCG AGATCGGCTTCCCGGCCGCGGAGTTGTTCGGTAAATTGTCACAACGCCGC GAATATAGTCTTTACCATGCCCTTGGCCACGCCCCTCTTTAATACGACGG GCAATTTGCACTTCAGAAAATGAAGAGTTTGCTTTAGCCATAACAAAAGT CCAGTATGCTTTTTCACAGCATAACTGGACTGATTTCAGTTTACAACTATT CTGTCTAGTTTAAGACTTTATTGTCATAGTTTAGATCTATTTTGTTCAGTT TAAGACTTTATTGTCCGCCCACACCCGCTTACGC

913

AAGATGACGGTTTGTCACATGGAGTTGGCAGGATGTTTGATTAAAAACAT AACAGGAAGAAAAATGCCCCGCTGTGGGCGGACAAAATAGTTGGGAACT GGGAGGGGTGGAAATGGAGTTTTTAAGGATTATTTAGGGAAGAGTGACA AAATAGATGGGAACTGGGTGTAGCGTCGTAAGCTAATACGAAAATTAAA AATGACAAAATAGTTTGGAACTAGATTTCACTTATCTGGTTCGGATCTCC TAGGCGATATCAGTGATCACGGATCTCGACCAATTGACATTATTGAAGCA ACTAGTATCGATTTTATCAGGGTTATTGTCTCAGACCACTCCCTCTATGCG CGCTCGCTCACTCACTCGGCCCTGGAGACCAAAGGTCTCCAGACTGCCGG CCTCTGGCCGGCAGGGCCGAGTGAGTGAGCGAGCGCGCATAGAGGGAGT GGGTACCTCCATCATCTAGGTTTGCCAGATCTGATATCGGCGCGCCCCTG GGCGCGCCCGAGTCCAACACCCGTGGGAATCCCATGGGCACCATGGCCC CTCGCTCCAAAAATGCTTTCGCGTCGCGCAGACACTGCTCGGTAGTTTCG GGGATCAGCGTTTGAGTAAGAGCCCGCGTCTGAACCCTCCGCGCCGCCCC GGCCCCAGTGGAAAGACGCGCAGGCAAAACGCACCACGTGACGGAGCG TGACCGCGCGCCGAGCGCGCGCCAAGGTCGGGCAGGAAGAGGGCCTATT TCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGA TAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACG TGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATG TTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTT CTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCACTAGAGTAAC AGTCTGACGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT

121

TTGCGGCCGCGCTTCGGCGCGCCCATATTTGCATGTCGCTATGTGTTCTG GGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGT TCTGTATGAGACCACGGTACACCGCACTAGAGTAACAGTCTGACGTTTAA GAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTA TCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGGCCGCTGAC GGCGCGCCCTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTG GATAGTGTCAAAACAGCCGGAAATCAAGTCCGTTTATCTCAAACTTTAGC ATTTTGGGAATAAATGATATTTGCTATGCTGGTTAAATTAGATTTTAGTTA AATTTCCTGCTGAAGCTCTAGTACGATAAGTAACTTGACCTAAGTGTAAA GTTGAGATTTCCTTCAGGTTTATATAGCTTGTGCGCCGCCTGGGTACACC GCACTAGAGTAACAGTCTGACGTTTAAGAGCTATGCTGGAAACAGCATA GCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG AGTCGGTGCTTTTTTTGCGGCCGCGCTGCGGCCGCCTCGAGTGATCAAAA AAACCAACACACGCTTCCAATGAAAATAAACGATCCTTTATTGCTAGCCT TTACTTGTACAGCTCGTCCATGCCGAGAGTGATCCCGGCGGCGGTCACGA ACTCCAGCAGGACCATGTGATCGCGCTTCTCGTTGGGGTCTTTGCTCAGG GCGGACTGGGTGCTCAGGTAGTGGTTGTCGGGCAGCAACACGGGGCCGT CGCCGATGGGGGTGTTCTGCTGGTAGTGGTCGGCGAGCTGCACGCTGCCG TCCTCGATGTTGTGGCGGATCTTGAAGTTCACCTTGATGCCGTTCTTCTGC TTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGTTGTACTCCAGCTT GTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCGA TGCGGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCGCGGGTCTTG TAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGACGTAGCCTTC GGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAG CGGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGTCACGAGGGTGGGCC AGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTT GCCGTAGGTGGCATCGCCCTCGCCCTCGCCGGACACGCTGAACTTGTGGC CGTTTACGTCGCCGTCCAGCTCGACCAGGATGGGCACCACCCCGGTGAAC AGCTCCTCGCCCTTGCTCACCATGGTGGCGACCGGTGGATCCCGGGCCCG CGGGGTGGCTTTACCAACAGTACCCGGAATGCCAAGCTTACTTAGATCGC AGTCTCGACGCTGGCTGGCTCCTGAGTGTCTGTCTGTGCTGTGGAGGTGG TGGTAGAATGAGGGCAGCCCCTGTGCCCCTGGGTTATATAGAGGAGCCT ACAGGGTGTGACTAGCCAGGAGGGGCTGTCCCCAGGGAGGGGCCCCTGA GAGCAGATGAGCTTTCAGCTCGTTGCCCGGGCACCGTGCCCACCCCGGAC CCAGGCGTGCAGCTTGCCCAGCCCCATGGCCTTGTATGGGCTGCCCCAAG GGCTGACTTGCTCACTGGTTCCTAAACTAAGTGCTGAGTCTAGCTGGCGG GGGACAGCTGGCCCTTCGCCGGGAACATGGCATGCAGGGTTATTTTTAGA GGCAGCAGGTGTTGGGGGGGGGGGGGCAGCCACATGTCTGGGTTAATTA TAACCAGGCATCTCGGGTGTCCCCAGGCCTTGCCTCCTTACATGGGCACG TCGACGATATCAGATCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCT CTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGT CGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAG AGGGAGTGGCATGCAAGCTGTAGCCAACCACTAGAACTATAGCTAGAGT CCTGGGCGAACAAACGATGCTCGCCTTCCAGAAAACCGAGGATGCGAAC CACTTCATCCGGGGTCAGCACCACCGGCAAGCGCCGCGACGGCCGAGGT CTTCCGATCTCCTGAAGCCAGGGCAGATCCGTGCACAGCACCTTGCCGTA GAAGAACAGCAAGGCCGCCAATGCCTGACGATGCGTGGAGACCGAAACC TTGCGCTCGTTCGCCAGCCAGGACAGAAATGCCTCGACTTCGCTGCTGCC CAAGGTTGCCGGGTGACGCACACCGTGGAAACGGATGAAGGCACGAACC CAGTTGACATAAGCCTGTTCGGTTCGTAAACTGTAATGCAAGTAGCGTAT

122

GCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAAC GGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCT atgcctcgggcatccaagcagcaagcgcgttacgccgtgggtcgatgttt GATGTTATGGAGCAGCAACGATGTTACGCAGCAGCAACGATGTTACGCA GCAGGGCAGTCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATC ATTCGCACATGTAGGCTCGGCCCTGACCAAGTCAAATCCATGCGGGCTGC TCTTGATCTTTTCGGTCGTGAGTTCGGAGACGTAGCCACCTACTCCCAAC ATCAGCCGGACTCCGATTACCTCGGGAACTTGCTCCGTAGTAAGACATTC ATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCGCTCTCGCGGC TTACGTTCTGCCCAAGTTTGAGCAGCCGCGTAGTGAGATCTATATCTATG ATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCACCGCGCT CATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATCT ACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAA GTTGGGCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACC GCCACCTAACAATTCGTTCAAGCCGAGATCGGCTTCCCGGCCGCGGAGTT GTTCGGTAAATTGTCACAACGCCGCGAATATAGTCTTTACCATGCCCTTG GCCACGCCCCTCTTTAATACGACGGGCAATTTGCACTTCAGAAAATGAAG AGTTTGCTTTAGCCATAACAAAAGTCCAGTATGCTTTTTCACAGCATAAC TGGACTGATTTCAGTTTACAACTATTCTGTCTAGTTTAAGACTTTATTGTC ATAGTTTAGATCTATTTTGTTCAGTTTAAGACTTTATTGTCCGCCCACACC CGCTTACGC

921

AAGATGACGGTTTGTCACATGGAGTTGGCAGGATGTTTGATTAAAAACAT AACAGGAAGAAAAATGCCCCGCTGTGGGCGGACAAAATAGTTGGGAACT GGGAGGGGTGGAAATGGAGTTTTTAAGGATTATTTAGGGAAGAGTGACA AAATAGATGGGAACTGGGTGTAGCGTCGTAAGCTAATACGAAAATTAAA AATGACAAAATAGTTTGGAACTAGATTTCACTTATCTGGTTCGGATCTCC TAGGCGATATCAGTGATCACGGATCTCGACCAATTGACATTATTGAAGCA ACTAGTATCGATTTTATCAGGGTTATTGTCTCAGACCACTCCCTCTATGCG CGCTCGCTCACTCACTCGGCCCTGGAGACCAAAGGTCTCCAGACTGCCGG CCTCTGGCCGGCAGGGCCGAGTGAGTGAGCGAGCGCGCATAGAGGGAGT GGGTACCTCCATCATCTAGGTTTGCCAGATCTGATATCGGCGCGCCCCTG GGCGCGCCCGAGTCCAACACCCGTGGGAATCCCATGGGCACCATGGCCC CTCGCTCCAAAAATGCTTTCGCGTCGCGCAGACACTGCTCGGTAGTTTCG GGGATCAGCGTTTGAGTAAGAGCCCGCGTCTGAACCCTCCGCGCCGCCCC GGCCCCAGTGGAAAGACGCGCAGGCAAAACGCACCACGTGACGGAGCG TGACCGCGCGCCGAGCGCGCGCCAAGGTCGGGCAGGAAGAGGGCCTATT TCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGA TAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACG TGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATG TTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTT CTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCACCAGAGTAAC AGTCTGACGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATA aggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttt TTGCGGCCGCGCTCGGCGCGCCCATATTTGCATGTCGCTATGTGTTCTGG GAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTT CTGTATGAGACCACGGTACACCGCACCAGAGTAACAGTCTGACGTTTAA

123

GAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTA TCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGGCCGCTGAC GGCGCGCCCTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTG GATAGTGTCAAAACAGCCGGAAATCAAGTCCGTTTATCTCAAACTTTAGC ATTTTGGGAATAAATGATATTTGCTATGCTGGTTAAATTAGATTTTAGTTA AATTTCCTGCTGAAGCTCTAGTACGATAAGTAACTTGACCTAAGTGTAAA GTTGAGATTTCCTTCAGGTTTATATAGCTTGTGCGCCGCCTGGGTACACC GCACCAGAGTAACAGTCTGACGTTTAAGAGCTATGCTGGAAACAGCATA GCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG AGTCGGTGCTTTTTTTGCGGCCGCGCTGTTTAAACCAACAAAATCAGCAG CTAATGAAGGCAAGTCAGCAGGTCACTCATCATTTTCCACTTCGGCAATG CAGTGGGATTATTCCAACAGAGGTTTTTCACAGCATTCCTTCAGTTAACT GGAGATCGAATCTTGATTTTCACAGATATACTTGGCAAGGTCCGCCCTGT CATCAGCACATTCAAGCAGATCTCCATGGCAGCATTCCGTGTGGACTTAG GTAAGATCTGTCACTAACTTGGAAACTTCTGCAAACTCAGCTTAGGGAAA TCTCTGGCTCAGGCGAGCTACTGCCTCAGCTTAGAAAGCTCTTTCTCCAA ATTATTGGAGACTGGCACACTTAAGTCCCTGTTAGGCAGACGAAGCCTTC CCTTCATCCCGAAGTTCATCGAGCTTTGGCAACAGGCAGGCAGCTTTATC AGCAGCTTGGCAACATTCTGTAAAAGCAGCTTTATACCTTTAAGCAAAGA AAAGGAGTTCCGGGGCATAAAAGTAAGGATGTCTTCTGGCAATTTATAA TAAGTATTTTTTCAAAAATGTCTCTTCATTGTCATGAAAAGCAGTGCATC ACACATCAACCTCTGGTCTCACCAATCGGGGGAGGTTTGGGTTGTTTACT TAGTGTTGCAAGAATTATTTTATTCTCTCAGGTTCTTGTTTTGCACAGCAG TCAGCTCATTCACCATAGGTTTCACGAAGAGTTGCTGCGGCCGCCTCGAT AATACGACTCACTATAGGGTCGACGATATCAGATCTAGGAACCCCTAGT GATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCG CCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGT GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCATGCAAGCTGTAGCCAAC CACTAGAACTATAGCTAGAGTCCTGGGCGAACAAACGATGCTCGCCTTCC AGAAAACCGAGGATGCGAACCACTTCATCCGGGGTCAGCACCACCGGCA AGCGCCGCGACGGCCGAGGTCTTCCGATCTCCTGAAGCCAGGGCAGATC CGTGCACAGCACCTTGCCGTAGAAGAACAGCAAGGCCGCCAATGCCTGA CGATGCGTGGAGACCGAAACCTTGCGCTCGTTCGCCAGCCAGGACAGAA ATGCCTCGACTTCGCTGCTGCCCAAGGTTGCCGGGTGACGCACACCGTGG AAACGGATGAAGGCACGAACCCAGTTGACATAAGCCTGTTCGGTTCGTA AACTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTG ACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTA TGACTGTTTTTTTGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGC GTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACG CAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTA GGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCA AGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAG ACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAAC TTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGC GGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAAGTTTGAGCAGCCGC GTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGG CAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGC GCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCG CAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTT GATATCGACCCAAGTACCGCCACCTAACAATTCGTTCAAGCCGAGATCG

124

GCTTCCCGGCCGCGGAGTTGTTCGGTAAATTGTCACAACGCCGCGAATAT AGTCTTTACCATGCCCTTGGCCACGCCCCTCTTTAATACGACGGGCAATT TGCACTTCAGAAAATGAAGAGTTTGCTTTAGCCATAACAAAAGTCCAGTA TGCTTTTTCACAGCATAACTGGACTGATTTCAGTTTACAACTATTCTGTCT AGTTTAAGACTTTATTGTCATAGTTTAGATCTATTTTGTTCAGTTTAAGAC TTTATTGTCCGCCCACACCCGCTTACGC

917

AAGATGACGGTTTGTCACATGGAGTTGGCAGGATGTTTGATTAAAAACAT AACAGGAAGAAAAATGCCCCGCTGTGGGCGGACAAAATAGTTGGGAACT GGGAGGGGTGGAAATGGAGTTTTTAAGGATTATTTAGGGAAGAGTGACA AAATAGATGGGAACTGGGTGTAGCGTCGTAAGCTAATACGAAAATTAAA AATGACAAAATAGTTTGGAACTAGATTTCACTTATCTGGTTCGGATCTCC TAGGCGATATCAGTGATCACGGATCTCGACCAATTGACATTATTGAAGCA ACTAGTATCGATTTTATCAGGGTTATTGTCTCAGACCACTCCCTCTATGCG CGCTCGCTCACTCACTCGGCCCTGGAGACCAAAGGTCTCCAGACTGCCGG CCTCTGGCCGGCAGGGCCGAGTGAGTGAGCGAGCGCGCATAGAGGGAGT GGGTACCTCCATCATCTAGGTTTGCCAGATCTGATATCGGCGCGCCCCTG GGCGCGCCCGAGTCCAACACCCGTGGGAATCCCATGGGCACCATGGCCC CTCGCTCCAAAAATGCTTTCGCGTCGCGCAGACACTGCTCGGTAGTTTCG GGGATCAGCGTTTGAGTAAGAGCCCGCGTCTGAACCCTCCGCGCCGCCCC GGCCCCAGTGGAAAGACGCGCAGGCAAAACGCACCACGTGACGGAGCG TGACCGCGCGCCGAGCGCGCGCCAAGGTCGGGCAGGAAGAGGGCCTATT TCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGA TAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACG TGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATG TTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTT CTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCACCAGAGTAAC AGTCTGAGGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT TTGCGGCCGCGCTCGGCGCGCCCATATTTGCATGTCGCTATGTGTTCTGG GAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTT CTGTATGAGACCACGGTACACCGCACCAGAGTAACAGTCTGAGGTTTAA GAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTA TCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGGCCGCTGAC GGCGCGCCCTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTG GATAGTGTCAAAACAGCCGGAAATCAAGTCCGTTTATCTCAAACTTTAGC ATTTTGGGAATAAATGATATTTGCTATGCTGGTTAAATTAGATTTTAGTTA AATTTCCTGCTGAAGCTCTAGTACGATAAGTAACTTGACCTAAGTGTAAA GTTGAGATTTCCTTCAGGTTTATATAGCTTGTGCGCCGCCTGGGTACACC GCACCAGAGTAACAGTCTGAGGTTTAAGAGCTATGCTGGAAACAGCATA GCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG agtcggtgctttttttgcggccgcgctgtttaaaccaacaaaatcagcag CTAATGAAGGCAAGTCAGCAGGTCACTCATCATTTTCCACTTCGGCAATG CAGTGGGATTATTCCAACAGAGGTTTTTCACAGCATTCCTTCAGTTAACT GGAGATCGAATCTTGATTTTCACAGATATACTTGGCAAGGTCCGCCCTGT CATCAGCACATTCAAGCAGATCTCCATGGCAGCATTCCGTGTGGACTTAG GTAAGATCTGTCACTAACTTGGAAACTTCTGCAAACTCAGCTTAGGGAAA TCTCTGGCTCAGGCGAGCTACTGCCTCAGCTTAGAAAGCTCTTTCTCCAA

