HK40100824A - Codon optimized rpgrorf 15 genes and uses thereof - Google Patents

Description

Codon-optimized RPGRORF15 gene and its applications

Cross-references to related applications

This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/073,843, filed September 2, 2020, the entire disclosure of which is incorporated herein by reference.

Submitting sequence lists via EFS-WEB

A computer-readable text file entitled “090400-5012-WO-Sequence-Listing” (approximately 37KB in size), created on or around August 11, 2021, contains the sequence list of this application, and is hereby incorporated herein by reference in its entirety.

Background of the Invention

X-linked retinitis pigmentosa (XLRP) is a relatively severe and genetically heterogeneous inherited retinal degeneration. Approximately 70% of XLRP cases are caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. The RPGR gene encodes several different alternative splicing transcripts that are widely expressed. The function of the encoded protein is not fully understood, but studies have shown that it plays an important role in cellular structures called cilia.

One RPGR isoform contains a unique 3' region called ORF15, a 567-amino acid-rich C-terminal domain rich in Gly and Glu. This version of the RPGR protein, containing exons 1-13 and the ORF15 region of the RPGR gene, is primarily expressed in photoreceptors in the retina. Mutations in the ORF15 region of RPGR account for approximately 60% of all XLRP cases.

Several preclinical studies support the use of wild-type cDNA of RPGRorf15 to rescue the XLRP disease phenotype. However, the poor sequence stability of the wild-type sequence poses a challenge to maintaining sequence integrity during vector production, and the suboptimal expression level of the wild-type sequence in human photoreceptors presents a challenge to gene therapy approaches for treating XLRP.

Invention Overview

Disclosed are codon-optimized nucleic acid molecules encoding a human retinitis pigmentosa GTPase regulatory factor (RPGR) protein. In one aspect, this disclosure provides a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1 or a nucleic acid comprising a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO:1, and said nucleic acid encoding a human RPGR polypeptide having the amino acid sequence of SEQ ID NO:2. In some embodiments, a nucleic acid comprising or composed of the nucleotide sequence of SEQ ID NO:1 is provided. In related embodiments, the nucleic acid is expressed at a higher level than in cells otherwise identical to those expressing a wild-type RPGR nucleic acid sequence (e.g., SEQ ID NO:3).

In some respects, the codon-optimized nucleic acid molecules described herein have a higher human codon fitness index relative to wild-type RPGRcDNA (GenBank search number NM_001034853; SEQ ID NO:3). In some embodiments, the codon-optimized nucleic acid molecules have a human codon fitness index of at least about 0.85, at least about 0.88, or at least about 0.89.

In some embodiments, the nucleic acid contains a higher percentage of G/C nucleotides compared to the percentage of G/C nucleotides in SEQ ID NO:3. In other embodiments, the nucleic acid contains a G/C nucleotide percentage of up to about 59%, up to about 58%, or up to about 57%. In some aspects, the average G/C content of the nucleic acid is from about 55% to about 59%, or from about 56% to about 58%. In some preferred embodiments, the average G/C content is about 57%.

In other embodiments, the nucleic acid relative to SEQ ID NO:3 includes one or more optimized parameters selected from the removal of negative cis-acting sites (including but not limited to TATA boxes and splice sites) and the enhancement of optimal codon frequencies.

In another embodiment, the nucleic acid is operatively linked to at least one transcriptional control sequence, preferably a transcriptional control sequence heterologous to the nucleic acid. In some aspects, the transcriptional control sequence is a cell- or tissue-specific promoter that results in cell-specific expression of the nucleic acid, such as in photoreceptor-specific human G protein-coupled receptor rhodopsin kinase 1 (hGRK) or human inter-photoreceptor retinol-binding protein (IRBP) promoters. In a preferred embodiment, the transcriptional control sequence comprises a human rod photoreceptor-specific human G protein-coupled receptor rhodopsin kinase 1 (hGRK) promoter. In other aspects, the transcriptional control sequence is a constitutive promoter that results in similar levels of nucleic acid expression in many cell types (e.g., CAG, CBA, CMV, or PGK promoters). In a preferred embodiment, the transcriptional control sequence comprises a human G protein-coupled receptor kinase (hGRK, also known as rhodopsin kinase) promoter as described in Young et al., Investigative Ophthalmology and Visual Science, 44(9):4076-4085 (2003). In a particularly preferred embodiment, the hGRK promoter comprises the sequence of SEQ ID NO:4 or comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

GGGCCCCAGAAGCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGTGAGGCGGCCCCT

TGGAGGAAGGGGCCGGGCAGAATGATCTAATCGGATTCCAAGCAGCTCAGGGGATTGTC

TTTTTCTAGCACCTTCTTGCCACTCCTAAGCGTCCTCCGTGACCCCGGCTGGGATTTAGCCTGGTGCTGTGTCAGCCCCGGG (SEQ ID NO: 4).

In related embodiments, this document provides an expression cassette comprising a nucleic acid operatively linked to an expression control sequence, said nucleic acid comprising the nucleotide sequence of SEQ ID NO:1 or a nucleotide sequence having at least 90% identity with it.

In related embodiments, vectors comprising nucleic acids are provided herein, said nucleic acids comprising the nucleotide sequence of SEQ ID NO:1 or a nucleotide sequence having at least 90% identity with it. In a preferred embodiment, the vector is a recombinant gland-associated (rAAV) expression vector. In some embodiments, the rAAV vector comprises a natural capsid (e.g., a capsid of AAV serotype 2, AAV serotype 5, or AAV serotype 8). In other embodiments, the rAAV vector comprises a capsid modified relative to the natural AAV capsid (e.g., comprising one or more peptide insertions and/or one or more amino acid substitutions (e.g., tyrosine to phenylalanine) and/or amino acid insertions or deletions) (e.g., comprising one or more modifications relative to the AAV capsid of serotype 2, 5, or 8).

In another embodiment, a host cell comprising nucleic acid is provided herein, said nucleic acid comprising the nucleotide sequence of SEQ ID NO:1 or a nucleotide sequence having at least 90% identity with it. In some aspects, the host cell is a mammalian cell, including but not limited to CHO cells, HEK293 cells, HeLa cells, BHK21 cells, Vero cells, or V27 cells. In related aspects, the host cell is selected from CHO cells, HEK293 cells, HEK293T cells, HeLa cells, BHK21 cells, and Vero cells. In other aspects, the host cell is a photoreceptor cell (e.g., rods; cones), retinal ganglion cells (RGCs), glial cells (e.g., Müller glial cells, microglia), bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelium (RPE) cells. In related embodiments, this disclosure provides a method for enhancing the expression of the polypeptide of SEQ ID NO:2, comprising culturing host cells under conditions in which the polypeptide of SEQ ID NO:2 is expressed in a nucleic acid molecule, wherein the expression of the polypeptide is enhanced relative to host cells cultured under the same conditions containing a reference nucleic acid containing the nucleotide sequence of SEQ ID NO:3 (comparison sequence).

In another embodiment, this disclosure provides a method for enhancing the expression of the polypeptide of SEQ ID NO:2 in a human subject, comprising administering to the subject an isolated nucleic acid molecule comprising a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO:1 and encoding a polypeptide having the amino acid sequence of SEQ ID NO:2, or a vector comprising such a nucleotide sequence, wherein the expression of the polypeptide is enhanced relative to a reference nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:3 (comparison sequence) or a vector comprising a reference nucleic acid molecule.

In some embodiments, this disclosure provides a method for treating an ocular condition associated with insufficient RGRP ORF15 activity in human subjects, comprising administering to the subject a nucleic acid molecule or vector disclosed herein. In some embodiments, the retinal condition is X-linked retinitis pigmentosa.