125

ATTATTGGAGACTGGCACACTTAAGTCCCTGTTAGGCAGACGAAGCCTTC CCTTCATCCCGAAGTTCATCGAGCTTTGGCAACAGGCAGGCAGCTTTATC AGCAGCTTGGCAACATTCTGTAAAAGCAGCTTTATACCTTTAAGCAAAGA AAAGGAGTTCCGGGGCATAAAAGTAAGGATGTCTTCTGGCAATTTATAA TAAGTATTTTTTCAAAAATGTCTCTTCATTGTCATGAAAAGCAGTGCATC ACACATCAACCTCTGGTCTCACCAATCGGGGGAGGTTTGGGTTGTTTACT TAGTGTTGCAAGAATTATTTTATTCTCTCAGGTTCTTGTTTTGCACAGCAG TCAGCTCATTCACCATAGGTTTCACGAAGAGTTGCTGCGGCCGCCTCGAT AATACGACTCACTATAGGGTCGACGATATCAGATCTAGGAACCCCTAGT GATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCG CCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGT GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCATGCAAGCTGTAGCCAAC CACTAGAACTATAGCTAGAGTCCTGGGCGAACAAACGATGCTCGCCTTCC AGAAAACCGAGGATGCGAACCACTTCATCCGGGGTCAGCACCACCGGCA AGCGCCGCGACGGCCGAGGTCTTCCGATCTCCTGAAGCCAGGGCAGATC CGTGCACAGCACCTTGCCGTAGAAGAACAGCAAGGCCGCCAATGCCTGA CGATGCGTGGAGACCGAAACCTTGCGCTCGTTCGCCAGCCAGGACAGAA ATGCCTCGACTTCGCTGCTGCCCAAGGTTGCCGGGTGACGCACACCGTGG AAACGGATGAAGGCACGAACCCAGTTGACATAAGCCTGTTCGGTTCGTA AACTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTG ACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTA TGACTGTTTTTTTGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGC GTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACG CAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTA GGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCA AGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAG ACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAAC TTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGC GGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAAGTTTGAGCAGCCGC GTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGG CAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGC GCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCG CAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTT GATATCGACCCAAGTACCGCCACCTAACAATTCGTTCAAGCCGAGATCG GCTTCCCGGCCGCGGAGTTGTTCGGTAAATTGTCACAACGCCGCGAATAT AGTCTTTACCATGCCCTTGGCCACGCCCCTCTTTAATACGACGGGCAATT TGCACTTCAGAAAATGAAGAGTTTGCTTTAGCCATAACAAAAGTCCAGTA TGCTTTTTCACAGCATAACTGGACTGATTTCAGTTTACAACTATTCTGTCT AGTTTAAGACTTTATTGTCATAGTTTAGATCTATTTTGTTCAGTTTAAGAC TTTATTGTCCGCCCACACCCGCTTACGC

In some embodiments of the gene editing constructs of the disclosure, including those embodiments encompassing SEQ IDNOs: 910 to 921, the construct comprises or consists ofa first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence 5 encoding a gRNA, a third promoter, and a third sequence encoding a gRNA. Exemplary

126 sequences encoding gRNAs of the disclosure are SEQ ID NO. 383-705, 709-711, 715-717, 790-862, 864. In some embodiments, the sequence encoding the gRNA is CACTAGAGTAACAGTCTGAC (SEQ ID NO. 708). In some embodiments, the sequence encoding the gRNA is CACCAGAGTAACAGTCTGAG (SEQ ID NO. 714). In some embodiments, the sequence encoding the gRNA is CACCAGAGTAACAGTCTGAG (SEQ ID NO. 863). In some embodiments, the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA of SEQ ID NO. 708, a second promoter, a second sequence encoding a gRNA of SEQ IDNO. 708, a third promoter, and a third sequence encoding a gRNA of SEQ ID NO. 708. In some embodiments, the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA of SEQ ID NO. 714, a second promoter, a second sequence encoding a gRNA of SEQ ID NO. 714, a third promoter, and a third sequence encoding a gRNA of SEQ ID NO. 714. In some embodiments, the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA of SEQ ID NO. 863, a second promoter, a second sequence encoding a gRNA of SEQ ID NO. 863, a third promoter, and a third sequence encoding a gRNA of SEQ ID NO. 863. Exemplary promoters of the disclosure include the U6 promoter having a sequence of CGAGTCCAACACCCGTGGGAATCCCATGGGCACCATGGCCCCTCGCTCCAAAAA TGCTTTCGCGTCGCGCAGACACTGCTCGGTAGTTTCGGGGATCAGCGTTTGAGTA AGAGCCCGCGTCTGAACCCTCCGCGCCGCCCCGGCCCCAGTGGAAAGACGCGCA GGCAAAACGCACCACGTGACGGAGCGTGACCGCGCGCCGAGCGCGCGCCAAGGT CGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATAC AAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTA GTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAA AATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGAT TTCTTGGCTTTATATATCTTGTGGAAAGGACGAAA (SEQ ID NO: 922), the H1 promoter having a sequence of

GCTCGGCGCGCCCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAAC GTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACGGTA (SEQ ID. No923), and the 7SK promoter having a sequence of TGACGGCGCGCCCTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGG ATAGTGTCAAAACAGCCGGAAATCAAGTCCGTTTATCTCAAACTTTAGCATTTTG GGAATAAATGATATTTGCTATGCTGGTTAAATTAGATTTTAGTTAAATTTCCTGCT GAAGCTCTAGTACGATAAGTAACTTGACCTAAGTGTAAAGTTGAGATTTCCTTCA

127

GGTTTATATAGCTTGTGCGCCGCCTGGGTA (SEQ ID NO. 924). In some embodiments, the first, second, and third promoter are each individually selected from the U6 promoter (SEQ ID NO: 922), the H1 promoter (SEQ ID NO: 923), and the 7SK promoter (SEQ ID NO: 924). In some embodiments, the first, second, and third promoter are each individually selected from the U6 promoter (SEQ ID NO: 922), and the H1 promoter (SEQ ID NO: 923). In some embodiments, the construct comprises, from 5’ to 3’, a U6 promoter, a first sequence encoding a gRNA, a H1 promoter, a second sequence encoding a gRNA, a 7SK promoter, and a third sequence encoding a gRNA. In some embodiments, the construct comprises, from 5’ to 3’, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the H1 promoter, a second sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, and a third sequence encoding a gRNA of SEQ ID NO: 708. In some embodiments, the construct comprises, from 5’ to 3’, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 714, the H1 promoter, a second sequence encoding a gRNA of SEQ ID NO: 714, the 7SK promoter, and a third sequence encoding a gRNA of SEQ ID NO: 714. In some embodiments, the construct comprises, from 5’ to 3’, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 863, the H1 promoter, a second sequence encoding a gRNA of SEQ ID NO: 863, the 7SK promoter, and a third sequence encoding a gRNA of SEQ ID NO: 863. In some embodiments, the construct comprising a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, and a third sequence encoding a gRNA further comprises at least two inverted terminal repeat (ITR) sequences. In some embodiments, the construct comprising a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, and a third sequence encoding a gRNA further comprises at least two ITR sequences isolated or derived from an AAV of serotype 2 (AAV2). In some embodiments, the construct comprising a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, and a third sequence encoding a gRNA further comprises at least two ITR sequences, wherein the first ITR sequence is isolated or derived from an AAV of serotype 4 (AA V4) and the second ITR sequence is isolated or derived from an AAV of serotype 2 (AAV2). Exemplary ITR sequences are CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCG GGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAG AGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO: 881), CCACTCCCTCTATGCGCGCTCGCTCACTCACTCGGCCCTGGAGACCAAAGGTCTC

128

CAGACTGCCGGCCTCTGGCCGGCAGGGCCGAGTGAGTGAGCGAGCGCGCATAGA GGGAGTGGGTACCTCCATCATCTAGGTTTGCC (SEQ ID NO. 883), AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCAC TGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTC AGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGG (SEQ ID NO. 946), and AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCAC TGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTC AGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG (SEQ ID 882). In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a fïrst ITR, a U6 promoter, a first sequence encoding a gRNA, a Hl promoter, and a second sequence encoding a gRNA, a 7SK promoter, a third sequence encoding a gRNA, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a second promoter, and the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 708, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a second promoter, and the sequence encoding a gRNA of SEQ ID NO: 714, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 714, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a second promoter, and the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA of SEQ ID NO.: 863, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a U6, the sequence encoding a gRNA of SEQ ID NO: 708, a Hl promoter, and the sequence encoding a gRNA of SEQ ID NO: 708, a 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a U6, the sequence encoding a gRNA of SEQ ID NO: 714, a Hl promoter, and the sequence encoding a gRNA of SEQ ID NO: 714, a 7SK promoter, the sequence encoding a gRNA of SEQ IDNO: 714, and a second ITR. In some embodiments, the construct comprising, from 5’ to 3’ a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, a third

129 sequence encoding a gRNA, and a second ITR, further comprises a poly A sequence. In some embodiments, the polyA sequence comprises or consists of a minipolyA sequence. Exemplary minipolyA sequences of the disclosure comprise or consist of a nucléotide sequence of TAGCAATAAAGGATCGTTTATTTTCATTGGAAGCGTGTGTTGGTTTTTTGATCAGG CGCG (SEQ ID NO: 903). In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding s gRNA, a third promoter, a third sequence encoding a gRNA, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, a first sequence encoding a gRNA, the H1 promoter, a second sequence encoding a gRNA, the 7SK promoter, a third sequence encoding a gRNA, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a second promoter, the sequence encoding a gRNA of SEQ ID NO. 714, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA of SEQ IDNO: 863, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the H1 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 714, the H1 promoter, the sequence encoding a gRNA of SEQ ID NO: 714, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprising, from 5’ to 3’ a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, a minipolyA sequence, and a second ITR further comprises transposable element inverted repeats. Exemplary

130 transposable element inverted repeats of the disclosure comprise or consist of a nucléotide sequence of

TGTGGGCGGACAAAATAGTTGGGAACTGGGAGGGGTGGAAATGGAGTTTTTAAG GATTATTTAGGGAAGAGTGACAAAATAGATGGGAACTGGGTGTAGCGTCGTAAG CTAATACGAAAATTAAAAATGACAAAATAGTTTGGAACTAGATTTCACTTATCTG GTT (SEQ ID NO: 907) and/or a nucléotide sequence of GAATATAGTCTTTACCATGCCCTTGGCCACGCCCCTCTTTAATACGACGGGCAAT TTGCACTTCAGAAAATGAAGAGTTTGCTTTAGCCATAACAAAAGTCCAGTATGCT TTTTCACAGCATAACTGGACTGATTTCAGTTTACAACTATTCTGTCTAGTTTAAGA CTTTATTGTCATAGTTTAGATCTATTTTGTTCAGTTTAAGACTTTATTGTCCGCCCA CA (SEQ ID NO: 908). In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, the U6 promoter, a first sequence encoding a gRNA, the H1 promoter, a second sequence encoding a gRNA, the 7SK promoter, a third sequence encoding a gRNA, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprises

131 or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the H1 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ IDNO: 714, the H1 promoter, the sequence encoding a gRNA of SEQ ID NO: 714, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprising a first transposable element inverted repeat, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, a minipolyA sequence, a second ITR, a second transposable element inverted repeat, further comprises a regulatory sequence. Exemplary regulatory sequences of the disclosure comprise or consist of a nucléotide sequence of

CATGCAAGCTGTAGCCAACCACTAGAACTATAGCTAGAGTCCTGGGCGAACAAA CGATGCTCGCCTTCCAGAAAACCGAGGATGCGAACCACTTCATCCGGGGTCAGC ACCACCGGCAAGCGCCGCGACGGCCGAGGTCTTCCGATCTCCTGAAGCCAGGGC AGATCCGTGCACAGCACCTTGCCGTAGAAGAACAGCAAGGCCGCCAATGCCTGA CGATGCGTGGAGACCGAAACCTTGCGCTCGTTCGCCAGCCAGGACAGAAATGCC TCGACTTCGCTGCTGCCCAAGGTTGCCGGGTGACGCACACCGTGGAAACGGATG AAGGCACGAACCCAGTTGACATAAGCCTGTTCGGTTCGTAAACTGTAATGCAAGT AGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTA ACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATG CCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTAT GGAGCAGCAACGATGTTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGC CCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCG GCCCTGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTT CGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAA CTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTT GTTGGCGCTCTCGCGGCTTACGTTCTGCCCAAGTTTGAGCAGCCGCGTAGTGAGA TCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCAC CGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATC

132

TACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAAGTTGG GCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAAC AATTCGTTCAAGCCGAGATCGGCTTCCCGGCCGCGGAGTTGTTCGGTAAATTGTC ACAACGCCG (SEQ ID NO: 909). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, a first sequence encoding a gRNA, the H1 promoter, a second sequence encoding a gRNA, the 7SK promoter, a third sequence encoding a gRNA, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the H1 promoter, the sequence encoding a gRNA of SEQ ID NO. 708, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or

133 consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, a sequence encoding a gRNA of SEQ ID NO: 714, the H1 promoter, a sequence encoding a gRNA of SEQ ID NO: 714, the 7SK promoter, a sequence encoding a gRNA of SEQ ID NO: 714, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat In some embodiments, the construct comprising a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, a minipolyA sequence, and a second ITR, further comprises a stuffer sequence. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, a first sequence encoding a gRNA, the H1 promoter, a second sequence encoding a gRNA, the 7SK promoter, a third sequence encoding a gRNA, a stuffer sequence, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a stuffer sequence a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a third promoter, the sequence encoding a gRNA of SEQ ID NO. 714, a stuffer sequence, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a stuffer sequence, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the H1 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a stuffer sequence, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 714, the H1 promoter, the sequence encoding a gRNA of SEQ ID ΝΌ: 714, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a stuffer sequence, a minipolyA sequence, and a second ITR In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, a second sequence

134 encoding a gRNA, a third promoter, a third sequence encoding a gRNA, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, a first sequence encoding a gRNA, the FU promoter, a second sequence encoding a gRNA, the 7SK promoter, a third sequence encoding a gRNA, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the H1 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, a sequence encoding a gRNA of SEQ ID NO: 714, the H1 promoter, a sequence encoding a gRNA of SEQ ID NO: 714, the 7SK promoter, a sequence encoding a gRNA of SEQ ID NO: 714, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some

135 embodiments, the construct may further comprise one or more spacer sequences. Exemplary spacer sequences of the disclosure hâve length from 1-1500 nucléotides, inclusive of ail ranges therebetween. In some embodiments, the spacer sequences may be located at a position that is 5’ to or 3’ to an ITR, a promoter, a sequence encoding a gRNA, a polyA sequence, a transposable element inverted repeat, a stuffer sequence, and/or a regulator element.

V. Pharmaceutical Compositions and Delivery Methods

For clinical applications, pharmaceutical compositions are prepared in a form appropriate for the intended application. Generally, this entails preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animais.

Appropriate salts and buffers are used to render drugs, proteins or delivery vectors stable and allow for uptake by target cells. Aqueous compositions of the présent disclosure comprise an effective amount of the drug, vector or proteins, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase “pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergie, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier” includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonie and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is not incompatible with the active ingrédients of the présent disclosure, its use in therapeutic compositions may be used. Supplementary active ingrédients also can be incorporated into the compositions, provided they do not inactivate the vectors or cells of the compositions.

In some embodiments, the active compositions of the présent disclosure may include classic pharmaceutical préparations. Administration of these compositions according to the présent disclosure may be via any common route so long as the target tissue is available via that route, but generally including systemic administration. This includes oral, nasal, or buccal. Alternatively, administration may be by intradermal, subeutaneous, intramuscular, intraperitoneal or intravenous injection, or by direct injection into muscle tissue. Such

136 compositions would normally be admimstered as pharmaceutically acceptable compositions, as described supra.

The active compounds may also be administered parenterally or intraperitoneally. By way of illustration, solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these préparations generally contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include, for example, stérile aqueous solutions or dispersions and stérile powders for the extemporaneous préparation of stérile injectable solutions or dispersions. Generally, these préparations are stérile and fluid to the extent that easy injectability exists. Préparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Appropriate solvents or dispersion media may contain, for example, water, éthanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prévention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phénol, sorbic acid, thimerosal, and the like. In many cases, it will be préférable to include isotonie agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Stérile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingrédients (for example as enumerated above) as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingrédients into a stérile vehicle which contains the basic dispersion medium and the desired other ingrédients, e.g., as enumerated above. In the case of stérile powders for the préparation of stérile injectable solutions, the preferred methods of préparation include vacuum-drying and freeze-drying techniques which

137 yield a powder of the active ingredient(s) plus any additional desired ingrédient from a previously sterile-filtered solution thereof.

In some embodiments, the compositions of the présent disclosureare formulated in a neutral or sait form. Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferrie hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine) and the like.

Upon formulation, solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parentéral administration in an aqueous solution, for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonie for example with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subeutaneous and intraperitoneal administration. Preferably, stérile aqueous media are employed as is known to those of skill in the art, particularly in light of the présent disclosure. By way of illustration, a single dose may be dissolved in 1 ml of isotonie NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, Remington's Pharmaceutical Sciences 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, détermine the appropriate dose for the individual subject. Moreover, for human administration, préparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.

In some embodiments, the Cas9 or Cpfl and gRNAs described herein may be delivered to the patient using adoptive cell transfer (ACT). In adoptive cell transfer, one or more expression constructs are provided ex vivo to cells which hâve originated from the patient (autologous) or from one or more individual(s) other than the patient (allogeneic). The cells are subsequently introduced or reintroduced into the patient. Thus, in some embodiments, one

or more nucleic acids encoding Cas9 or Cpfl and a guide RNA that targets a dystrophin splice site are provided to a cell ex vivo before the cell is introduced or reintroduced to a patient.

The following tables provide exemplary primer and genomic targeting sequences for use in connection with the compositions and methods disclosed herein.

139

TABLE 6 - Genomic Target Sequences

Targeted gRNA Exon	Guide #	Strand	Genomic Target Sequence*	PAM	SEQ ID NO.
Human-Exon 51	4	1	tctttttcttcttttttccttttt	tttt	60
Human-Exon 51	5	1	ctttttcttcttttttcctttttG	tttt	61
Human-Exon 51	6	1	tttttcttcttttttcctttttGC	tttc	62
Human-Exon 51	7	1	tcttcttttttcctttttGCAAAA	tttt	63
Human-Exon 51	8	1	cttcttttttcctttttGCAAAAA	tttt	64
Human-Exon 51	9	1	ttcttttttcctttttGCAAAAAC	tttc	65
Human-Exon 51	10	1	ttcctttttGCAAAAACCCAAAAT	tttt	66
Human-Exon 51	11	1	tcctttttGCAAAAACCCAAAATA	tttt	67
Human-Exon 51	12	1	cctttttGCAAAAACCCAAAATAT	tttt	68
Human-Exon 51	13	1	ctttttGCAAAAACCCAAAATATT	tttc	69
Human-Exon 51	14	1	tGCAAAAACCCAAAATATTTTAGC	tttt	70
Human-Exon 51	15	1	GCAAAAACCCAAAATATTTTAGCT	tttt	71
Human-Exon 51	16	1	CAAAAACCCAAAATATTTTAGCTC	tttG	72
Human-Exon 51	17	1	AGCTCCTACTCAGACTGTTACTCT	TTTT	73
Human-Exon 51	18	1	GCTCCTACTCAGACTGTTACTCTG	TTTA	74
Human-Exon 51	19	-1	CTTAGTAACCACAGGTTGTGTCAC	TTTC	75
Human-Exon 51	20	-1	GAGATGGCAGTTTCCTTAGTAACC	TTTG	76
Human-Exon 51	21	-1	TAGTTTGGAGATGGCAGTTTCCTT	TTTC	77
Human-Exon 51	22	-1	TTCTCATACCTTCTGCTTGATGAT	TTTT	78
Human-Exon 51	23	-1	TCATTTTTTCTCATACCTTCTGCT	TTTA	79
Human-Exon 51	24	-1	ATCATTTTTTCTCATACCTTCTGC	TTTT	80
Human-Exon 51	25	-1	AAGAAAAACTTCTGCCAACTTTTA	TTTA	81
Human-Exon 51	26	-1	AAAGAAAAACTTCTGCCAACTTTT	TTTT	82
Human-Exon 51	27	1	TCTTTAAAATGAAGATTTTCCACC	TTTT	83
Human-Exon 51	28	1	CTTTAAAATGAAGATTTTCCACCA	TTTT	84
Human-Exon 51	29	1	TTTAAAATGAAGATTTTCCACCAA	TTTC	85

140

Human-Exon 51	30	1	AAATGAAGATTTTCCACCAATCAC	TTTA	86
Human-Exon 51	31	1	CCACCAATCACTTTACTCTCCTAG	TTTT	87
Human-Exon 51	32	1	CACCAATCACTTTACTCTCCTAGA	TTTC	88
Human-Exon 51	33	1	CTCTCCTAGACCATTTCCCACCAG	TTTA	89
Human-Exon 45	1	-1	agaaaagattaaacagtgtgctac	tttg	90
Human-Exon 45	2	-1	tttgagaaaagattaaacagtgtg	TTTa	91
Human-Exon 45	3	-1	atttgagaaaagattaaacagtgt	TTTT	92
Human-Exon 45	4	-1	T atttgagaaaagattaaacagtg	TTTT	93
Human-Exon 45	5	1	atcttttctcaaatAAAAAGACAT	ttta	94
Human-Exon 45	6	1	ctcaaatAAAAAGACATGGGGCTT	tttt	95
Human-Exon 45	7	1	tcaaatAAAAAGACATGGGGCTTC	tttc	96
Human-Exon 45	8	1	TGTTTTGCCTTTTTGGTATCTTAC	TTTT	97
Human-Exon 45	9	1	GTTTTGCCTTTTTGGTATCTTACA	TTTT	98
Human-Exon 45	10	1	TTTTGCCTTTTTGGTATCTTACAG	TTTG	99
Human-Exon 45	11	1	GCCTTTTTGGTATCTTACAGGAAC	TTTT	100
Human-Exon 45	12	1	CCTTTTTGGTATCTTACAGGAACT	TTTG	101
Human-Exon 45	13	1	TGGTATCTTACAGGAACTCCAGGA	TTTT	102
Human-Exon 45	14	1	GGTATCTTACAGGAACTCCAGGAT	TTTT	103
Human-Exon 45	15	-1	AGGATTGCTGAATTATTTCTTCCC	TTTG	104
Human-Exon 45	16	-1	GAGGATTGCTGAATTATTTCTTCC	TTTT	105
Human-Exon 45	17	-1	TGAGGATTGCTGAATTATTTCTTC	TTTT	106
Human-Exon 45	18	-1	CTGTAGAATACTGGCATCTGTTTT	TTTC	107
Human-Exon 45	19	-1	CCTGTAGAATACTGGCATCTGTTT	TTTT	108
Human-Exon 45	20	-1	TCCTGTAGAATACTGGCATCTGTT	TTTT	109
Human-Exon 45	21	-1	CAGACCTCCTGCCACCGCAGATTC	TTTG	110
Human-Exon 45	22	-1	TGTCTGACAGCTGTTTGCAGACCT	TTTC	111
Human-Exon 45	23	-1	CTGTCTGACAGCTGTTTGCAGACC	TTTT	112
Human-Exon 45	24	-1	TCTGTCTGACAGCTGTTTGCAGAC	TTTT	113
Human-Exon 45	25	-1	TTCTGTCTGACAGCTGTTTGCAGA	TTTT	114

141

Human-Exon 45	26	-1	ATTCCTATTAGATCTGTCGCCCTA	TTTC	115
Human-Exon 45	27	-1	CATTCCTATTAGATCTGTCGCCCT	TTTT	116
Human-Exon 45	28	1	AGCAGACTTTTTAAGCTTTCTTTA	TTTT	117
Human-Exon 45	29	1	GCAGACTTTTTAAGCTTTCTTTAG	TTTA	118
Human-Exon 45	30	1	TAAGCTTTCTTTAGAAGAATATTT	TTTT	119
Human-Exon 45	31	1	AAGCTTTCTTTAGAAGAATATTTC	TTTT	120
Human-Exon 45	32	1	AGCTTTCTTTAGAAGAATATTTCA	TTTA	121
Human-Exon 45	33	1	TTTAGAAGAATATTTCATGAGAGA	TTTC	122
Human-Exon 45	34	1	GAAGAATATTTCATGAGAGATTAT	TTTA	123
Human-Exon 44	1	1	TCAGTATAACCAAAAAATATACGC	TTTG	124
Human-Exon 44	2	1	acataatccatctatttttcttga	tttt	125
Human-Exon 44	3	1	cataatccatctatttttcttgat	ttta	126
Human-Exon 44	4	1	tcttgatccatatgcttttACCTG	tttt	127
Human-Exon 44	5	1	cttgatccatatgcttttACCTGC	tttt	128
Human-Exon 44	6	1	ttgatccatatgcttttACCTGCA	tttc	129
Human-Exon 44	7	-1	TCAACAGATCTGTCAAATCGCCTG	TTTC	130
Human-Exon 44	8	1	ACCTGCAGGCGATTTGACAGATCT	tttt	131
Human-Exon 44	9	1	CCTGCAGGCGATTTGACAGATCTG	tttA	132
Human-Exon 44	10	1	ACAGATCTGTTGAGAAATGGCGGC	TTTG	133
Human-Exon 44	11	-1	TATCATAATGAAAACGCCGCCATT	TTTA	134
Human-Exon 44	12	1	CATTATGATATAAAGATATTTAAT	TTTT	135
Human-Exon 44	13	-1	TATTTAGCATGTTCCCAATTCTCA	TTTG	136
Human-Exon 44	14	-1	GAAAAAACAAATCAAAGACTTACC	TTTC	137
Human-Exon 44	15	1	ATTTGTTTTTTCGAAATTGTATTT	TTTG	138
Human-Exon 44	16	1	TTTTTTCGAAATTGTATTTATCTT	TTTG	139
Human-Exon 44	17	1	TTCGAAATTGTATTTATCTTCAGC	TTTT	140
Human-Exon 44	18	1	TCGAAATTGTATTTATCTTCAGCA	TTTT	141
Human-Exon 44	19	1	CGAAATTGTATTTATCTTCAGCAC	TTTT	142
Human-Exon 44	20	1	GAAATTGTATTTATCTTCAGCACA	TTTC	143

142

Human-Exon 44	21	-1	AGAAGTTAAAGAGTCCAGATGTGC	TTTA	144
Human-Exon 44	22	1	TCTTCAGCACATCTGGACTCTTTA	TTTA	145
Human-Exon 44	23	-1	CATCACCCTTCAGAACCTGATCTT	TTTC	146
Human-Exon 44	24	1	ACTTCTTAAAGATCAGGTTCTGAA	TTTA	147
Human-Exon 44	25	1	GACTGTTGTTGTCATCATTATATT '	TTTT	148
Human-Exon 44	26	1	ACTGTTGTTGTCATCATTATATTA	TTTG	149
Human-Exon 53	1	-1	AACTAGAATAAAAGGAAAAATAAA	TTTC	150
Human-Exon 53	2	1	CTACTATATATTTATTTTTCCTTT	TTTA	151
Human-Exon 53	3	1	TTTTTCCTTTTATTCTAGTTGAAA	TTTA	152
Human-Exon 53	4	1	TCCTTTTATTCTAGTTGAAAGAAT	TTTT	153
Human-Exon 53	5	1	CCTTTTATTCTAGTTGAAAGAATT	TTTT	154
Human-Exon 53	6	1	CTTTTATTCTAGTTGAAAGAATTC	TTTC	155
Human-Exon 53	7	1	ATTCTAGTTGAAAGAATTCAGAAT	TTTT	156
Human-Exon 53	8	1	TTCTAGTTGAAAGAATTCAGAATC	TTTA	157
Human-Exon 53	9	-1	ATTCAACTGTTGCCTCCGGTTCTG	TTTC	158
Human-Exon 53	10	-1	ACATTTCATTCAACTGTTGCCTCC	TTTA	159
Human-Exon 53	11	-1	CTTTTGGATTGCATCTACTGTATA	TTTT	160
Human-Exon 53	12	-1	TGTGATTTTCTTTTGGATTGCATC	TTTC	161
Human-Exon 53	13	-1	ATACTAACCTTGGTTTCTGTGATT	TTTG	162
Human-Exon 53	14	-1	AAAAGGTATCTTTGATACTAACCT	TTTA	163
Human-Exon 53	15	-1	AAAAAGGTATCTTTGATACTAACC	TTTT	164
Human-Exon 53	16	-1	TTTTAAAAAGGTATCTTTGATACT	TTTA	165
Human-Exon 53	17	-1	ATTTTAAAAAGGTATCTTTGATAC	TTTT	166
Human-Exon 46	1	-1	TTAATGCAAACTGGGACACAAACA	TTTG	167
Human-Exon 46	2	1	TAAATTGCCATGTTTGTGTCCCAG	TTTT	168
Human-Exon 46	3	1	AAATTGCCATGTTTGTGTCCCAGT	TTTT	169
Human-Exon 46	4	1	AATTGCCATGTTTGTGTCCCAGTT	TTTA	170
Human-Exon 46	5	1	TGTCCCAGTTTGCATTAACAAATA	TTTG	171
Human-Exon 46	6	-1	CAACATAGTTCTCAAACTATTTGT	tttC	172

143

Human-Exon 46	7	-1	ZCAACATAGTTCTCAAACTATTTG	tttt	173
Human-Exon 46	8	-1	CCAACATAGTTCTCAAACTATTT	tttt	174
Human-Exon 46	9	-1	ttCCAACATAGTTCTCAAACTAT	tttt	175
Human-Exon 46	10	-1	ttttCCAACATAGTTCTCAAACTA	tttt	176
Human-Exon 46	11	-1	tttttCCAACATAGTTCTCAAACT	tttt	177
Human-Exon 46	12	1	CATTAACAAATAGTTTGAGAACTA	TTTG	178
Human-Exon 46	13	1	AGAACTATGTTGGaaaaaaaaaTA	TTTG	179
Human-Exon 46	14	-1	GTTCTTCTAGCCTGGAGAAAGAAG	TTTT	180
Human-Exon 46	15	1	ATTCTTCTTTCTCCAGGCTAGAAG	TTTT	181
Human-Exon 46	16	1	TTCTTCTTTCTCCAGGCTAGAAGA	TTTA	182
Human-Exon 46	17	1	TCCAGGCTAGAAGAACAAAAGAAT	TTTC	183
Human-Exon 46	18	-1	AAATTCTGACAAGATATTCTTTTG	TTTG	184
Human-Exon 46	19	-1	CTTTTAGTTGCTGCTCTTTTCCAG	TTTT	185
Human-Exon 46	20	-1	AGAAAATAAAATTACCTTGACTTG	TTTG	186
Human-Exon 46	21	-1	TGCAAGCAGGCCCTGGGGGATTTG	TTTA	187
Human-Exon 46	22	1	ATTTTCTCAAATCCCCCAGGGCCT	TTTT	188
Human-Exon 46	23	1	TTTTCTCAAATCCCCCAGGGCCTG	TTTA	189
Human-Exon 46	24	1	CTCAAATCCCCCAGGGCCTGCTTG	TTTT	190
Human-Exon 46	25	1	TCAAATCCCCCAGGGCCTGCTTGC	TTTC	191
Human-Exon 46	26	1	TTAATTCAATCATTGGTTTTCTGC	TTTT	192
Human-Exon 46	27	1	TAATTCAATCATTGGTTTTCTGCC	TTTT	193
Human-Exon 46	28	1	AATTCAATCATTGGTTTTCTGCCC	TTTT	194
Human-Exon 46	29	1	ATTCAATCATTGGTTTTCTGCCCA	TTTA	195
Human-Exon 46	30	-1	GCAAGGAACTATGAATAACCTAAT	TTTA	196
Human-Exon 46	31	1	CTGCCCATTAGGTTATTCATAGTT	TTTT	197
Human-Exon 46	32	1	TGCCCATTAGGTTATTCATAGTTC	TTTC	198
Human-Exon 52	1	-1	TAGAAAACAATTTAACAGGAAATA	TTTA	199
Human-Exon 52	2	1	CTGTTAAATTGTTTTCTATAAACC	TTTC	200
Human-Exon 52	3	-1	GAAATAAAAAAGATGTTACTGTAT	TTTA	201

144

Human-Exon 52	4	-1	AGAAATAAAAAAGATGTTACTGTA	TTTT	202
Human-Exon 52	5	1	CTATAAACCCTTATACAGTAACAT	TTTT	203
Human-Exon 52	6	1	TATAAACCCTTATACAGTAACATC	TTTC	204
Human-Exon 52	7	1	TTATTTCTAAAAGTGTTTTGGCTG	TTTT	205
Human-Exon 52	8	1	TATTTCTAAAAGTGTTTTGGCTGG	TTTT	206
Human-Exon 52	9	1	ATTTCTAAAAGTGTTTTGGCTGGT	TTTT	207
Human-Exon 52	10	1	TTTCTAAAAGTGTTTTGGCTGGTC	TTTA	208
Human-Exon 52	1 1	1	TAAAAGTGTTTTGGCTGGTCTCAC	TTTC	209
Human-Exon 52	12	-1	CATAATACAAAGTAAAGTACAATT	TTTA	210
Human-Exon 52	13	-1	ACATAATACAAAGTAAAGTACAAT	TTTT	21 1
Human-Exon 52	14	1	GGCTGGTCTCACAATTGTACTTTA	TTTT	212
Human-Exon 52	15	1	GCTGGTCTCACAATTGTACTTTAC	TTTG	213
Human-Exon 52	16	1	CTTTGTATTATGTAAAAGGAATAC	TTTA	214
Human-Exon 52	17	1	TATTATGTAAAAGGAATACACAAC	TTTG	215
Human-Exon 52	18	1	TTCTTACAGGCAACAATGCAGGAT	TTTG	216
Human-Exon 52	19	1	GAACAGAGGCGTCCCCAGTTGGAA	TTTG	217
Human-Exon 52	20	-1	GGCAGCGGTAATGAGTTCTTCCAA	TTTG	218
Human-Exon 52	21	-1	TCAAATTTTGGGCAGCGGTAATGA	TTTT	219
Human-Exon 52	22	1	AAAAACAAGACCAGCAATCAAGAG	TTTG	220
Human-Exon 52	23	-1	TGTGTCCCATGCTTGTTAAAAAAC	TTTG	221
Human-Exon 52	24	1	TTAACAAGCATGGGACACACAAAG	TTTT	222
Human-Exon 52	25	1	TAACAAGCATGGGACACACAAAGC	TTTT	223
Human-Exon 52	26	1	AACAAGCATGGGACACACAAAGCA	TTTT	224
Human-Exon 52	27	1	ACAAGCATGGGACACACAAAGCAA	TTTA	225
Human-Exon 52	28	-1	TTGAAACTTGTCATGCATCTTGCT	TTTA	226
Human-Exon 52	29	-1	ATTGAAACTTGTCATGCATCTTGC	TTTT	227
Human-Exon 52	30	-1	TATTGAAACTTGTCATGCATCTTG	TTTT	228
Human-Exon 52	31	1	AATAAAAACTTAAGTTCATATATC	TTTC	229
Human-Exon 50	1	-1	GTGAATATATTATTGGATTTCTAT	TTTG	230

145

Human-Exon 50	2	-1	\ AGATA ATTC ATG A AC ATCTTA AT	TTTG	231
Hum an-Exon 50	3	-1	ÂCAGAAAAGCATACACATTACTTA	TTTA	232
Human-Exon 50	4	1	ZTGTTAAAGAGGAAGTTAGAAGAT	TTTT	233
Human-Exon 50	5	1	TGTTAAAGAGGAAGTTAGAAGATC	TTTC	234
Human-Exon 50	6	-1	CCGCCTTCCACTCAGAGCTCAGAT	TTTA	235
Human-Exon 50	7	-1	CCCTCAGCTCTTGAAGTAAACGGT	TTTG	236
Human-Exon 50	8	1	CTTCAAGAGCTGAGGGCAAAGCAG	TTTA	237
Human-Exon 50	9	-1	AACAAATAGCTAGAGCCAAAGAGA	TTTG	238
Human-Exon 50	10	-1	GAACAAATAGCTAGAGCCAAAGAG	TTTT	239
Human-Exon 50	11	1	GCTCTAGCTATTTGTTCAAAAGTG	TTTG	240
Human-Exon 50	12	1	TTCAAAAGTGCAACTATGAAGTGA	TTTG	241
Human-Exon 50	13	-1	TCTCTCACCCAGTCATCACTTCAT	TTTC	242
Human-Exon 50	14	-1	CTCTCTCACCCAGTCATCACTTCA	TTTT	243
Human-Exon 43	1	1	tatatatatatatatTTTTCTCTT	TTTG	244
Human-Exon 43	2	1	TCTCTTTCTATAGACAGCTAATTC	tTTT	245
Human-Exon 43	3	1	CTCTTTCTATAGACAGCTAATTCA	TTTT	246
Human-Exon 43	4	-1	AAACAGTAAAAAAATGAATTAGCT	TTTA	247
Human-Exon 43	5	1	TCTTTCTATAGACAGCTAATTCAT	TTTC	248
Human-Exon 43	6	-1	AAAACAGTAAAAAAATGAATTAGC	TTTT	249
Human-Exon 43	7	1	TATAG AC AGCT A ATTC ATTTTTTT	TTTC	250
Human-Exon 43	8	-1	TATTCTGTAATATAAAAATTTTAA	TTTA	251
Human-Exon 43	9	-1	ATATTCTGTAATATAAAAATTTTA	TTTT	252
Human-Exon 43	10	1	TTTACTGTTTTAAAATTTTTATAT	TTTT	253
Human-Exon 43	11	1	TTACTGTTTTAAAATTTTTATATT	TTTT	254
Human-Exon 43	12	1	TACTGTTTTAAAATTTTTATATTA	TTTT	255
Human-Exon 43	13	1	ACTGTTTTAAAATTTTTATATTAC	TTTT	256
Human-Exon 43	14	1	CTGTTTTAAAATTTTTATATTACA	TTTA	257
Human-Exon 43	15	1	AAAATTTTTATATTACAGAATATA	TTTT	258
Human-Exon 43	16	1	AAATTTTTATATTACAGAATATAA	TTTA	259