Attached Figure Description

Figure 1 shows the gel electrophoresis of the restriction digests from pAAV-GRK-cohRPGRorf15-SV40. Large-scale DNA preparations (Maxiprep DNA) were prepared by digestion with various enzymes and analyzed by agarose gel electrophoresis: lane 1 = 2-log ladder; lane 2 = BsrGI-H + BglII; lane 3 = Pml + Sph-HF; lane 4 = HindIII-HF + Sph-HF; lane 5 = Pst. The resulting restriction fragments matched the predicted fragments in all digests (lanes 2: 3.9, 2.5, 0.6 kb; lane 3: 3.7, 2.1, 1.3 kb; lane 4: 3.9, 1.7, 1.5 kb; lane 5: 4.6, 1.4, 1.2 kb). The dimensions of the prominent 2-log ladder in kilobase pairs are shown on the left side of the gel.

Figure 2 shows a Western blot of cell lysates from HEK293T cells transfected with pAAV-GRK-cohRPGRorf15-SV40. Human RPGrrf15 protein expression in HEK293 cells was assessed using the specified primary antibodies (Sigma; CT-15; Polyglut GT335). For each antibody, lane 1 = untransfected control; lane 2 = pAAV-GRK-cohRPGRorf15-SV40; lane 3 = pAAV-PGK-cohRPGRorf15-SV40. Arrows indicate hRPGRorf15 protein. Molecular weight markers (in kilodaltons) are shown on the left.

Figure 3 shows that transduction of recombinant AAV (rAAV) viral particles containing codon-optimized RPGrrf15 (SEQ ID NO:1) under the control of the hGRK1 promoter resulted in a strong increase in cohRPGRorf15 (SEQ ID NO:1) transcript levels in XLRP-iPSC-derived photoreceptor cultures. Thirty days post-transduction, RNA extracted from XLRP-iPSC-derived photoreceptor cultures was subjected to droplet digital PCR after transduction with rAAV containing a capsid of pAAV-GRK-cohRPGRorf15-SV40 and SEQ ID NO:9 at an MOI of 50,000. hRPGR1-19 (internal control) and cohRPGRorf15 transcript levels were determined and quantified as copies/mL above a set threshold, plotted on a logarithmic scale. Following transduction, codon-optimized hRPGRorf15 (SEQ ID NO:1) transcript levels were statistically greater than hRPGR1-19. NT = Untransduced, MOI = Multiple of infection, hRPGR1-19 = Exons 1-19 of human retinitis pigmentosa GTPase regulator, constitutive isoform, cohRPGRorf15 = Codon-optimized open reading frame 15 of human retinitis pigmentosa GTPase regulator, retina-specific isoform of SEQ ID NO:1. * p ≤ 0.05 compared with MOI 50,000 hRPGR1-19, p ≤ 0.05 compared with NT cohRPGRorf15. Error bars ± standard deviation. n = 3 per patient. Y-axis is expressed on a logarithmic scale.

Figure 4. Transduction with rAAV of RPGORf15, codon-optimized to SEQ ID NO:1 and controlled by the hGRK1 promoter, increased hRPGRorf15 protein levels in XLRP photoreceptor cultures. XLRP-iPSC-derived photoreceptor cultures were transduced at 50,000 MOI, and protein lysates were harvested 30 days post-transduction. SDS-PAGE and Western blot analysis showed an increase in hRPGRorf15 at 127 kDa (normalized relative to loaded control α-tubulin) in both patients compared to untransduced cells (NT). Band intensities were quantified and averaged across patients. Transduction with rAAV produced a significant increase in hRPGRorf15 protein. *p ≤ 0.05 compared to NT. Error bars ± standard deviation. n = 3 per patient.

Figure 5. Glutonylation of hRPGRorf15 after rAAV transduction with codon-optimized RPGRorf15 containing the hGRK promoter in XLRP photoreceptor cultures. XLRP-iPSC-derived photoreceptor cultures were transduced at an MOI of 50,000, and protein lysates were harvested 30 days post-transduction. SDS-PAGE and Western blot analysis showed increased glutonylation of the 127 kDa protein hRPGRorf15 (normalized relative to loaded control α-tubulin) in both patients compared to the untransduced (NT) control, with quantified band intensities averaged across patients. The significant increase in hRPGRorf15 glutonylation was observed during rAAV transduction. GT335 = anti-glutamylating antibody, NT = untransduced, MOI = multiplicity of infection, hRPGRorf15 = human retinitis pigmentosa GTPase regulator open reading frame 15, retina-specific isoform. * p ≤ 0.05 compared to NT. Error bars ± standard deviation. n = 3 per patient.