146

Human-Exon 43	17	-1	TTGTAGACTATCTTTTATATTCTG	TTTG	260
Human-Exon 43	18	1	TATATTACAGAATATAAAAGATAG	TTTT	261
Human-Exon 43	19	1	ATATTACAGAATATAAAAGATAGT	TTTT	262
Human-Exon 43	20	1	TATTACAGAATATAAAAGATAGTC	TTTA	263
Human-Exon 43	21	-1	CAATGCTGCTGTCTTCTTGCTATG	TTTG	264
Human-Exon 43	22	1	CAATGGGAAAAAGTTAACAAAATG	TTTC	265
Human-Exon 43	23	-1	TGCAAGTATCAAGAAAAATATATG	TTTC	266
Human-Exon 43	24	1	TCTTGATACTTGCAGAAATGATTT	TTTT	267
Human-Exon 43	25	1	CTTG ATAC TTGC AG AA ATG ATTTG	TTTT	268
Human-Exon 43	26	1	TTGATACTTGCAGAAATGATTTGT	TTTC	269
Human-Exon 43	27	1	TTTTCAGGGAACTGTAGAATTTAT	TTTG	270
Human-Exon 43	28	-1	CATGGAGGGTACTGAAATAAATTC	TTTC	271
Human-Exon 43	29	-1	CCATGGAGGGTACTGAAATAAATT	TTTT	272
Human-Exon 43	30	1	CAGGGAACTGTAGAATTTATTTCA	TTTT	273
Human-Exon 43	31	-1	TCCATGGAGGGTACTGAAATAAAT	TTTT	274
Human-Exon 43	32	1	AGGGAACTGTAGAATTTATTTCAG	TTTC	275
Human-Exon 43	33	-1	TTCCATGGAGGGTACTGAAATAAA	TTTT	276
Human-Exon 43	34	-1	CCTGTCTTTTTTCCATGGAGGGTA	TTTC	277
Human-Exon 43	35	-1	CCCTGTCTTTTTTCCATGGAGGGT	TTTT	278
Human-Exon 43	36	-1	TCCCTGTCTTTTTTCCATGG AGGG	TTTT	279
Human-Exon 43	37	1	TTTCAGTACCCTCCATGGAAAAAA	TTTA	280
Human-Exon 43	38	1	AGTACCCTCCATGGAAAAAAGACA	TTTC	281
Human-Exon 6	1	1	AGTTTGCATGGTTCTTGCTCAAGG	TTTA	282
Human-Exon 6	2	-1	ATAAGAAAATGCATTCCTTGAGCA	TTTC	283
Human-Exon 6	3	-1	CATAAGAAAATGCATTCCTTGAGC	TTTT	284
Human-Exon 6	4	1	CATGGTTCTTGCTCAAGGAATGCA	TTTG	285
Human-Exon 6	5	-1	ACCTACATGTGGAAATAAATTTTC	TTTG	286
Human-Exon 6	6	-1	GACCTACATGTGGAAATAAATTTT	TTTT	287
Human-Exon 6	7	-1	TGACCTACATGTGGAAATAAA TTT	TTTT	288

147

Human-Exon 6	8	1	2TTATGAAAATTTATTTCCACATG	TTTT	289
Human-Exon 6	9	1	ETATGAAAATTTATTTCCACATGT	TTTC	290
Human-Exon 6	10	-1	YTTACATTTTTGACCTACATGTGG	TTTC	291
Human-Exon 6	11	-1	CATTACATTTTTGACCTACATGTG	TTTT	292
Human-Exon 6	12	-1	TCATTACATTTTTGACCTACATGT	TTTT	293
Human-Exon 6	13	1	TTTCCACATGTAGGTCAAAAATGT	TTTA	294
Human-Exon 6	14	1	CACATGTAGGTCAAAAATGTAATG	TTTC	295
Human-Exon 6	15	-1	TTGCAATCCAGCCATGATATTTTT	TTTG	296
Human-Exon 6	16	-1	ACTGTTGGTTTGTTGCAATCCAGC	TTTC	297
Human-Exon 6	17	-1	CACTGTTGGTTTGTTGCAATCCAG	TTTT	298
Human-Exon 6	18	1	AATGCTCTCATCCATAGTCATAGG	TTTG	299
Human-Exon 6	19	-1	ATGTCTCAGTAATCTTCTTACCTA	TTTA	300
Human-Exon 6	20	-1	CAAGTTATTTAATGTCTCAGTAAT	TTTA	301
Human-Exon 6	21	-1	ACAAGTTATTTAATGTCTCAGTAA	TTTT	302
Human-Exon 6	22	1	GACTCTGATGACATATTTTTCCCC	TTTA	303
Human-Exon 6	23	1	TCCCCAGTATGGTTCCAGATCATG	TTTT	304
Human-Exon 6	24	1	CCCCAGTATGGTTCCAGATCATGT	TTTT	305
Human-Exon 6	25	1	CCCAGTATGGTTCCAGATCATGTC	TTTC	306
Human-Exon 7	1	1	TATTTGTCTTtgtgtatgtgtgta	TTTA	307
Human-Exon 7	2	1	TCTTtgtgtatgtgtgtatgtgta	TTTG	308
Human-Exon 7	3	1	tgtatgtgtgtatgtgtatgtgtt	TTtg	309
Human-Exon 7	4	1	AGGCCAGACCTATTTGACTGGAAT	ttTT	310
Human-Exon 7	5	1	GGCCAGACCTATTTGACTGGAATA	tTTA	311
Human-Exon 7	6	1	ACTGGAATAGTGTGGTTTGCCAGC	TTTG	312
Human-Exon 7	7	1	CCAGCAGTCAGCCACACAACGACT	TTTG	313
Human-Exon 7	8	-1	TCTATGCCTAATTGATATCTGGCG	TTTC	314
Human-Exon 7	9	-1	CCAACCTTCAGGATCGAGTAGTTT	TTTA	315
Human-Exon 7	10	1	TGGACTACCACTGCTTTTAGTATG	TTTC	316
Human-Exon 7	11	1	AGTATGGTAGAGTTTAATGTTTTC	TTTT	317

148

Human-Exon 7	12	1	□TATGGTAGAGTTTAATGTTTTCA	TTTA	318
Human-Exon 8	1	-1	AGACTCTAAAAGGATAATGAACAA	TTTG	319
Human-Exon 8	2	1	ACTTTGATTTGTTCATTATCCTTT	TTTA	320
Human-Exon 8	3	-1	TATATTTGAGACTCTAAAAGGATA	TTTC	321
Human-Exon 8	4	1	ATTTGTTCATTATCCTTTTAGAGT	TTTG	322
Human-Exon 8	5	-1	gtttctatatttgagactctaaaa	TTTG	323
Human-Exon 8	6	-1	GGTTTCTATATTTGAGACTCTAAA	TTTT	324
Human-Exon 8	7	-1	TGGTTTCTATATTTGAGACTCTAA	TTTT	325
Human-Exon 8	8	1	TTCATTATCCTTTTAGAGTCTCAA	TTTG	326
Human-Exon 8	9	1	AGAGTCTCAAATATAGAAACCAAA	TTTT	327
Human-Exon 8	10	1	GAGTCTCAAATATAGAAACCAAAA	TTTA	328
Human-Exon 8	11	-1	CACTTCCTGGATGGCTTCAATGCT	TTTC	329
Human-Exon 8	12	1	GCCTCAACAAGTGAGCATTGAAGC	TTTT	330
Human-Exon 8	13	1	CCTCAACAAGTGAGCATTGAAGCC	TTTG	331
Human-Exon 8	14	-1	GGTGGCCTTGGCAACATTTCCACT	TTTA	332
Human-Exon 8	15	-1	GTCACTTTAGGTGGCCTTGGCAAC	TTTA	333
Human-Exon 8	16	-1	ATGATGTAACTGAAAATGTTCTTC	TTTG	334
Human-Exon 8	17	-1	CCTGTTGAGAATAGTGCATTTGAT	TTTA	335
Human-Exon 8	18	1	CAGTTACATCATCAAATGCACTAT	TTTT	336
Human-Exon 8	19	1	AGTTACATCATCAAATGCACTATT	TTTC	337
Human-Exon 8	20	-1	CACACTTTACCTGTTGAGAATAGT	TTTA	338
Human-Exon 8	21	1	CTGTTTTATATGCATTTTTAGGTA	TTTT	339
Human-Exon 8	22	1	TGTTTTATATGCATTTTTAGGTAT	TTTC	340
Human-Exon 8	23	1	ATATGCATTTTTAGGTATTACGTG	TTTT	341
Human-Exon 8	24	1	TATGCATTTTTAGGTATTACGTGC	TTTA	342
Human-Exon 8	25	1	TAGGTATTACGTGCACatatatat	TTTT	343
Human-Exon 8	26	1	AGGTATTACGTGCACatatatata	TTTT	344
Human-Exon 8	27	1	GGTATTACGTGCACatatatatat	TTTA	345
Human-Exon 55	1	-1	AGCAACAACTATAATATTGTGCAG	TTTA	346

149

Human-Exon 55	2	1	GTTCCTCCATCTTTCTCTTTTTAT	TTTA	347
Human-Exon 55	3	1	TCTTTTTATGGAGTTCACTAGGTG	TTTC	348
Human-Exon 55	4	1	TATGGAGTTCACTAGGTGCACCAT	TTTT	349
Human-Exon 55	5	1	ATGGAGTTCACTAGGTGCACCATT	TTTT	350
Human-Exon 55	6	1	TGGAGTTCACTAGGTGCACCATTC	TTTA	351
Human-Exon 55	7	1	ATAATTGCATCTGAACATTTGGTC	TTTA	352
Human-Exon 55	8	1	GTCCTTTGCAGGGTGAGTGAGCGA	TTTG	353
Human-Exon 55	9	-1	TTCCAAAGCAGCCTCTCGCTCACT	TTTC	354
Human-Exon 55	10	1	CAGGGTGAGTGAGCGAGAGGCTGC	TTTG	355
Human-Exon 55	11	1	GAAGAAACTCATAGATTACTGCAA	TTTG	356
Human-Exon 55	12	-1	CAGGTCCAGGGGGAACTGTTGCAG	TTTC	357
Human-Exon 55	13	-1	CCAGGTCCAGGGGGAACTGTTGCA	TTTT	358
Human-Exon 55	14	-1	AGCTTCTGTAAGCCAGGCAAGAAA	TTTC	359
Human-Exon 55	15	1	TTGCCTGGCTTACAGAAGCTGAAA	TTTC	360
Human-Exon 55	16	-1	CTTACGGGTAGCATCCTGTAGGAC	TTTC	361
Human-Exon 55	17	-1	CTCCCTTGGAGTCTTCTAGGAGCC	TTTA	362
Human-Exon 55	18	-1	ACTCCCTTGGAGTCTTCTAGGAGC	TTTT	363
Human-Exon 55	19	-1	ATCAGCTCTTTTACTCCCTTGGAG	TTTC	364
Human-Exon 55	20	1	CGCTTTAGCACTCTTGTGGATCCA	TTTC	365
Human-Exon 55	21	1	GCACTCTTGTGGATCCAATTGAAC	TTTA	366
Human-Exon 55	22	-1	TCCCTGGCTTGTCAGTTACAAGTA	TTTG	367
Human-Exon 55	23	-1	GTCCCTGGCTTGTCAGTTACAAGT	TTTT	368
Human-Exon 55	24	-1	TTTTGTCCCTGGCTTGTCAGTTAC	TTTG	369
Human-Exon 55	25	-1	GTTTTGTCCCTGGCTTGTCAGTTA	TTTT	370
Human-Exon 55	26	1	TACTTGTAACTGACAAGCCAGGGA	TTTG	371
Human-G 1 -exon51		1	gCTCCTACTCAGACTGTTACTCTG	TTTA	372
Human-G2-exon51		1	taccatgtattgctaaacaaagta	TTTC	373

150

Human-G3-exon51		-1	attgaagagtaacaatttgagcca	TTTA	374
mouse-Exon23-G 1		1	aggctctgcaaagttctTTGAAAG	TTTG	375
mouse-Exon23-G2		1	AAAGAGCAACAAAATGGCttcaac	TTTG	376
mouse-Exon23-G3		1	AAAGAGCAATAAAATGGCttcaac	TTTG	377
mouse-Exon23-G4		-1	AAAGAACTTTGCAGAGCctcaaaa	TTTC	378
mouse-Exon23-G5		-1	ctgaatatctatgcattaataact	TTTA	379
mouse-Exon23-G6		-1	tattatattacagggcatattata	TTTC	380
mouse-Exon23-G7		1	Aggtaagccgaggtttggccttta	TTTC	381
mouse-Exon23-G8		1	cccagagtccttcaaagatattga	TTTA	382

* In this table, upper case letters represent nucléotides that align to the exon sequence of the gene. Lower case letters represent nucléotides that align to the intron sequence of the gene.

TABLE 7 - gRNA sequences

Targeted gRNA Exon	Guide #	Strand	gRNA sequence*	PAM	SEQ ID NO.
Human-Exon 51	4	1	aaaaaggaaaaaagaagaaaaaga	tttt	383
Human-Exon 51	5	1	C aaaaaggaaaaaagaagaaaaag	tttt	384
Human-Exon 51	6	1	GCaaaaaggaaaaaagaagaaaaa	tttc	385
Human-Exon 51	7	1	UUUUGCaaaaaggaaaaaagaaga	tttt	386
Human-Exon 51	8	1	U UUUU GC aaaaaggaaaaaagaag	tttt	387
Human-Exon 51	9	1	GUUUUUGC aaaaaggaaaaaagaa	tttc	388
Human-Exon 51	10	1	AUUUUGGGUUUUUGCaaaaaggaa	tttt	389
Human-Exon 51	11	1	UAUUUUGGGUUUUUGCaaaaagga	tttt	390
Human-Exon 51	12	1	AUAUUUUGGGUUUUUGCaaaaagg	tttt	391

Human-Exon 51	13	1	A AU AUU UUGGGUUUUUGCaaaaag	tttc	392
Human-Exon 51	14	1	GCUAAAAUAUUUUGGGUUUUUGCa	tttt	393
Human-Exon 51	15	1	AGCUAAAAUAUUUUGGGUUU U UGC	tttt	394
Human-Exon 51	16	1	GAGC UAAAAUAUUUUGGGUUUUUG	tttG	395
Human-Exon 51	17	1	AGAGUAACAGUCUGAGUAGGAGCU	TTTT	396
Human-Exon 51	18	1	CAGAGUAACAGUCUGAGUAGGAGC	TTTA	397
Human-Exon 51	19	-1	GUGACACAACCUGUGGUUACUAAG	TTTC	398
Human-Exon 51	20	-1	GGUUACUAAGGAAACUGCCAUCU	TTTG	399
Human-Exon 51	21	-1	AAGGAAACUGCCAUCUCCAAACUA	TTTC	400
Human-Exon 51	22	-1	AUCAUCAAGCAGAAGGUAUGAGAA	TTTT	401
Human-Exon 51	23	-1	AGCAGAAGGUAUGAGAAAAAAUGA	TTTA	402
Human-Exon 51	24	-1	GCAGAAGGUAUGAGAAAAAAUGAU	TTTT	403
Human-Exon 51	25	-1	UAAAAGUUGGCAGAAGUUUUUCUU	TTTA	404
Human-Exon 51	26	-1	AAAAGUUGGCAGAAGUUUUUCUUU	TTTT	405
Human-Exon 51	27	1	GGUGGAAAAUCUUCAUUUUAAAGA	TTTT	406
Human-Exon 51	28	1	UGGUGGAAAAUCUUCAUUUUAAAG	TTTT	407
Human-Exon 51	29	1	UUGGUGGAAAAUCUUCAUUUUAAA	TTTC	408
Human-Exon 51	30	1	GUGAUUGGUGGAAAAUCUUCAUUU	TTTA	409
Human-Exon 51	31	1	CUAGGAGAGUAAAGUGAUUGGUGG	TTTT	410
Human-Exon 51	32	1	UCUAGGAGAGUAAAGUGAUUGGUG	TTTC	411
Human-Exon 51	33	1	CUGGUGGGAAAUGGUCUAGGAGA	TTTA	412
Human-Exon 45	1	-1	guagcacacuguuuaaucuuuucu	tttg	413
Human-Exon 45	2	-1	cacacuguuuaaucuuuucucaaa	TTTa	414
Human-Exon 45	3	-1	acacuguuuaaucuuuucucaaau	TTTT	415
Human-Exon 45	4	-1	cacuguuuaaucuuuucucaaauA	TTTT	416
Human-Exon 45	5	1	A UGUC UUUUUauuugagaaaagau	ttta	417
Human-Exon 45	6	1	AAGCCCCAUGUCUUUUUauuugag	tttt	418
Human-Exon 45	7	1	GAAGCCCCAUGUCUUUUUauuuga	tttc	419
Human-Exon 45	8	1	GUAAGAUACCAAAAAGGCAAAACA	TTTT	420

152

Human-Exon 45	9	1	UGUAAGAUACCAAAAAGGCAAAAC	TTTT	421
Human-Exon 45	10	1	CUGUAAGAUACCAAAAAGGCAAAA	TTTG	422
Human-Exon 45	11	1	GUUCCUGUAAGAUACCAAAAAGGC	TTTT	423
Human-Exon 45	12	1	AGUUCCUGUAAGAUACCAAAAAGG	TTTG	424
Human-Exon 45	13	1	UCCUGGAGUUCCUGUAAGAUACCA	TTTT	425
Human-Exon 45	14	1	AUCCUGGAGUUCCUGUAAGAUACC	TTTT	426
Human-Exon 45	15	-1	GGGAAGAAAUAAUUCAGCAAUCCU	TTTG	427
Human-Exon 45	16	-1	GGAAGAAAUAAUUCAGCAAUCCUC	TTTT	428
Human-Exon 45	17	-1	GAAGAAAUAAUUCAGCAAUCCUCA	TTTT	429
Human-Exon 45	18	-1	AAAACAGAUGCCAGUAUUCUACAG	TTTC	430
Human-Exon 45	19	-1	AAACAGAUGCCAGUAUUCUACAGG	TTTT	431
Human-Exon 45	20	-1	AACAGAUGCCAGUAUUCUACAGGA	TTTT	432
Human-Exon 45	21	-1	GAAUCUGCGGUGGCAGGAGGUCUG	TTTG	433
Human-Exon 45	22	-1	AGGUCUGCAAACAGCUGUCAGACA	TTTC	434
Human-Exon 45	23	-1	GGUCUGCAAACAGCUGUCAGACAG	TTTT	435
Human-Exon 45	24	-1	GUCUGCAAACAGCUGUCAGACAGA	TTTT	436
Human-Exon 45	25	-1	UCUGCAAACAGCUGUCAGACAGAA	TTTT	437
Human-Exon 45	26	-1	UAGGGCGACAGAUCUAAUAGGAAU	TTTC	438
Human-Exon 45	27	-1	AGGGCGACAGAUCUAAUAGGAAUG	TTTT	439
Human-Exon 45	28	1	UAAAGAAAGCUUAAAAAGUCUGCU	TTTT	440
Human-Exon 45	29	1	CUAAAGAAAGCUUAAAAAGUCUGC	TTTA	441
Human-Exon 45	30	1	AAAUAUUCUUCUAAAGAAAGCUUA	TTTT	442
Human-Exon 45	31	1	GAAAUAUUCUUCUAAAGAAAGCUU	TTTT	443
Human-Exon 45	32	1	UGAAAUAUUCUUCUAAAGAAAGCU	TTTA	444
Human-Exon 45	33	1	UCUCUCAUGAAAUAUUCUUCUAAA	TTTC	445
Human-Exon 45	34	1	AUAAUCUCUCAUGAAAUAUUCUUC	TTTA	446
Human-Exon 44	1	1	GCGUAUAUUUUUUGGUUAUACUGA	TTTG	447
Human-Exon 44	2	1	ucaagaaaaauagauggauuaugu	tttt	448
Human-Exon 44	3	1	aucaagaaaaauagauggauuaug	ttta	449

153

Human-Exon 44	4	1	CAGGUaaaagcauauggaucaaga	tttt	450
Human-Exon 44	5	1	GC AGG U aaaagcauauggaucaag	tttt	451
Human-Exon 44	6	1	UGCAGG U aaaagcauauggaucaa	tttc	452
Human-Exon 44	7	-1	CAGGCGAUUUGACAGAUCUGUUGA	TTTC	453
Human-Exon 44	8	1	AGAUCUGUCAAAUCGCCUGCAGGU	tttt	454
Human-Exon 44	9	1	CAGAUCUGUCAAAUCGCCUGCAGG	tttA	455
Human-Exon 44	10	1	GCCGCCAUUUCUCAACAGAUCUGU	TTTG	456
Human-Exon 44	11	-1	AAUGGCGGCGUUUUCAUUAUGAUA	TTTA	457
Human-Exon 44	12	1	AUUAAAUAUCUUUAUAUCAUAAUG	TTTT	458
Human-Exon 44	13	-1	UGAGAAUUGGGAACAUGCGAAAUA	TTTG	459
Human-Exon 44	14	-1	GGUAAGUCUUUGAUUUGUUUUUUC	TTTC	460
Human-Exon 44	15	1	AAAUACAAUUUCGAAAAAACAAAU	TTTG	461
Human-Exon 44	16	1	AAGAUAAAUACAAUUUCGAAAAAA	TTTG	462
Human-Exon 44	17	1	GCUGAAGAUAAAUACAAUUUCGAA	TTTT	463
Human-Exon 44	18	1	UGCUGAAGAUAAAUACAAUUUCGA	TTTT	464
Human-Exon 44	19	1	GUGCUGAAGAUAAAUACAAUUUCG	TTTT	465
Human-Exon 44	20	1	UGUGCUGAAGAUAAAUACAAUUUC	TTTC	466
Human-Exon 44	21	-1	GCACAUCUGGACUCUUUAACUUCU	TTTA	467
Human-Exon 44	22	1	UAAAGAGUCCAGAUGUGCUGAAGA	TTTA	468
Human-Exon 44	23	-1	AAGAUCAGGUUCUGAAGGGUGAUG	TTTC	469
Human-Exon 44	24	1	UUCAGAACCUGAUCUUUAAGAAGU	TTTA	470
Human-Exon 44	25	1	AAUAUAAUGAUGACAACAACAGUC	TTTT	471
Human-Exon 44	26	1	UAAUAUAAUGAUGACAACAACAGU	TTTG	472
Human-Exon 53	1	-1	UUUAUUUUUCCUUUUAUUCUAGUU	TTTC	473
Human-Exon 53	2	1	AAAGGAAAAAUAAAUAUAUAGUAG	TTTA	474
Human-Exon 53	3	1	UUUCAACUAGAAUAAAAGGAAAAA	TTTA	475
Human-Exon 53	4	1	AUUCUUUCAACUAGAAUAAAAGGA	TTTT	476
Human-Exon 53	5	1	AAUUCUUUCAACUAGAAUAAAAGG	TTTT	477
Human-Exon 53	6	1	GAAUUCUUUCAACUAGAAUAAAAG	TTTC	478

154

Human-Exon 53	7	1	AUUCUGAAUUCUUUCAACUAGAAU	TTTT	479
Human-Exon 53	8	1	GAUUCUGAAUUCUUUCAACUAGAA	TTTA	480
Human-Exon 53	9	-1	CAGAACCGGAGGCAACAGUUGAAU	TTTC	481
Human-Exon 53	10	-1	GGAGGCAACAGUUGAAUGAAAUGU	TTTA	482
Human-Exon 53	11	-1	UAUACAGUAGAUGCAAUCCAAAAG	TTTT	483
Human-Exon 53	12	-1	GAUGCAAUCCAAAAGAAAAUCACA	TTTC	484
Human-Exon 53	13	-1	AAUCACAGAAACCAAGGUUAGUAU	TTTG	485
Human-Exon 53	14	-1	AGGUUAGUAUCAAAGAUACCUUU	TTTA	486
Human-Exon 53	15	-1	GGUUAGUAUCAAAGAUACCUUUUU	TTTT	487
Human-Exon 53	16	-1	AGUAUCAAAGAUACCUUUUUAAAA	TTTA	488
Human-Exon 53	17	-1	G UAUCAAAGAUACCUUUUUAAAA U	TTTT	489
Human-Exon 46	1	-1	UGUUUGUGUCCCAGUUUGCAUUAA	TTTG	490
Human-Exon 46	2	1	CUGGGACACAAACAUGGCAAUUUA	TTTT	491
Human-Exon 46	3	1	ACUGGGACACAAACAUGGCAAUUU	TTTT	492
Human-Exon 46	4	1	AACUGGGACACAAACAUGGCAAUU	TTTA	493
Human-Exon 46	5	1	UAUUUGUUAAUGCAAACUGGGACA	TTTG	494
Human-Exon 46	6	-1	ACAAAUAGUUUGAGAACUAUGUUG	tttC	495
Human-Exon 46	7	-1	CAAAUAGUUUGAGAACUAUGUUGG	tttt	496
Human-Exon 46	8	-1	AAAUAGUUUGAGAACUAUGUUGGa	tttt	497
Human-Exon 46	9	-1	AUAGUUUGAGAACUAUGUUGGaaa	tttt	498
Human-Exon 46	10	-1	U AGUUUGAGA AC U AUGUUGGaaaa	tttt	499
Human-Exon 46	11	-1	AGUUUG AG AAC U A UGUUGGaaaaa	tttt	500
Human-Exon 46	12	1	UAGUUCUCAAACUAUUUGUUAAUG	TTTG	501
Human-Exon 46	13	1	UAuuuuuuuuuCCAACAUAGUUCU	TTTG	502
Human-Exon 46	14	-1	CUUCUUUCUCCAGGCUAGAAGAAC	TTTT	503
Human-Exon 46	15	1	CUUCUAGCCUGGAGAAAGAAGAAU	TTTT	504
Human-Exon 46	16	1	UCUUCUAGCCUGGAGAAAGAAGAA	TTTA	505
Human-Exon 46	17	1	AUUCUUUUGUUCUUCUAGCCUGGA	TTTC	506
Human-Exon 46	18	-1	CAAAAGAAUAUCUUGUCAGAAUUU	TTTG	507

155

Human-Exon 46	19	-1	CUGGAAAAGAGCAGCAACUAAAAG	TTTT	508
Human-Exon 46	20	-1	CAAGUCAAGGUAAUUUUAUUUUCU	TTTG	509
Human-Exon 46	21	-1	CAAAUCCCCCAGGGCCUGCUUGCA	TTTA	510
Human-Exon 46	22	1	AGGCCCUGGGGGAUUUGAGAAAAU	TTTT	511
Human-Exon 46	23-	1	CAGGCCCUGGGGGAUUUGAGAAAA	TTTA	512
Human-Exon 46	24	1	CAAGCAGGCCCUGGGGGAUUUGAG	TTTT	513
Human-Exon 46	25	1	GCAAGCAGGCCCUGGGGGAUUUGA	TTTC	514
Human-Exon 46	26	1	GCAGAAAACCAAUGAUUGAAUUAA	TTTT	515
Human-Exon 46	27	1	GGCAGAAAACCAAUGAUUGAAUUA	TTTT	516
Human-Exon 46	28	1	GGGCAGAAAACCAAUGAUUGAAUU	TTTT	517
Human-Exon 46	29	1	UGGGCAGAAAACCAAUGAUUGAAU	TTTA	518
Human-Exon 46	30	-1	AUUAGGUUAUUCAUAGUUCCUUGC	TTTA	519
Human-Exon 46	31	1	AACUAUGAAUAACCUAAUGGGCAG	TTTT	520
Human-Exon 46	32	1	GAACUAUGAAUAACCUAAUGGGCA	TTTC	521
Human-Exon 52	1	-1	UAUUUCCUGUUAAAUUGUUUUCUA	TTTA	522
Human-Exon 52	2	1	GGUUUAUAGAAAACAAUUUAACAG	TTTC	523
Human-Exon 52	3	-1	AUACAGUAACAUCUUUUUUAUUUC	TTTA	524
Human-Exon 52	4	-1	UACAGUAACAUCUUUUUUAUUUCU	TTTT	525
Human-Exon 52	5	1	AUGUUACUGUAUAAGGGUUUAUAG	TTTT	526
Human-Exon 52	6	1	GAUGUUACUGUAUAAGGGUUUAUA	TTTC	527
Human-Exon 52	7	1	CAGCCAAAACACUUUUAGAAAUAA	TTTT	528
Human-Exon 52	8	1	CCAGCCAAAACACUUUUAGAAAUA	TTTT	529
Human-Exon 52	9	1	ACCAGCCAAAACACUUUUAGAAAU	TTTT	530
Human-Exon 52	10	1	GACCAGCCAAAACACUUUUAGAAA	TTTA	531
Human-Exon 52	11	1	GUGAGACCAGCCAAAACACUUUUA	TTTC	532
Human-Exon 52	12	-1	AAUUGUACUUUACUUUGUAUUAUG	TTTA	533
Human-Exon 52	13	-1	AUUGUACUUUACUUUGUAUUAUGU	TTTT	534
Human-Exon 52	14	1	UAAAGUACAAUUGUGAGACCAGCC	TTTT	535
Human-Exon 52	15	1	GUAAAGUACAAUUGUGAGACCAGC	TTTG	536

156

Human-Exon 52	16	1	GUAUHCCUUUUACAUAAUACAAAG	TTTA	537
Human-Exon 52	17	1	GUUGUGUAUUCCUUUUACAUAAUA	TTTG	538
Human-Exon 52	18	1	AUCCUGCAUUGUUGCCUGUAAGAA	TTTG	539
Human-Exon 52	19	1	UUCCAACUGGGGACGCCUCUGUUC	TTTG	540
Human-Exon 52	20	-1	UUGGAAGAACUCAUUACCGCUGCC	TTTG	541
Human-Exon 52	21	-1	UCAUUACCGCUGCCCAAAAUUUGA	TTTT	542
Human-Exon 52	22	1	CUCUUGAUUGCUGGUCUUGUUUUU	TTTG	543
Human-Exon 52	23	-1	G UUUUUUAACAAGCAUGGGACACA	TTTG	544
Human-Exon 52	24	1	CUUUGUGUGUCCCAUGCUUGUUAA	TTTT	545
Human-Exon 52	25	1	GCUUUGUGUGUCCCAUGCUUGUUA	TTTT	546
Human-Exon 52	26	1	UGCUUUGUGUGUCCCAUGCUUGUU	TTTT	547
Human-Exon 52	27	1	UUGCUUUGUGUGUCCCAUGCUUGU	TTTA	548
Human-Exon 52	28	-1	AGCAAGAUGCAUGACAAGUUUCAA	TTTA	549
Human-Exon 52	29	-1	GCAAGAUGCAUGACAAGUUUCAAU	TTTT	550
Human-Exon 52	30	-1	CAAGAUGCAUGACAAGUUUCAAUA	TTTT	551
Human-Exon 52	31	1	GAUAUAUGAACUUAAGUUUUUAUU	TTTC	552
Human-Exon 50	1	-1	AUAGAAAUCCAAUAAUAUAUUCAC	TTTG	553
Human-Exon 50	2	-1	AUUAAGAUGUUCAUGAAUUAUCUU	TTTG	554
Human-Exon 50	3	-1	UAAGUAAUGUGUAUGCUUUUCUGU	TTTA	555
Human-Exon 50	4	1	AUCUUCUAACUUCCUCUUUAACAG	TTTT	556
Human-Exon 50	5	1	GAUCUUCUAACUUCCUCUUCAACA	TTTC	557
Human-Exon 50	6	-1	AUCUGAGCUCUGAGUGGAAGGCGG	TTTA	558
Human-Exon 50	7	-1	ACCGUUUACUUCAAGAGCUGAGGG	TTTG	559
Human-Exon 50	8	1	CUGCUUUGCCCUCAGCUCUUGAAG	TTTA	560
Human-Exon 50	9	-1	UCUCUUUGGCUCUAGCUAUUUGUU	TTTG	561
Human-Exon 50	10	-1	CUCUUUGGCUCUAGCUAUUUGUUC	TTTT	562
Human-Exon 50	1 1	1	CACUUUUGAACAAAUAGCUAGAGC	TTTG	563
Human-Exon 50	12	1	UCACUUCAUAGUUGCACUUUUGAA	TTTG	564
Human-Exon 50	13	-1	AUGAAGUGAUGACUGGGUGAGAGA	TTTC	565

157

Human-Exon 50	14	-1	UGAAGUGAUGACUGGGUGAGAGAG	TTTT	566
Human-Exon 43	1	1	AAGAGAAAAauauauauauauaua	TTTG	567
Human-Exon 43	2	1	GAAUUAGCUGUCUAUAGAAAGAGA	tTTT	568
Human-Exon 43	3	1	UGAAUUAGCUGUCUAUAGAAAGAG	TTTT	569
Human-Exon 43	4	-1	AGCUAAUUCAUUUUUUUACUGUUU	TTTA	570
Human-Exon 43	5	1	AUGAAUUAGCUGUCUAUAGAAAGA	TTTC	571
Human-Exon 43	6	-1	GCUAAUUCAUUUUUUUACUGUUUU	TTTT	572
Human-Exon 43	7	1	AAAAAAAUGAAUUAGCUGUCUAUA	TTTC	573
Human-Exon 43	8	-1	UUAAAAUUUUUAUAUUACAGAAUA	TTTA	574
Human-Exon 43	9	-1	UAAAAUUUUUAUAUUACAGAAUAU	TTTT	575
Human-Exon 43	10	1	AUAUAAAAAUUUUAAAACAGUAAA	TTTT	576
Human-Exon 43	11	1	AAUAUAAAAAUUUUAAAACAGUAA	TTTT	577
Human-Exon 43	12	1	UAAUAUAAAAAUUUUAAAACAGUA	TTTT	578
Human-Exon 43	13	1	GUAAUAUAAAAAUUUUAAAACAGU	TTTT	579
Human-Exon 43	14	1	UGUAAUAUAAAAAUUUUAAAACAG	TTTA	580
Human-Exon 43	15	1	UAUAUUCUGUAAUAUAAAAAUUUU	TTTT	581
Human-Exon 43	16	1	UUAUAUUCUGUAAUAUAAAAAUUU	TTTA	582
Human-Exon 43	17	-1	CAGAAUAUAAAAGAUAGUCUACAA	TTTG	583
Human-Exon 43	18	1	CUAUCUUUUAUAUUCUGUAAUAUA	TTTT	584
Human-Exon 43	19	1	ACUAUCUUUUAUAUUCUGUAAUAU	TTTT	585
Human-Exon 43	20	1	GACUAUCUUUUAUAUHCUGUAAUA	TTTA	586
Human-Exon 43	21	-1	CAUAGCAAGAAGACAGCAGCAUUG	TTTG	587
Human-Exon 43	22	1	CAUUUUGUUAACUUUUUCCCAUUG	TTTC	588
Human-Exon 43	23	-1	CAUAUAUUUUUCUUGAUACUUGCA	TTTC	589
Human-Exon 43	24	1	AAAUCAUUUCUGCAAGUA UCAAGA	TTTT	590
Human-Exon 43	25	1	CAAAUCAUUUCUGCAAGUAUCAAG	TTTT	591
Human-Exon 43	26	1	ACAAAUCAUUUCUGCAAGUAUCAA	TTTC	592
Human-Exon 43	27	1	AUAAAUUCUACAGUUCCCUGAAAA	TTTG	593
Human-Exon 43	28	-1	GAAUUUAUUUCAGUACCCUCCAUG	TTTC	594

158

Human-Exon 43	29	-1	AAUUUAUUUCAGUACCCUCCAUGG	TTTT	595
Human-Exon 43	30	1	UGAAAUAAAUUCUACAGUUCCCUG	TTTT	596
Human-Exon 43	31	-1	AUUUAUUUCAGUACCCUCCAUGGA	TTTT	597
Human-Exon 43	32	1	CUGAAAUAAAUUCUACAGUUCCCU	TTTC	598
Human-Exon 43	33	-1	UUUAUUUCAGUACCCUCCAUGGAA	TTTT	599
Human-Exon 43	34	-1	UACCCUCCAUGGAAAAAAGACAGG	TTTC	600
Human-Exon 43	35	-1	ACCCUCCAUGGAAAAAAGACAGGG	TTTT	601
Human-Exon 43	36	-1	CCCUCCAUGGAAAAAAGACAGGGA	TTTT	602
Human-Exon 43	37	1	UUUUUUCCAUGGAGGGUACUGAAA	TTTA	603
Human-Exon 43	38	1	UGUCUUUUUUCCAUGGAGGGUACU	TTTC	604
Human-Exon 6	1	1	CCUUGAGCAAGAACCAUGCAAACU	TTTA	605
Human-Exon 6	2	-1	UGCUCAAGGAAUGCAUUUUCUUAU	TTTC	606
Human-Exon 6	3	-1	GCUCAAGGAAUGCAUUUUCUUAUG	TTTT	607
Human-Exon 6	4	1	UGCAUUCCUUGAGCAAGAACCAUG	TTTG	608
Human-Exon 6	5	-1	GAAAAUUUAUUUCCACAUGUAGGU	TTTG	609
Human-Exon 6	6	-1	AAAAUUUAUUUCCACAUGUAGGUC	TTTT	610
Human-Exon 6	7	-1	AAAUUUAUUUCCACAUGUAGGUCA	TTTT	611
Human-Exon 6	8	1	CAUGUGGAAAUAAAUUUUCAUAAG	TTTT	612
Human-Exon 6	9	1	ACAUGUGGAAAUAAAUUUUCAUAA	TTTC	613
Human-Exon 6	10	-1	CCACAUGUAGGUCAAAAAUGUAAU	TTTC	614
Human-Exon 6	11	-1	CACAUGUAGGUCAAAAAUGUAAUG	TTTT	615
Human-Exon 6	12	-1	ACAUGUAGGUCAAAAAUGUAAUGA	TTTT	616
Human-Exon 6	13	1	ACAUUUUUGACCUACAUGUGGAAA	TTTA	617
Human-Exon 6	14	1	CAUUACAUUUUUGACCUACAUGUG	TTTC	618
Human-Exon 6	15	-1	AAAAAUAUCAUGGCUGGAUUGCAA	TTTG	619
Human-Exon 6	16	-1	GCUGGAUUGCAACAAACCAACAGU	TTTC	620
Human-Exon 6	17	-1	CUGGAUUGCAACAAACCAACAGUG	TTTT	621
Human-Exon 6	18	1	CCUAUGACUAUGGAUGAGAGCAUU	TTTG	622
Human-Exon 6	19	-1	UAGGUAAGAAGAUUACUGAGACAU	TTTA	623