Figure 6. Constitutive promoter-driven increase in hRPGRorf15 protein and glutamylation in XLRP photoreceptor cultures. XLRP-iPSC-derived photoreceptor cultures were transduced with rAAV of RPGRorf15 codon-optimized to SEQ ID NO:1, controlled by the PGK promoter, at MOIs of 5,000, 10,000, and 20,000. Protein lysates were harvested 30 days post-transduction. SDS-PAGE and Western blot analysis showed increased hRPGRorf15 and glutamylation at 127 kDa (normalized relative to loaded control α-tubulin) in patient 78 compared to the untransduced (NT) control. Band intensity was quantified. Transduction resulted in a significant increase in hRPGRorf15 protein. NT = Untransduced, MOI = Multiple of infection, hRPGRorf15 = Human retinitis pigmentosa GTPase regulatory factor open reading frame 15, retinal-specific isoform, GT335 = Anti-glutamylating antibody. *p≤0.05 compared to NT. Error bars ± standard deviation. n=3.

Figure 7 shows the codon-optimized sequence and encoded amino acid sequence of SEQ ID NO:1.

Figure 8 is a schematic diagram of the transgenic cassette contained in the rAAV described in the examples below. The transgenic cassette contains a 5'AAV2ITR, a human rhodopsin kinase (hGRK) promoter, codon-optimized human RPGRorf15 cDNA of SEQ ID NO:1, a late SV40 polyadenylation signal, and a 3'AAV2ITR, and has the nucleotide sequence of SEQ ID NO:5.

Figure 9 illustrates the safety of 4D-125 (containing the transgenic cassette shown in Figure 8 and the capsid protein of SEQ ID NO: 9) for quantifying ocular inflammation, as assessed by aqueous flare, aqueous cells, and vitreous cells. Transient, mild ocular inflammation with fundus signs was observed at high doses. These changes were in response to increased systemic steroid treatment. No adverse findings were considered to be associated with 4D-125. IOP values in all animals were within normal limits at different examination intervals. ERG values and OCT images including macular morphology were also within normal limits.

Figure 10 shows the vector genomic biodistribution in selected retinal, ocular, and non-ocular tissues at three autopsy time points after intravitreal administration of 4D-125 NHP. LOD = Limit of Detection; for visualization purposes, all samples are plotted as LOD values.

Figure 11 shows the expression of RPGR transgenic mRNA in selected retinal, ocular, and non-ocular tissues at three autopsy time points after intravitreal administration of 4D-125 NHP, as measured by RT-qPCR. LOD = Limit of Detection; for visualization purposes, all samples are plotted as LOD values.

Invention Details

definition

The term "codon fitness index" as used in this paper refers to a measure of codon usage bias. The codon fitness index (CAI) measures the deviation of a given protein-coding gene sequence from a reference gene set (Sharp PM and Li WH, Nucleic Acids Res. 15(3):1281-95(1987)). CAI is calculated by determining the geometric mean (in codon measure) of the weights associated with each codon along the gene sequence length:

For each amino acid, the weight of each of its codons (in CAI) is calculated as the ratio between the observed codon frequency (fi) and the synonymous codon frequency (fj) for that amino acid:

The term "isolated" refers to biological material (cells, nucleic acids, or proteins) that has been removed from its original environment (the environment in which it naturally exists). For example, polynucleotides that exist naturally in plants or animals are not isolated, but the same polynucleotides isolated from their naturally occurring neighboring nucleic acids are considered "isolated".

The term "4D-125" refers to recombinant AAV particles comprising (i) a capsid protein containing the amino acid sequence of SEQ ID NO:9 and a heterologous nucleic acid containing the nucleotide sequence of SEQ ID NO:5.

The term "R100" refers to the variant AAV capsid protein containing the amino acid sequence of SEQ ID NO:9.

As used herein, a “coding region” or “coding sequence” is a portion of a polynucleotide consisting of codons that can be translated into amino acids. While “stop codons” (TAG, TGA, or TAA) are not typically translated into amino acids, they can be considered part of a coding region, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of a coding region. The boundaries of a coding region are typically determined by the start codon at the 5' end of the amino terminus of the resulting polypeptide and the translation stop codon at the 3' end of the carboxyl terminus of the resulting polypeptide. Two or more coding regions can exist in a single polynucleotide construct, such as on a single vector, or in separate polynucleotide constructs, such as on separate (different) vectors. Therefore, a single vector can contain only a single coding region or contain two or more coding regions.

As used herein, the term "regulatory region" refers to a nucleotide sequence located upstream (5' non-coding sequence), inside, or downstream (3' non-coding sequence) of a coding region that influences transcription, RNA processing, stability, or translation of the relevant coding region. Regulatory regions may include promoters, translational leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. If the coding region is intended for expression in eukaryotic cells, the polyadenylation signal and transcription termination sequence will typically be located at the 3' end of the coding sequence.

The term “nucleic acid” as used in this article may be used interchangeably with “polynucleotide” or “nucleic acid molecule” and is intended to refer to a polymer of nucleotides.

Polynucleotides encoding gene products (e.g., polypeptides) may include promoters and/or other transcriptional or translational control elements operatively associated with one or more coding regions. In an operative association, the coding region of a gene product (e.g., a polypeptide) is associated with one or more regulatory regions in a manner that places the expression of the gene product under the influence or control of one or more regulatory regions. For example, the coding region and the promoter are “operatively associated” if induction of promoter function leads to transcription of mRNA encoding the gene product encoded by the coding region, and if the nature of the connection between the promoter and the coding region does not interfere with the promoter’s ability to direct gene product expression or the ability to transcribe a DNA template. In addition to promoters, other transcriptional control elements (e.g., enhancers, operons, repressors, and transcription termination signals) may also be operatively associated with coding regions to direct gene product expression.

"Transcriptional control sequences" refer to DNA regulatory sequences such as promoters, enhancers, and terminators that provide for the expression of coding sequences in host cells. Various transcriptional control regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (an immediate early promoter that binds to intron A), simian virus 40 (an early promoter), and retroviruses (such as Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes (such as actin, heat shock protein, bovine growth hormone, and rabbit β-globin), as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers, and lymphokine-inducible promoters (e.g., promoters that can be induced by interferon or interleukin).

Similarly, a variety of translation control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation start and stop codons, and elements derived from microRNAs (particularly internal ribosome entry sites or IRES, also known as CITE sequences).

As used herein, the term “expression” refers to the process by which polynucleotides produce gene products (e.g., RNA or polypeptides). This includes, but is not limited to, the transcription of polynucleotides into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and the translation of mRNA into polypeptides. Expression produces “gene products.” As used herein, gene products can be nucleic acids (e.g., messenger RNA produced through gene transcription) or polypeptides translated from transcripts. Gene products described herein further include nucleic acids with post-transcriptional modifications (e.g., polyadenylation or splicing) or polypeptides with post-translational modifications (e.g., methylation, glycosylation, addition of lipids, association with other protein subunits, or proteolytic cleavage).

"Vector" refers to any medium used to clone and/or transfer nucleic acids into host cells. A vector can be a replicon to which another nucleic acid segment can be ligated to induce replication of the ligated segment. The term "vector" includes viral and nonviral media for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. A large number of vectors are known and used in the art, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be achieved by ligating a suitable polynucleotide fragment into a selected vector having complementary sticky ends.

Vectors can be engineered to encode selective markers or reporters that provide selection or identification of cells incorporated into the vector. Expression of selective markers or reporters enables the identification and/or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selective marker genes known and used in the art include genes that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, diammonium phosphate herbicide, sulfonamides, etc.; and genes used as phenotypic markers, such as anthocyanin regulatory genes, isopentanyl transferase genes, etc. Examples of reporters known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selective markers can also be considered as reporters.