159

Human-Exon 6	20	-1	AUUACUGAGACAUUAAAUAACUUG	TTTA	624
Human-Exon 6	21	-1	UUACUGAGACAUUAAAUAACUUGU	TTTT	625
Human-Exon 6	22	1	GGGGAAAAAUAUGUCAUCAGAGUC	TTTA	626
Human-Exon 6	23	1	CAUGAUCUGGAACCAUACUGGGGA	TTTT	627
Human-Exon 6	24	1	ACAUGAUCUGGAACCAUACUGGGG	TTTT	628
Human-Exon 6	25	1	GACAUGAUCUGGAACCAUACUGGG	TTTC	629
Human-Exon 7	1	1	uacacacauacacaA AGAÇA AAUA	TTTA	630
Human-Exon 7	2	1	uacacauacacacauacacaAAGA	TTTG	631
Human-Exon 7	3	1	aacacauacacauacacacauaca	TTtg	632
Human-Exon 7	4	1	AUUCCAGUCAAAUAGGUCUGGCCU	ttTT	633
Human-Exon 7	5	1	UAUUCCAGUCAAAUAGGUCUGGCC	tTTA	634
Human-Exon 7	6	1	GCUGGCAAACCACACUAUUCCAGU	TTTG	635
Human-Exon 7	7	1	AGUCGUUGUGUGGCUGACUGCUGG	TTTG	636
Human-Exon 7	8	-1	CGCCAGAUAUCAAUHAGGCAUAGA	TTTC	637
Human-Exon 7	9	-1	AAACUACUCGAUCCUGAAGGUUGG	TTTA	638
Human-Exon 7	10	1	CAUACUAAAAGCAGUGGUAGUCCA	TTTC	639
Human-Exon 7	H	1	GAAAACAUUAAACUCUACCAUACU	TTTT	640
Human-Exon 7	12	1	UGAAAACAUUAAACUCUACCAUAC	TTTA	641
Human-Exon 8	1	-1	UUGUUCAUUAUCCUUUUAGAGUCU	TTTG	642
Human-Exon 8	2	1	AAAGGAUAAUGAACAAAUCAAAGU	TTTA	643
Human-Exon 8	3	-1	UAUCCUUUUAGAGUCUCAAAUAUA	TTTC	644
Human-Exon 8	4	1	ACUCUAAAAGGAUAAUGAACAAAU	TTTG	645
Human-Exon 8	5	-1	UUUUAGAGUCUCAAAUAUAGAAAC	TTTG	646
Human-Exon 8	6	-1	UUUAGAGUCUCAAAUAUAGAAACC	TTTT	647
Human-Exon 8	7	-1	UUAGAGUCUCAAAUAUAGAAACCA	TTTT	648
Human-Exon 8	8	1	UUGAGACUCUAAAAGGAUAAUGAA	TTTG	649
Human-Exon 8	9	1	UUUGGUUUCUAUAUUUGAGACUCU	TTTT	650
Human-Exon 8	10	1	UUUUGGUUUCUAUAUUUGAGACUC	TTTA	651
Human-Exon 8	11	-1	AGCAUUGAAGCCAUCCAGGAAGUG	TTTC	652

160

Human-Exon 8	12	1	GCUUCAAUGCUCACUUGUUGAGGC	TTTT	653
Human-Exon 8	13	1	GGCUUCAAUGCUCACUUGUUGAGG	TTTG	654
Human-Exon 8	14	-1	AGUGGAAAUGUUGCCAAGGCCACC	TTTA	655
Human-Exon 8	15	-1	GUUGCCAAGGCCACCUAAAGUGAC	TTTA	656
Human-Exon 8	16	-1	GAAGAACAUUUUCAGUUACAUCAU	TTTG	657
Human-Exon 8	17	-1	AUCAAAUGCACUAUUCUCAACAGG	TTTA	658
Human-Exon 8	18	1	AUAGUGCAUUUGAUGAUGUAACUG	TTTT	659
Human-Exon 8	19	1	AAUAGUGCAUUUGAUGAGGUAACU	TTTC	660
Human-Exon 8	20	-1	ACUAUUCUCAACAGGUAAAGUGUG	TTTA	661
Human-Exon 8	21	1	UACCUAAAAAUGCAUAUAAAACAG	TTTT	662
Human-Exon 8	22	1	AUACCUAAAAAUGCAUAUAAAACA	TTTC	663
Human-Exon 8	23	1	CACGUAAUACCUAAAAAUGCAUAU	TTTT	664
Human-Exon 8	24	1	GCACGUAAUACCUAAAAAUGCAUA	TTTA	665
Human-Exon 8	25	1	auauauauGUGCACGUAAUACCUA	TTTT	666
Human-Exon 8	26	1	uauauauauGUGCACGUAAUACCU	TTTT	667
Human-Exon 8	27	1	auauauauauGUGCACGUAAUACC	TTTA	668
Human-Exon 55	1	-1	CUGCACAAUAUUAUAGUUGUUGCU	TTTA	669
Human-Exon 55	2	1	AUAAAAAGAGAAAGAUGGAGGAAC	TTTA	670
Human-Exon 55	3	1	CACCUAGUGAACUCCAUAAAAAGA	TTTC	671
Human-Exon 55	4	1	AUGGUGCACCUAGUGAACUCCAUA	TTTT	672
Human-Exon 55	5	1	AAUGGUGCACCUAGUGAACUCCAU	TTTT	673
Human-Exon 55	6	1	GAAUGGUGCACCUAGUGAACUCCA	TTTA	674
Human-Exon 55	7	1	GACCAAAUGUUCAGAUGCAAUUAU	TTTA	675
Human-Exon 55	8	1	UCGCUCACUCACCCUGCAAAGGAC	TTTG	676
Human-Exon 55	9	-1	AGUGAGCGAGAGGCUGCUUUGGAA	TTTC	677
Human-Exon 55	10	1	GCAGCCUCUCGCUCACUCACCCUG	TTTG	678
Human-Exon 55	11	1	UUGCAGUAAUCUAUGAGUUUCUUC	TTTG	679
Human-Exon 55	12	-1	CUGCAACAGUUCCCCCUGGACCUG	TTTC	680
Human-Exon 55	13	-1	UGCAACAGUUCCCCCUGGACCUGG	TTTT	681

161

Human-Exon 55	14	-1	UUUCUUGCCUGGCUUACAGAAGCU	TTTC	682
Human-Exon 55	15	1	UUUCAGCUUCUGUAAGCCAGGCAA	TTTC	683
Human-Exon 55	16	-1	GUCCUACAGGAUGCUACCCGUAAG	TTTC	684
Human-Exon 55	17	-1	GGCUCCUAGAAGACUCCAAGGGAG	TTTA	685
Human-Exon 55	18	-1	GCUCCUAGAAGACUCCAAGGGAGU	TTTT	686
Human-Exon 55	19	-1	CUCCAAGGGAGUAAAAGAGCUGAU	TTTC	687
Human-Exon 55	20	1	UGGAUCCACAAGAGUGCUAAAGCG	TTTC	688
Human-Exon 55	21	1	GUUCAAUUGGAUCCACAAGAGUGC	TTTA	689
Human-Exon 55	22	-1	UACUUGUAACUGACAAGCCAGGGA	TTTG	690
Human-Exon 55	23	-1	ACUUGUAACUGACAAGCCAGGGAC	TTTT	691
Human-Exon 55	24	-1	GUAACUGACAAGCCAGGGACAAAA	TTTG	692
Human-Exon 55	25	-1	UAACUGACAAGCCAGGGACAAAAC	TTTT	693
Human-Exon 55	26	1	UCCCUGGCUUGUCAGUUACAAGUA	TTTG	694
Human-G 1 -exon51		1	CAGAGUAACAGUCUGAGUAGGAGc	TTTA	695
Human-G2-exon51		1	uacuuuguuuagcaauacauggua	TTTC	696
Human-G3-exon51		-1	uggcucaaauuguuacucuucaau	TTTA	697
mouse-Exon23-G 1		1	CUUUCAAagaacuuugcagagccu	TTTG	698
mouse-Exon23-G2		1	guugaaGCCAUUUUGUUGCUCUUU	TTTG	699
mouse-Exon23-G3		1	guugaaGCCAUUUUAUUGCUCUUU	TTTG	700
mouse-Exon23-G4		-1	uuuugagGCUCUGCAAAGUUCUUU	TTTC	701
mouse-Exon23-G5		-1	aguuauuaaugcauagauauucag	TTTA	702
mouse-Exon23-G6		-1	uauaauaugcccuguaauauaaua	TTTC	703
mouse-Exon23-G7		1	uaaaggccaaaccucggcuuaccU	TTTC	704
mouse-Exon23-G8		1	ucaauaucuuugaaggacucuggg	TTTA	705

* In this table, upper case letters represent sgRNA nucléotides that align to the exon sequence of the gene. Lower case letters represent sgRNA nucléotides that align to the intron sequence of the gene.

162

VI. Sequence Tables

Table 8: Genomic target sites for sgRNA in mouse Dmd Exon 51

ID sgRNA	Strand	Target site	SEQ ID NO:	PAM
Ex51-SA1	3’	AGAGTAACAGTCTGACTGG	706	CAG
Ex51-SD	5’	GAAATGATCATCAAACAGA	707	AGG
Ex51-SA-2	3’	CACTAGAGTAACAGTCTGAC	708	TGG

Table 9: gRNA sequences targeting mouse Dmd Exon 51

ID sgRNA	Strand	Target site	SEQ ID NO:	PAM
Ex51-SA1	3’	CCAGUCAGACUGUUACUCU	709	CAG
Ex51-SD	5’	UCUGUUGGAUGAUCAUUUC	710	AGG
Ex51-SA-2	3’	GUCAGACUGUUACUCUAGUG	711	TGG

Table 10: Genomic target sequences for sgRNAs targeting human Dmd Exon 51

ID sgRNA	Strand	Target site	SEQ ID NO:	PAM
Ex51-SA	3’	AGAGTAACAGTCTGAGTAG	712	GAG
Ex51-SD	5’	GAGATGATCATCAAGCAGA	713	AGG
Ex51-SA-2	3’	CACCAGAGTAACAGTCTGAG	714	TAG

Table 11: sgRNA sequences targeting human Dmt/Exon 51

ID sgRNA

Strand

Target site

SEQ ID NO:

PAM

163

Ex51-SA	3’	CUACUCAGACUGUUACUCU	715	GAG
Ex51-SD	5’	UCUGCUUGAUGAUCAUCUC	716	AGG
Ex51-SA-2	3’	CUCAGACUGUUACUCUGGUG	717	TAG

Table 12: Genomic target sequences for sgRNAs targeting sites in varions human Dmd

Exons

ID sgRNA	Strand	Target site	SEQ ID NO:	PAM
Exon51 -# 1	3’	CAGAGTAACAGTCTGAGTAG	947	GAG
Exon51-#2	3’	CACCAGAGTAACAGTCTGAG	718	TAG
Exon51-#3	3’	TATTTTGGGTTTTTGCAAAA	719	AGG
Exon51 -#4	3’	AGTAGGAGCTAAAATATTTT	720	GGG
Exon51 -#5	3’	GAGTAGGAGCTAAAATATTT	721	TGG
Exon51-#6	3’	ACCAGAGTAACAGTCTGAGT	722	AGG
Exon51-#7	5’	TCCTACTCAGACTGTTACTC	723	TGG
Exon51-#8	5’	TACTCTGGTGACACAACCTG	724	TGG
Exon51 -#9	3’	GCAGTTTCCTTAGTAACCAC	725	AGG
Exon51-#10	5’	GACACAACCTGTGGTTACTA	726	AGG
Exon51-#11	3’	TGTCACCAGAGTAACAGTCT	727	GAG
Exon51-#12	3’	AGGTTGTGTCACCAGAGTAA	728	CAG
Exon51 -# 13	3’	AACCACAGGTTGTGTCACCA	729	GAG
Exon51-#14	3’	GTAACCACAGGTTGTGTCAC	730	CAG
Exon53-#1	5’	ATTTATTTTTCCTTTTATTC	731	TAG

164

Exon53-#2	5’	TTTCCTTTTATTCTAGTTGA	732	AAG
Εχοη53-#3	3’	TGATTCTGAATTCTTTCAAC	733	TAG
Exon 5 3-#4	3’	AATTCTTTCAACTAGAATAA	734	AAG
Εχοη53-#6	5’	TTATTCTAGTTGAAAGAATT	735	CAG
Εχοη53-#7	5’	TAGTTGAAAGAATTCAGAAT	736	CAG
Εχοη53-#8	5’	AATTCAGAATCAGTGGGATG	737	AAG
Εχοη53-#9	3’	ATTCTTTCAACTAGAATAAA	738	AGG
Εχοη53-#10	5’	TTGAAAGAATTCAGAATCAG	739	TGG
Εχοη53-#11	5’	TGAAAGAATTCAGAATCAGT	740	GGG
Εχοη53-#12	3’	ACTGTTGCCTCCGGTTCTGA	741	AGG
Εχοη44-#1	3’	CAGATCTGTCAAATCGCCTG	742	CAG
Εχοη44-#2	3’	AAAACGCCGCCATTTCTCAA	743	CAG
Εχοη44-#3	3’	AGATCTGTCAAATCGCCTGC	744	AGG
Εχοη44-#4	3’	TATGGATCAAGAAAAATAGA	745	TGG
Εχοη44-#5	3’	CGCCTGCAGGTAAAAGCATA	746	TGG
Εχοη44-#6	5’	ATCCATATGCTTTTACCTGC	747	AGG
Εχοη44-#8	5’	TTGACAGATCTGTTGAGAAA	748	TGG
Εχοη44-#9	5’	ACAGATCTGTTGAGAAATGG	749	CGG
Εχοη44-# 11	5’	GGCGATTTGACAGATCTGTT	750	GAG
Εχοη44-#13	5’	GGCGTTTTCATTATGATATA	751	AAG
Εχοη44-#14	5’	ATGATATAAAGATATTTAAT	752	CAG
Εχοη44-# 15	5’	GATATTTAATCAGTGGCTAA	753	CAG
Εχοη44-#16	5’	ATTTAATCAGTGGCTAACAG	754	AAG
Εχοη44-#17	3’	AGAAACTGTTCAGCTTCTGT	755	TAG

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Exon43-#1	5’	GTTTTAAAATTTTTATATTA	756	CAG
Exon43-#2	5’	TTTTATATTACAGAATATAA	757	AAG
Exon43-#3	5’	ATATTACAGAATATAAAAGA	758	TAG
Exon45-#1	3’	GTTCCTGTAAGATACCAAAA	759	AGG
Exon45-#2	5’	TTGCCTTTTTGGTATCTTAC	760	AGG
Exon45-#3	5’	TGGTATCTTACAGGAACTCC	761	AGG
Exon45-#4	5’	ATCTTACAGGAACTCCAGGA	762	TGG
Exon45-#5	3’	GCCGCTGCCCAATGCCATCC	763	TGG
Exon45-#6	5’	CAGGAACTCCAGGATGGCAT	764	TGG
Exon45-#7	5’	AGGAACTCCAGGATGGCATT	765	GGG
Exon45-#8	5’	TCCAGGATGGCATTGGGCAG	766	CGG
Exon45-#9	5’	GTCAGAACATTGAATGCAAC	767	TGG
Exon45-#10	3’	AGTTCCTGTAAGATACCAAA	768	AAG
Exon45-#11	3’	TGCCATCCTGGAGTTCCTGT	769	AAG
Exon45-#12	5’	TTGGTATCTTACAGGAACTC	770	CAG
Exon45-#13	3’	CGCTGCCCAATGCCATCCTG	771	GAG
Exon45-#14	5’	AACTCCAGGATGGCATTGGG	772	CAG
Exon45-#15	5’	GGGCAGCGGCAAACTGTTGT	773	CAG
Exon52-#1	3’	AGATCTGTCAAATCGCCTGC	774	AGG
Exon52-#2	3’	AATCCTGCATTGTTGCCTGT	775	AAG
Exon52-#3	5’	CGCTGAAGAACCCTGATACT	776	AAG
Exon52-#4	3’	GAACAAATATCCCTTAGTAT	777	CAG
Exon52-#5	3’	CTGTAAGAACAAATATCCCT	778	TAG
Exon52-#6	5’	CTAAGGGATATTTGTTCTTA	779	CAG

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Exon52-#8	5’	TGTTCTTACAGGCAACAATG	780	GAG
Exon52-#9	5’	CAACAATGCAGGATTTGGAA	781	CAG
Exon52-#10	5’	ACAATGCAGGATTTGGAACA	782	GAG
Exon52-#11	5’	ATTTGGAACAGAGGCGTCCC	783	CAG
Exon52-#12	5’	ACAGAGGCGTCCCCAGTTGG	784	AAG
Exon2-#1	5’	TATTTTTTTATTTTGCATTT	785	TAG
Exon2-#2	5’	TTATTTTGCATTTTAGATGA	786	AAG
Exon2-#3	5’	ATTTTGCATTTTAGATGAAA	787	GAG
Exon2-#4	5’	TTGCATTTTAGATGAAAGAG	788	AAG
Exon2-#5	5’	ATGAAAGAGAAGATGTTCAA	789	AAG

Table 13: gRNA sequences for targeting sites in various human Dmd Exons

ID sgRNA	Strand	Target site	SEQ ID NO:	PAM
Exon51-#1	3’	CUACUCAGACUGUUACUCUG	790	GAG
Exon51-#2	3’	CUCAGACUGUUACUCUGGUG	791	TAG
Exon51-#3	3’	UUUUGCAAAAACCCAAAAUA	792	AGG
Exon51-#4	3’	AAAAUAUUUUAGCUCCUACU	793	GGG
Exon51-#5	3’	AAAUAUUUUAGCUCCUACUC	794	TGG
Exon51 -#6	3’	ACUCAGACUGUUACUCUGGU	795	AGG
Exon51-#7	5’	GAGUAACAGUCUGAGUAGGA	796	TGG
Exon51-#8	5’	CAGGUUGUGUCACCAGAGUA	797	TGG
Exon51-#9	3’	GUGGUUACUAAGGAAACUGC	798	AGG
Exon51 -# 10	5’	UAGUAACCACAGGUUGUGUC	799	AGG

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Exon51-#11	3’	AGACUGUUACUCUGGUGACA	800	GAG
Εχοη51-#12	3’	UUACUCUGGUGACACAACCU	801	CAG
Εχοη51-#13	3’	UGGUGACACAACCUGUGGUU	802	GAG
Εχοη51 -#14	3’	GUGACACAACCUGUGGUUAC	803	CAG
Εχοη53-#1	5’	GAAUAAAAGGAAAAAUAAAU	804	TAG
Εχοη53-#2	5’	UCAACUAGAAUAAAAGGAAA	805	AAG
Εχοη53-#3	3’	GUUGAAAGAAUUCAGAAUCA	806	TAG
Εχοη53-#4	3’	UUAUUCUAGUUGAAAGAAUU	807	AAG
Εχοη53-#6	5’	AAUUCUUUCAACUAGAAUAA	808	CAG
Εχοη53-#7	5’	AUUCUGAAUUCUUUCAACUA	809	CAG
Εχοη53-#8	5’	CAUCCCACUGAUUCUGAAUU	810	AAG
Εχοη53-#9	3’	UUUAUUCUAGUUGAAAGAAU	811	AGG
Εχοη53-#Ι0	5’	CUGAUUCUGAAUUCUUUCAA	812	TGG
Εχοη53-#11	5’	ACUGAUUCUGAAUUCUUUCA	813	GGG
Εχοη53-#12	3’	UCAGAACCGGAGGCAACAGU	814	AGG
Εχοη44-#1	3’	CAGGCGAUUUGACAGAUCUG	815	CAG
Εχοη44-#2	3’	UUGAGAAAUGGCGGCGUUUU	816	CAG
Εχοη44-#3	3’	GCAGGCGAUUUGACAGAUCU	817	AGG
Εχοη44-#4	3’	UCUAUUUUUCUUGAUCCAUA	818	TGG
Εχοη44-#5	3’	UAUGCUUUUACCUGCAGGCG	819	TGG
Εχοη44-#6	5’	GCAGGUAAAAGCAUAUGGAU	820	AGG
Εχοη44-#8	5’	UUUCUCAACAGAUCUGUCAA	821	TGG
Εχοη44-#9	5’	CCAUUUCUCAACAGAUCUGU	822	CGG
Εχοη44-# 11	5’	AACAGAUCUGUCAAAUCGCC	823	GAG

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Exon44-#13	5’	UAUAUCAUAAUGAAAACGCC	824	AAG
Εχοη44-#14	5’	AUUAAAUAUCUUUAUAUCAU	825	CAG
Εχοη44-#15	5’	UUAGCCACUGAUUAAAUAUC	826	CAG
Εχοη44-#16	5’	CUGUUAGCCACUGAUUAAAU	827	AAG
Εχοη44-#17	3’	ACAGAAGCUGAACAGUUUCU	828	TAG
Εχοη43-#1	5’	UAAUAUAAAAAUUUUAAAAC	829	CAG
Εχοη43-#2	5’	UUAUAUUCUGUAAUAUAAAA	830	AAG
Εχοη43-#3	5’	UCUUUUAUAUUCUGUAAUAU	831	TAG
Εχοη45-#1	3’	UUUUGGUAUCUUACAGGAAC	832	AGG
Εχοη45-#2	5’	GUAAGAUACCAAAAAGGCAA	833	AGG
Εχοη45-#3	5’	GGAGUUCCUGUAAGAUACCA	834	AGG
Εχοη45-#4	5’	UCCUGGAGUUCCUGUAAGAU	835	TGG
Εχοη45-#5	3’	GGAUGGCAU UGGGCAGCGGC	836	TGG
Εχοη45-#6	5’	AUGCCAUCCUGGAGUUCCUG	837	TGG
Εχοη45-#7	5’	AAUGCCAUCCUGGAGUUCCU	838	GGG
Εχοη45-#8	5’	CUGCCCAAUGCCAUCCUGGA	839	CGG
Εχοη45-#9	5’	GUUGCAUUCAAUGUUCUGAC	840	TGG
Εχοη45-#10	3’	UUUGGUAUCUUACAGGAACU	841	AAG
Εχοη45-#11	3’	ACAGGAACUCCAGGAUGGCA	842	AAG
Εχοη45-#12	5’	GAGUUCCUGUAAGAUACCAA	843	CAG
Εχοη45-#13	3’	CAGGAUGGCAUUGGGCAGCG	844	GAG
Εχοη45-#14	5’	CCCAAUGCCAUCCUGGAGUU	845	CAG
Εχοη45-#15	5’	ACAACAGUUUGCCGCUGCCC	846	CAG
				1

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Exon52-#1	3’	GCAGGCGAUUUGACAGAUCU	847	AGG
Exon52-#2	3’	ACAGGCAACAAUGCAGGAUU	848	AAG
Exon52-#3	5’	AGUAUCAGGGUUCUUCAGCG	849	AAG
Exon52-#4	3’	AUACUAAGGGAUAUUUGUUC	850	CAG
Exon52-#5	3’	AGGGAUAUUUGUUCUUACAG	851	TAG
Exon52-#6	5’	UAAGAACAAAUAUCCCUUAG	852	CAG
Exon52-#8	5’	CAUUGUUGCCUGUAAGAACA	853	CAG
Exon52-#9	5’	UUCCAAAUCCUGCAUUGUUG	854	CAG
Exon52-#10	5’	UGUUCCAAAUCCUGCAUUGU	855	GAG
Exon52-#11	5’	GGGACGCCUCUGUUCCAAAU	856	CAG
Exon52-#12	5’	CCAACUGGGGACGCCUCUGU	857	AAG
Exon2-#1	5’	ACAGAGGCGUCCCCAGUUGG	858	TAG
Exon2-#2	5’	UCAUCUAAAAUGCAAAAUAA	859	AAG
Exon2-#3	5’	UUUCAUCUAAAAUGCAAAAU	860	GAG
Exon2-#4	5’	CUCUUUCAUCUAAAAUGCAA	861	AAG
Exon2-#5	5’	UUGAACAUCUUCUCUUUCAU	862	AAG

Table 14: Genomic targeting sequence for sgRNAs targeting dog Dmd Exon 51

ID sgRNA	Strand	Target site	SEQ ID NO:	PAM
Ex51-SA-2	3’	CACCAGAGTAACAGTCTGAC	863	TGG

Table 15: gRNA sequence for targeting dog Dmd Exon 51

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ID sgRNA	Strand	Target site	SEQ ID NO:	PAM
Ex51-SA-2	3’	GUCAGACUGUUACUCUGGUG	864	TGG

VII. Examples

The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventer to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the présent disclosure, appreciate that many changes can be made in the spécifie embodiments which are disclosed and still obtain a like or similar resuit without departing from the spirit and scope ofthe disclosure.

EXAMPLE 1 - Materials and Methods

Study Approval. Ail experimental procedures involving animais in this study were reviewed and approved by the University of Texas Southwestern Medical Center s Institutional Animal Care and Use Committee.

CRISPR/Cas9-mediated exon 50 délétion in mice. Two single-guide RNA (sgRNA) spécifie intronic région surrounding exon 50 sequence of the mouse Dmd locus were cloned into vector px330 using the following primers: Dmd exon 50_Fl. 5

CACCGAAATGATGAGTGAAGTTATAT-3’ (SEQ ID NO: 926); Dmd exon 5O_R1: 5’AAACATATAACTTCACTCATCATTTC-3’ (SEQ ID NO: 927); Dmd exon 50_F2: 5’ CACCGGTTTGTTCAAAAGCGTGGCT-3’ (SEQ ID NO: 928); Dmd exon 50_R2: 5’AAACAGCCACGCTTTTGAACAAAC-3’ (SEQ IDNO: 929).

For the in vitro transcription of sgRNA, T7 promoter sequence was added to the sgRNA template by PCR using the following primers: Dmd exon 5O_T7-F1 : GAATTGTAATACGACTCACTATAGGAATGATGAGTGAAGTTATAT (SEQ IDNO: 930); Dmd exon 50_T7-F2:

GAATTGTAATACGACTCACTATAGGGTTTGTTCAAAAGCGTGGCT (SEQ ID NO: 931); Dmdexon 50_T7-Rv: AAAAGCACCGACTCGGTGCCAC (SEQ IDNO: 932).

171

The gel purified PCR products were used as template for in vitro transcription using the MEGAshortscript T7 Kit (Life Technologies). sgRNA were purified by MEGAclear kit (Life Technologies) and eluted with nuclease-free water (Ambion). The concentration of guide RNA was measured by a NanoDrop instrument (Thermo Scientific).

Genotyping of AEx50 Mice. AEx50 mice were genotyped using primers encompassing the targeted région: Geno50-F: 5’-GGATTGACTGAAATGATGGCCAAGG-3’ (SEQ ID NO: 937); Geno50-R: 5’-CTGCCACGATTACTCTGCTTCCAG-3’ (SEQ ID NO: 938). Tail biopsies were digested in 100 pL of 25-mM NaOFl, 0.2-mM EDTA (pH 12) for 20 min at 95 °C. Tails were briefly centrifuged followed by addition of 100 pL of40-mM Tris-HCl (pH 5) and mixed to homogenize. Two microliters of this reaction was used for subséquent PCR reactions with the primers below, followed by gel electrophoresis.

Plasmids. The pSpCas9(BB)-2A-GFP (PX458) plasmid containing the human codon optimized SpCas9 gene with 2A-EGFP and the backbone of sgRNA was purchased from Addgene (Plasmid #48138). AAV TRIPSR plasmids were obtained from Dr. Dirk Grimm (Heidelberg University Hospital). Cloning of sgRNA was donc using a Bbsl site. pGL3-CK8e plasmid was obtained from Dr. Stephen Hauschka (Department of Biochemistry, University of Washington, Seattle, USA). AAV-miniCMV-Cas9-shortPolyA plasmid was obtained from Dr. Dirk Grimm (Heidelberg University Hospital). To generate the final AAV9-CK8CRISPR/Cas9 vector used in this manuscript, AAV-miniCMV-Cas9-short-PolyA was digested with Pacl and Nhel enzyme to remove the miniCMV promoter. CK8 promoter was amplified from pGL3-CK8e plasmid using primers containing Pacl and Nhel site sequence and cloned into digested vector to generate AAV-CK8-Cas9-shortPolyA plasmid.

sgRNA Identification and Cloning for skipping exon 51. Dmd exon 51 guide RNAs were defined using crispr.mit.edu. Guide sequences were cloned into Addgene plasmid #42230 (6), a gift from Feng Zhang, using the following primers: Dmd exon 51_F1: 5'CACCGAGAGTAACAGTCTGACTGG -3’ (SEQ ID NO: 942); Dmd exon 51_R1: 5'AAACGTCAGACTGTTACTCTAGTGC-3' (SEQ ID NO; 943); Dmd exon 51_F2: 5'CACCGCACTAGAGTAACAGTCTGAC -3' (SEQ ID NO: 944); Dmd exon 51_R2: 5'AAACCCAGTCAGACTGTTACTCTC -3' (SEQ ID NO: 945). Guide sequences were tested in culture using 10T1 /2 cells before cloning into the AAV backbone.

Triplicate sgRNA assembly in AAV backbone using Golden Gâte System. The assembly of the AAV TRISPR backbone cloning System relies on two consecutive steps of

172

Golden Gâte Assembly. First step assembly of gRNA into donor plasmid. Annealing of oligonucleotides is performed by heating a reaction containing 2.5 μΐ of each oligo (0.5 μΜ), 5 μΐ NEBuffer 2 (NEB) and 40 μΕ ddH₂O to 95 °C for 5 minutes using heating block. For the assembly reaction into donor plasmid mix 40 fmol (~100 ng) destination backbone, 1 pL annealed, diluted oligos, 0.75 pL of Esp3I, 1 pL buffer tango (both Thermo Scientific), 1 pL of T4 DNA Ligase (400 U/pL) (NEB) as well as ATP and DTT at a final concentration of 1 mM in 10 pL total volume. Using a thermocycler, conduct 25 to 50 cycles ot 37°C/3 min followed by 20 °C/5 minutes. Dénaturé restriction enzyme and ligase by heating to 80 °C for 20 minutes. Use 3 pL of this reaction for transformation of chemo-competent bacteria, recover in SOC (37 °C, 800 rpm, 40 min) and spread on LB-Agar plates containing chloramphenicol (25 pg/mL). Annealed oligonucleotides encoding for the sgRNA are cloned into donor plasmids that carry the négative sélection marker ccdB (to reduce background during cloning) as well as the chloramphenicol résistance gene. To test the correct assembly the plasmid are sequenced using the primer Dono-R-5’-GTATGTTGTGTGGAATTGTGAG-3 (SEQ ID NO. 948). Second step is that three ofthese donor plasmids driving expression of one sgRNA under transcriptional control of U6, H1 or 7SK promoter are pooled in a second Golden Gâte assembly along with a récipient plasmid that carries AAV ITRs. The assembly reaction will contain ail four plasmids: donor plasmid-#l-U6-sgRNA, donor plasmid-#2-Hl-sgRNA, donor plasmid-#3-7SK-sgRNA and récipient plasmid containing the ITR. Digest with Bbsl will generate unique overhangs for each fragment (U6, Hl, 7SK, récipient backbone). During the ligation procedure, these overhangs anneal a circularized plasmid is only obtained, when the three cassettes match each other.

Sérum creatine kinase (CK) measurement. Mouse sérum CK was measured by the Metabolic Phenotyping Core at UT Southwestern Medical Center. Blood was collected from the submandibular vein and sérum CK level was measured by VITROS Chemistry 7 Products CK Slides to quantitatively measure CK activity using VITROS 250 Chemistry System.

In vivo canine studies. Intra-muscular (IM) injections were performed in 1 month old ΔΕ50 MD dogs under ventilated general anaesthesia. One single injection was performed at 4 different sites of the left cranial tibialis muscle. Ail dogs received premedication of an opiate (buprénorphine), perioperative antibiosis and 4 days of postoperative analgesia (carprofen). Two affected dogs received rAAV-mixed with myoediting components, AAV-Cas9 and AAVsgRNA-ex51, which were prepared in lactated Ringer’s solution to a final concentration of lE13vg per injected muscle.

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EXAMPLE 2 - Results

A humanized model of DMD. The most common hot spot mutation région in DMD patients is the région between exon 45 to 51, and skipping of exon 51 could be used to treat the largest group (13-14%) of patients. To investigate CRlSPR/Cas9-mediated exon 51 skipping in vivo, the inventors generated a mouse model that mimics the human “hot spot” région by deleting exon 50 using the CR1SPR/Cas9 System directed by 2 sgRNAs (FIG. 1 A). The délétion of exon 50 was confirmed by DNA sequencing (FIG. IB). Délétion of exon 50 placed the dystrophin gene out of frame leading to the absence of dystrophin protein in skeletal muscle and heart (FIGS. 1C-E). Mice lacking exon 50 showed pronounced dystrophie muscle changes by 2 months of âge (FIG. 1E). Sérum analysis of delta-exon 50 mice showed a significant increase in creatine kinase (CK) levels, indicative of muscle damage (FIG. 1 F). Taken together, dystrophin protein expression, muscle histology and sérum CK levels validated the dystrophie phenotype of the ΔΕχ50 mouse model.

Restoration of dystrophin expression using a single eut strategy to skip exon 51. S. pyogenes Cas9 requires NAG/NGG as a PAM sequence to generate a double-strand DNA break. Interestingly, the universal splice acceptor and donor sites of exons contain NAG or NGG (FIG. 3B). Therefore, to correct the reading frame and dystrophin expression in the ΔΕχ50 mouse model, the inventors generated sgRNA that targeted splice acceptor and donor sites of exon 51 to delete it, thereby recreating the in-frame dystrophin protein. To test whether the sgRNA guides were able to efficiently eut, the inventors first evaluated their effectiveness in mouse and human cell lines (FIG. 5). To détermine the most efficient way to correct the DMD reading frame, the inventors compared 2 different strategies: (1) double-guide strategy in which one copy of a first sgRNA targeting splice acceptor site (sgRNA-SA) and one copy of a second sgRNA targeting donor acceptor site (sgRNA-SD), were cloned into the rAAV9sgRNA vector; (2) triplicate strategy in which the inventors cloned 3 copies ofthe same sgRNA (sgRNA-SA) into the rAAV9-sgRNA vector (FIG. 3C). Expression of each copy of sgRNASA was driven by a different RNA promoter (U6, Hl and 7SK). The inventors generated AAVCas9 using an AAV-Cas9 vector (CK8-Cas9-shortPolyA), which employs a CK8 promoter to drive expression of the humanized SpCas9 specifically in skeletal muscle and heart tissues. Following intra-muscular (IM) injection of PI2 mice with AAVs, muscle tissues were analyzed. RT-PCR of RNA from ΔΕχ50 mice injected with AAV-Tri-SA and AAV-SA+SD showed that délétion of exon 51 (ΔΕχ50-51) allowed splicing from exon 49 to 52 (FIG. 2A, lower band). Sequencing of RT-PCR products of the ΔΕχ50-51 band confirmed that exon 49

174 spliced to exon 52 (FIG. 2B). Unexpectedly, the RT-PCR analysis showed that a single eut strategy using a triplicate version of sgRNA-SA (AAV-Tri-SA) is as efficient as using two sgRNAs - sgRNA-SA and sgRNA-SD (AAV-SA+SD).