Eukaryotic viral vectors that can be used include, but are not limited to, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, poxviruses (e.g., vaccinia virus vectors), baculovirus vectors, or herpesvirus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytotransfectants), DNA-protein complexes, and biopolymers.

The terms "promoter" and "promoter sequence" are used interchangeably and refer to the DNA sequence that controls the expression of a coding sequence or functional RNA. Typically, the coding sequence is located at the 3' end of the promoter sequence. Promoters can be entirely derived from natural genes, or composed of different elements from different promoters found in nature, or even contain synthetic DNA segments. Those skilled in the art will understand that different promoters can direct gene expression in different tissues or cell types, at different developmental stages, or in response to different environmental or physiological conditions. Promoters that cause gene expression in most cell types most of the time are generally called "constitutive promoters." Promoters that cause gene expression in specific cell types are generally called "cell-specific promoters" or "tissue-specific promoters." Promoters that cause gene expression at specific developmental or cell differentiation stages are generally called "development-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced to express genes after exposure or treatment of cells with promoter-inducing agents, biomolecules, chemicals, ligands, light, etc., are generally called "inducible promoters" or "regulatory promoters." It was further recognized that, since the exact boundaries of the regulatory sequence are not fully determined in most cases, DNA fragments of different lengths can have the same promoter activity.

The term "plasmid" refers to an extrachromosomal element that typically carries a gene that is not part of the cell's central metabolism and is usually in the form of a circular double-stranded DNA molecule. Such elements can be linear, circular, or supercoiled autonomously replicating sequences, genome-integrated sequences, bacteriophage sequences, or nucleotide sequences derived from any source of single-stranded or double-stranded DNA or RNA. Many of these nucleotide sequences have been linked or reassembled into a unique construct capable of introducing promoter fragments and the DNA sequence of selected gene products into the cell along with appropriate 3' untranslated sequences.

A certain percentage of “sequence identity” between a polynucleotide or polypeptide and another polynucleotide or polypeptide means that, during alignment, that percentage of bases or amino acids are identical when comparing the two sequences. Sequence similarity can be determined in many different ways. To determine sequence identity, sequences can be aligned using methods and computer programs including BLAST, which is available online at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, which is available in the Genetics Computing Group (GCG) package from Madison, Wis., USA. Other techniques for alignment are described in Methods in Enzymology, Vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc. Of particular interest are alignment procedures that allow for gaps in the sequence. Smith-Waterman is one type of algorithm that allows for gaps in sequence alignment. See Meth.Mol.Biol.70:173-187 (1997). Additionally, the GAP procedure using the Needleman and Wunsch alignment methods can be used for sequence alignment. See J.Mol.Biol.48:443-453 (1970).

In one embodiment, the present invention provides a modified nucleic acid molecule comprising the nucleotide sequence encoding the polypeptide (human RGPGR ORF15) of SEQ ID NO:2, wherein the nucleic acid sequence has been codon-optimized. In another embodiment, the starting nucleic acid sequence encoding the polypeptide of SEQ ID NO:2 and codon-optimized has the nucleotide sequence shown in SEQ ID NO:3. In a preferred embodiment, the sequence encoding the polypeptide of SEQ ID NO:2 is codon-optimized for human expression. SEQ ID NO:1 is a codon-optimized version of SEQ ID NO:3 optimized for human expression: ATGAGAGAACCCGAGGAACTGATGCCCGACTCTGGCGCCGTGTTTACCTTCGGCAAGAGCAAGTTCGCCGAGAACAACCCCGGCAAGTTCTGGTTCAAGAACGACGTGCCAGTGCACCTGAGCTGCGGAGATGAACACTCTGCCGTGGTCACCGGCAACAACAAGCTGTACATGTTCGGCAGCAACAACTGGGGCCAGCTCGGCCTGGGATCTAAGTCTGCCATCAGCAAGCCTACCTGCGTGAAGGCCCTGAAGCCTGAGAAAGTGAAACTGGC CGCCTGCGGCAGAAATCACACCCTGGTTTCTACCGAAGGCGGCAATGTGTATGCCACCGGCGGAAACAATGAGGGACAGCTTGGACTGGGCGACACCGAGGAAAGAAACACCTTCCACGTGATCAGCTTTTTTCACCAGCGAGCACAAGATCAAGCAGCT GAGCGCCGGCTCTAATACCTCTGCCGCTCTGACAGAGGACGGCAGACTGTTTATGTGGGGCGACAATTCTGAGGGCCAGATCGGACTGAAGAACGTGTCCAATGTGGTGCGTGCCCCAGCAAGTGACAATCGGCAAGCCTGTGTCTTGGATCAGCTGCGG CTACTACCACAGCGCCTTTGTGACAACCGATGGCGAGCTGTATGTGTTCGGCGAGCCAGAGAATGGCAAGCTGGGACTGCCTAACCAGCTGCTGGGCAATCACAGAACCCCTCAGCTGGTGTCTGAGATCCCCGAAAAAGTGATCCAGGTGGCCTGTGG CGGAGAGCACACAGTGGTGCTGACAGAGAATGCCGTGTACACCTTTGGCCTGGGCCAGTTTGGACAACTCGGACTGGGAACCTTCCTGTTCGAGACAAGCGAGCCCAAAGTGATCGAGAACATCCGGGACCAGACCATCAGCTACATCAGCTGTGGCGA GAACCACACAGCCCTGATCACAGACATCGGCCTGATGTACACATTCGGCGACGGAAGGCATGGAAAGCTCGGACTTGGCCTGGAAAACTTCACCAACCACTTCATCCCTACGCTGTGCAGCAACTTCCTGCGGTTTCATTGTGAAGCTGGTGGCCTGCGG AGGATGCCACATGGTGGTTTTTGCTGCCCCTCACAGAGGCGTGGCCAAAGAGATTGAGTTCGACGAGATCAACGATAACCTGCCTGAGCGTGGCCACCTTCCTGCCTTACAGCAGCCTGACATCTGGCAACGTGCTGCAGAGGACACTGAGCGCCAGAAT