To further assess the efficiency of the AAV-Tri-SA editing strategy, the inventors performed histological analysis of injected muscle to evaluate the number of fibers that express dystrophin throughout entire muscle sections. Interestingly, dystrophin immunostaining of muscle cryosections from AEx50 mice injected with AAV-Tri-SA revealed significantly higher numbers of dystrophin-positive fibers (average of 43 ± 0.9%) compared to the muscle from AEx50 mice injected with AAV-SA+SD (average of 31 ± 0.1%) (FIGS. 3D-E, FIG. 6). Western blot analysis confirmed the restoration of dystrophin expression in skeletal muscle. (FIGS. 3F-G). Hematoxylin and eosin (H&E) staining of muscle showed that histopathologie hallmarks of muscular dystrophy, such as necrotic myofibers, were diminished in TA muscle at 3-weeks post-AAV delivery (FIG. 4A). Quantitative analysis of the distribution of myofiber areas showed a clear increase in fiber size for both AAV-Tri-SA and AAV-SA-SD treated muscles compared to AEx50 muscles (FIG. 4B). However, AAV-Tri-SA treated muscles revealed a higher decrease in the frequency of small fibers (<500 pm) compared to AAVSA+SD treated muscles. Together, these results demonstrate that targeting the splice acceptor site of exon 51 with one single eut using AAV-Tri-SA is highly efficient in restoring dystrophin expression in DMD. This approach has usefulness for many disorders that can be corrected by exon skipping.

Tailoring of the single DNA eut genome editing strategy. S. pyogenes Cas9 guided by sgRNAs binds to the targeted genomic locus next to a PAM and generates a double-strand DNA break (DSB) 3 nucléotides preceding the PAM sequence. To further assess the efficiency of the method by targeting the splice acceptor site, the inventors designed a second sgRNA triple guide construct (sgRNA-ex51-SA2), targeting a région adjacent to the exon 51 splice acceptor site. This gRNA uses a PAM sequence 3 nucléotides further into the exon in order to generate the DSB close to the splice acceptor site for exon 51 (FIG. 7A-FIG. 7B). Cutting in the vicinity of the splice acceptor région and within the exon sequence resulted in reframing events and exon skipping events. Moreover, designing the sgRNA in the exon sequence that shows higher conservation than intron sequence across species facilitâtes translation of the sgRNA to other species.

175

The DNA cutting activity of Cas9 coupled with sgRNA-ex51-SA2 was evaluated in 10T1 /2 mouse fibroblasts using the mismatch-specific T7 endonuclease I (T7E1) assay (FIG. 8A). To investigate the type of mutations generated by Cas9 coupled with sgRNA-51-SA2, genomic deep-sequencing analysis was performed. The sequencing analysis revealed that 9.3% of mutations contained a single adenosine (A) insertion located 3 nucléotides 3’ of the PAM sequence. In addition, 7.3% of mutations contained délétions covering the splice accepter site and a highly-predicted exonic splicing enhancer site for exon 51 (FIG. 8B). The sgRNA-ex51SA2 corresponds to a highly conserved région of the Dmd gene (FIGS. 8C-D), and the inventors tested the ability of Cas9 and human sgRNA-51 to eut the human Dmd locus in 293T cells. The T7E1 assay revealed clear cleavage at the predicted site (FIG. 8E). Similarly, sequence analysis revealed that Cas9 coupled with human sgRNA-ex51-SA2 generated the same adenosine (A) insertion and a different range of délétions around the cleavage site (FIG. 8F).

For the in vivo delivery of Cas9 and sgRNA-ex51-SA2 to skeletal muscle and heart tissue, adeno-associated virus 9 (AAV9) was used, which displays preferential tropism for these tissues. To further enhance muscle-specific expression, an AAV9-Cas9 vector (CK8eCas9-shortPolyA) was employed, which contains a muscle-specific CK regulatory cassette, referred to as the CK8e promoter, which is highly spécifie for expression in muscle and heart (FIG. 9A). Together, this 436 bp muscle-specific cassette and the 4101 bp Cas9 cDNA are within the packaging limit of AAV9. Expression of each sgRNA was driven by one of three RNA polymerase III promoters (U6, H1 and 7SK) (FIG. 9B).

Correction of the dystrophin reading frame in AEx50 mice by a single DNA eut. The sgRNA-ex51-SA2 was delivered to mice in triple copy (AAV-Tri-SA2), along with a Cas9 (AAV-Cas9), by intra-muscular (IM) injection. Following the injection, muscle tissues were analyzed. In vivo targeting efficiency was estimated by RT-PCR with primers for sequences in exons 48 and 53 and the T7E1 assay for the targeted genomic régions. To investigate whether efficient target cleavage was achieved, the inventors amplified a 771 bp région spanning the target site and analyzed it using the T7EI assay (FIG. 10A). The activity of SpCas9 with the corresponding sgRNA was analyzed on the target site. T7EI assays revealed mutagenesis of the Dmd locus after delivery of AAV-Cas9 and AAV9-sgRNA-51-SA2 (FIG. 10A). To investigate the type of mutations generated in ΔΕχ50 mice injected with Cas9 and sgRNAexpressing AAV9s, genomic PCR amplification products spanning the target site were analyzed by amplicon deep-sequencing analysis. Deep sequencing of the targeted région

176 indicated that 27.9% of total reads contained changes at the targeted genomic site (FIG. 10B). On average, 15% of the identified mutations contained the same A insertion seen in mouse 10T1/2 and human 293 cells in vitro. The délétions identified using this method encompassed a highly-predicted exonic splicing enhancer site for exon 51 (FIG. 10B).

RT-PCR products of RNA from muscle of ΔΕχ50 mice injected intramuscularly with

AAV9-Cas9 and AAV9-sgRNA-51 showed that délétion of exon 51 (AEx50-51) allowed splicing from exon 49 to 52 (FIG. 11 A, lower band). By sequencing RT-PCR products of the ΔΕχ50-51 band, it was confirmed that exon 49 was spliced to exon 52. To further define the mutations introduced by our gene editing strategy, RT-PCR amplification products from 4 10 samples were directly subjected to topoisomerase-based thymidine to adenosine (TOPO-TA) cloning without gel purification, then sequenced. Surprisingly, sequence analysis of 40 clones from each sample showed that in addition to exon 51-skipped cDNA products (ΔΕχ50-51) identified in 15% of sequenced clones, ΔΕχ50 mice injected with AAV9-Cas9 and AAV9sgRNA-51 showed a high frequency of reframing events. Of sequenced clones, 63% contained 15 a single nucléotide insertion in the sequence of exon 51 (FIGS. 11 B-C). The most dominant insertion mutation seen was an A insertion.

On gels, the A insertion was indistinguishable in size from non-edited cDNA products, so deep-sequencing analysis was performed to détermine the abundance of this insertion compared to other small insertions. Deep-sequencing of the upper band containing the non20 edited cDNA product and reframed cDNA products indicated that 69.22% of total reads contained reframed cDNA products with an A insertion, 17.71% contained non-edited cDNA product, and the rest contained small délétions and insertions (FIG. 11 D). The deep-sequencing analysis of uninjected ΔΕχ50 mice confirmed that the A insertion is a resuit of Cas9-generated editing. These amplicon deep-sequencing results confirmed the results from TOPO-TA cloning 25 and sequencing. Taken together, RT-PCR analysis revealed that ΔΕχ50 mice injected with AAV9-Cas9 and AAV9-sgRNA-51-SA2 showed a high frequency of reframing events with cDNA products containing an A insertion in the sequence of exon 51 in addition to exon 51 skipping events resulting from délétion in a highly conserved exonic splicing enhancer région.

Restoration of dystrophin expression after intramuscular AAV9 delivery of Cas9 30 and sgRNA-51-SA2. Remarkably, dystrophin immunostaining of muscle cryosections from ΔΕχ50 mice injected with AAV-Tri-SA2 revealed significantly higher numbers of dystrophinpositive fibers with an average of 99% restoration of normal fibers (FIG. 12A, FIG. 13).

177

Western blot analysis confirmed the restoration of dystrophin expression in skeletal muscle. (FIG. 12C, FIG. 12D). Hematoxylin and eosin (H&E) staining of muscle showed that histopathologie hallmarks of muscular dystrophy, such as necrotic myofibers, were corrected in TA muscle at 3-weeks post-AAV delivery (FIG. 12B and FIG. 14). This method, using a distinct sgRNA design, represents a major advance in efficiency of DMD correction with direct applicability to the patients with the most common dystrophin mutations.

Rescue of dystrophin expression following intramuscular injections of AAV9-Cas9 combined with different AAV9s expressing single copy or triple copy of sgRNA. To evaluate the benefit of triple promoter expression of sgRNA-ex51-SA2 in vivo, different constructs were investigated, where sgRNA expression was driven by a single RNA polymerase III promoter (U6 or H1 or 7SK) and, separately, by three RNA polymerase III promoters (U6, H1 and 7SK) (FIG. 15A). The inventors delivered the sgRNA-ex51-SA2 in single copy driven separately by the U6 promoter (AAV9-U6-sgRNA-51-SA2), the H1 promoter (AAV9-Hl-sgRNA-51-SA2), the 7SK promoter (AAV9-7SK-sgRNA-51-SA2) and triple copy (AAV9-Triple-sgRNA-51-SA2). Following intra-muscular (IM) injection of P12 mice with AAV9s, muscle tissues were analyzed. Unexpectedly, dystrophin immunostaining of muscle cryosections from AEx50 mice injected with AAV9-Triple-sgRNA-51-SA2 revealed significantly higher numbers of dystrophin-positive fibers with an average of 95% restoration of normal fibers compared to AEx50 mice injected with AAV9-U6-sgRNA-51-SA2, AAV-H1 sgRNA-51-SA2 and AAV-7SK-sgRNA-51-SA2 with an average of 70%; 40% and 50% restoration of normal fibers respectively (FIGS. 15B).

Rescue of muscle structure and function following systemic delivery of AAV9Cas9 and AAV9-sgRNA-51-SA2. Systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51SA2 to P4 AEx50 mice yielded widespread dystrophin expression in the heart, triceps, tibialis anterior (TA) muscle, and diaphragm in gene-edited AEx50 mice at 4 and 8 weeks postinjection (FIG. 16A and FIG. 17A). Western blot analysis confirmed the restoration of dystrophin expression in skeletal and heart muscles (FIG. 16B and FIG. 17B). Grip strength testing also showed a significant increase in muscle strength of AEx50 mice at 4 weeks postintraperitoneal AAV9 injection compared to AEx50 control mice (wildtype control 92.6±1.63; AEx50 control 50.5±1.85; AEx50-AAV9-sgRNA-51 79.7±2.63) (FIG. 18A). Consistently, AAV9-sgRNA-51-SA2 gene-edited AEx50 mice also showed significant réductions in sérum CK concentrations compared to AEx50 control mice (FIG. 18B).

178

Correction of dystrophin expression in a dog model of Duchenne muscular dystrophy. To further assess the efficiency and therapeutic potential ofthis new approach, the inventors investigated the correction the disease-causing mutation in a dog model of DMD, the AE50-MD dog, which harbors a missense mutation in the 5’ donor splice site of exon 50 that results in délétion of exon 50 ((Walmsley et al., 2010). The AE50-MD dog is an idéal canine model for the investigation of gene-editing as an approach to permanently correct the most common DMD mutations in humans.

The inventors used sgRNA (sgRNA-ex51-SA2) targeting the same genomic locus of the splice accepter région. The sgRNA-ex51-SA2 sequence is highly conserved. The target sequences of the mouse sgRNA-ex51-SA2 and the dog sgRNA-ex51-SA2 (sgRNA-D-ex51 SA2) differ only by one single nucléotide (Table 4). The inventors delivered the sgRNA-Dex51-SA2 in triple copy (AAV-D-Tri-SA2). Following intra-muscular (IM) injection of 1 month old dogs with AAVs, muscle tissues were analyzed. Remarkably, dystrophin immunostaining of muscle cryosections from AEx50 mice injected with AAV-Tri-SA2 revealed significantly higher numbers of dystrophin-positive fibers (FIG. 19). Western blot analysis confirmed the restoration of dystrophin expression in skeletal muscle (FIG. 20). Hematoxylin and eosin (H&E) staining of muscle showed that histopathologie hallmarks of muscular dystrophy, such as necrotic myofibers, were diminished in cranial tibialis muscle at 6-weeks post-AAV delivery (FIG. 21). This method, using a distinct sgRNA design, represents a major advance in efficiency of DMD correction with direct applicability to the patients with the most common dystrophin mutations.

*************

B 179

Ail ofthe compositions and/or methods disclosed and claimed herein can be made and executed without undue expérimentation in light of the présent disclosure. While the compositions and methods of this disclosure hâve been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to 5 the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope ofthe disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. Ail such similar substitutes and modifications apparent to 10 those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended daims.

180

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Claims (39)

1. A nucleic acid encoding a single guide RNA (sgRNA), wherein the nucleic acid comprises a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises SEQ ID NO: 714.

2. A nucleic acid encoding a sgRNA, wherein the nucleic acid comprises a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises SEQ ID NO: 762.

3. A vector comprising the nucleic acid of claim 1 or 2.

4. The vector of claim 3, wherein the scaffold sequence is integrated into the backbone of the vector.

5. The vector of claim 3 or 4, wherein the vector comprises three copies of the nucleic acid encoding the sgRNA.

6. A composition comprising:

(i) the vector of any one of daims 3-5; and (ii) a second vector comprising a nucleic acid sequence encoding a Cas9 nuclease.

20

7. The composition of claim 6, wherein the vector and the second vector are both AAV vectors.

8. Use of (i) the vector of any one of daims 3-5; and (ii) a second vector comprising a nucleic acid sequence encoding a Cas9 nuclease

25 in the manufacture of a compositionjor treating muscular dystrophy in a subject in need thereof.

9. A nucleic acid comprising:

a sequence encoding a first DMD sgRNA targeting a first genomic target sequence, a sequence encoding a second DMD sgRNA targeting a second genomic target sequence, 30 a sequence encoding a first promoter, wherein the first promoter drives expression of the sequence encoding the first DMD sgRNA, and a sequence encoding a second promoter, wherein the second promoter drives expression of the sequence encoding the second DMD sgRNA,

190 ^wherein the first genomic target sequence and the second genomic target sequence each comprise a dystrophin splice acceptor site.

10. The nucleic acid of claim 9, wherein the nucleic acid further comprises:

5 a sequence encoding a third DMD sgRNA targeting a third genomic target sequence, and a sequence encoding a third promoter, wherein the third promoter drives expression of the sequence encoding the third DMD sgRNA, wherein the third genomic target sequence comprises a dystrophin splice acceptor site.

10 H. The nucleic acid of claim 10, wherein the sequences encoding the first, second, and third DMD sgRNA are the same, and wherein the first, second, and third genomic target sequences are the same.

12. The nucleic acid of claim 10, wherein the sequence encoding the first DMD sgRNA, the sequence encoding the second DMD sgRNA, or the sequence encoding the third DMD sgRNA comprises the 15 sequence of any one of SEQ ID NOs. 60-382 and 706-864.

13. The nucleic acid of claim 10, wherein the sequence encoding the first DMD sgRNA, the sequence encoding the second DMD sgRNA, or the sequence encoding the third DMD sgRNA comprises the sequence of SEQ ID NO: 714.

14. The nucleic acid of claim 10, wherein the sequence encoding the first DMD sgRNA, the sequence encoding the second DMD sgRNA, and the sequence encoding the third DMD sgRNA comprises the sequence of SEQ ID NO: 714.

25

15. The nucleic acid of claim 10, wherein the sequence encoding the first DMD sgRNA, the sequence encoding the second DMD sgRNA, or the sequence encoding the third DMD sgRNA comprises the sequence of SEQ ID NO: 762.

16. The nucleic acid of claim 9, wherein the sequence encoding the first DMD sgRNA, the sequence

30 encoding the second DMD sgRNA, and the sequence encoding the third DMD sgRNA comprises the sequence of SEQ ID NO: 762.

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17. The nucleic acid of any one of daims 10-16, wherein the first promoter, the second promoter, and the third promoter are each independently selected from the U6 promoter, the Hl promoter, and the 7SK promoter.

18. The nucleic acid of anyone of daims 10-17, wherein the first promoter is the U6 promoter, the second promoter is the Hl promoter, and the third promoter is the 7SK promoter.

19. The nucleic acid of any one of daims 9-18, wherein the nucleic acid further comprises one or more sequences encoding an inverted terminal repeat (ITR).

20. A vector comprising the nucleic acid of any one of daims 9-19.

21. The vector of claim 20, wherein the vector is a viral vector.

22. The vector of claim 21, wherein the viral vector is an adeno-associated viral (AAV) vector.

23. The vector of claim 22, wherein the AAV vector is an AAV9 vector.

24. A composition comprising the vector of any one of daims 20-23.

25. The composition of claim 24, further comprising a pharmaceutically acceptable carrier.

26. A cell comprising the composition of claim 24 or 25.

27. A nucleic acid comprising a promoter sequence and a sequence encoding a Cas9 protein or a nuclease domain thereof, wherein the promoter sequence is a muscle-specific promoter sequence.

28. The nucleic acid of claim 27, wherein the muscle-specific promoter is the CK8e promoter.

29. A vector comprising the nucleic acid of claim 27 or 28.

30. The vector of claim 29, wherein the vector is an AAV9 vector.

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31. A composition comprising the vector of claim 29 or 30.

32. A cell comprising the vector of claim 29 or 30.

33. A composition comprising:

a first nucleic acid comprising a nucleic acid of any one of claims 9-19 and a second nucleic acid comprising a nucleic acid of any one of claims 27-28.

34. A composition comprising a first vector comprising a nucleic acid of any one of claims 9-19, and a second vector comprising a nucleic acid of any one of claims 27-28.

35. A composition comprising a vector of any one of claims 20-23, and a vector of claim 29 or 30.

36. The composition of any one of claims 33-35, wherein the composition further comprises a pharmaceutically acceptable carrier.

37. Use of the vector of any one of claims 20-23; or the vector of claim 29 or 30; or a first nucleic acid comprising a nucleic acid of any one of claims 9-19 and a second nucleic acid comprising a nucleic acid of any one of claims 27-28; or a first vector comprising a nucleic acid of any one of claims 9-19 and a second vector comprising a nucleic acid of any one of claims 27-28; or a vector of any one of claims 20-23 and a vector of claim 29 or 30, in the manufacture of a composition for treating muscular dystrophy in a subject in need thereof.

38. Use of a first AAV vector comprising the nucleic acid of any one of claims 9-19 and a second AAV vector comprising the nucleic acid of any one of claims 29-30 in the manufacture of a composition for treating muscular dystrophy in a subject in need thereof.

39. The use of claim 38, wherein the first and the second AAV vector are each AAV9 vectors.

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A composition according to claim 6 or claim 7 for use in a method of treating muscular dystrophy in a subject in need thereof.

5 41. A composition according to any one of daims 24-25, 31, or 33-36, for use in a method of treating muscular dystrophy in a subject in need thereof.

42. A first AAV vector comprising the nucleic acid of any one of daims 9-19 and a second AAV vector comprising the nucleic acid of any one of daims 29-30 for use in a method of treating muscular dystrophy 10 in a subject in need thereof.