GCGCAGACGGGAAAGAGAGAGAAGCCCCGACAGCTTCAGCATGAGAAGAACCCTGCC

TCCAATCGAGGGCACACTGGGCCTGTCTGCCTGCTTTCTGCCTAACAGCGTGTTCCCCA

GATGCAGCGAGAGAAACCTGCAAGAGAGCGTGCTGAGCGAGCAGGATCTGATGCAGCC

TGAGGAACCCGACTACCTGCTGGACGAGATGACCAAAGAGGCCGAGATCGACAACAGC

AGCACAGTGGAAAGCCTGGGCGAGACAACCGACATCCTGAACATGACCCACATCATGA

GCCTGAACAGCAACGAGAAGTCTCTGAAGCTGAGCCCCGTGCAGAAGCAGAAGAAGC

AGCAGACCATCGGCGAGCTGACACAGGATACTGCCCTGACCGAGAACGACGACAGCGA

CGAGTACGAAGAGATGAGCGAGATGAAGGAAGGCAAGGCCTGCAAGCAGCACGTGTC

CCAGGGCATCTTTATGACCCAGCCTGCCACCACCATCGAGGCCTTTTCCGACGAGGAAG

TGGAAATCCCCGAGGAAAAAGAGGGCGCCGAGGACAGCAAAGGCAACGGCATTGAGG

AACAAGAGGTGGAAGCCAACGAAGAGAACGTGAAGGTGCACGGCGGACGGAAAGAA

AAGACCGAGATCCTGAGCGACGACCTGACCGATAAGGCCGAGGTTTCCGAGGGCAAAG

CCAAGTCTGTGGGAGAAGCCGAGGATGGACCTGAAGGCCGCGGAGATGGAACCTGTGA

AGAAGGATCTAGCGGAGCCGAGCACTGGCAGGATGAGGAACGCGAGAAGGGCGAGAA

AGACAAAGGCAGAGGCGAGATGGAAAGACCCGGCGAGGGCGAAAAAGAGCTGGCCG

AGAAAGAGGAATGGAAGAAACGCGACGGCGAAGAACAAGAGCAGAAAGAAAGAGAG

CAGGCCACCAGAAAGAACGGAATCAAGAGATGGAAGAAGGCGGCGAGGAAGAACAC

GGCGAAGGGGAAGAAGAGGAAGGCGACCGAGAGGAAGAAGAAGAGAAAGAAGGCG

AAGGCAAAGAAGAAGGCGAGGGCGAAGAGGTGGAAGGCGAGCGTGAAAAAGAAGAG

GGCGAACGCAAGAAAGAAGAACGCGCCGGAAAAGAGGAAAAAGGCGAGGAAGAGGG

CGACCAAGGCGAAGGCGAGGAAGAAGAAACTGAAGGCAGAGGGGAAGAGAAAGAGG

AAGGCGGCGAAGTCGAAGGCGGAGAGGTTGAAGAAGGCAAAGGCGAGCGAGAAGAG

GAAGAAGAAGAAGGCGAAGGCGAGGAAGAGGAAGGCGAAGGCGAAGAGGAAGAAG

GCGAAGGGGAAGAAGAAGAAGGCGAAGGCAAGGGCGAAGAGGAGGGCGAAGAAGGC

GAGGGCGAAGAGGAGGGCGAAGAAGGCGAAGGCGAGGGCGAAGAAGAAGAAGGCGA

AGGCGAAGGCGAGGAAGAAGGCGAAGGCGAAGGGGAAGAAGAAGGAAGCGAAGGCG

AAGGCGAAGAAGAAGGCGAAGGCGAGGGCGAAGAGGAAGAAGGCGAAGGCAAAGGG

GAAGAAGAAGGCGAGGAAGGCGAAGGCGAAGGCGAGGAAGAAGAAGGCGAAGGCGA

GGGCGAAGATGGCGAAGGCGAAGGCGAAGAGGAAGAGGGCGAGTGGGAGGGCGAAG

AAGAGGAAGGCGAAGGCGAGGGCGAAGAGGAAGGCGAAGGCGAGGGCGAAGAAGGC

GAAGGCGAAGGCGAGGAAGAGGAAGGCGAAGGCGAAGGGGAAGAAGAAGAGGGCG

AAGAAGAAGGCGAAGAGGAAGGCGAAGGGGAAGAAGAAGGCGAAGGCGAAGGCGA

AGAAGAGGAAGAGGGCGAAGTTGAAGGCGAGGTTGAGGGCGAAGAAGGCGAAGGCG

AAGGGGAAGAAGAAGAAGGCGAGGAAGAAGGGGAAGAGAGAGAAAAAGAAGGCGA

GGGCGAAGAAAACCGCCGGAACCGCGAAGAGGAAGAGGAAGAAGAGGGCAAGTACC

AAGAGACTGGCGAGGAAGAGAACGAGCGGCAGGATGGCGAAGAGTACAAGAAGGTGT

CCAAGATCAAGGGCAGCGTGAAGTACGGCAAGCACAAGACCTACCAGAAGAAGTCCGT

CACCAACACGCAAGGCAATGGAAAAGAACAGCGGAGCAAGATGCCCGTGCAGTCCAA

GAGGCTGCTGAAGAATGGCCCTAGCGGCAGCAAGAAATTCTGGAACAATGTGCTGCCCCACTACCTCGAGCTGAAGTGA (SEQ ID NO: 1).

In some implementations, a codon-optimized sequence encoding human RPGR ORF15 is provided, which lacks the TGA stop codon of SEQ ID NO:1 (i.e., consists of nucleotides 1-3456 of SEQ ID NO:1).

In one aspect, this disclosure provides a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1, or a polynucleotide comprising a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO:1, and said polynucleotide encoding a human RPGR polypeptide having the amino acid sequence of SEQ ID NO:2:

MREPEELMPDSGAVFFTFGKSKFAENNPGKFWFKNDVPVHLSCGDEHSAVVTGNNKLYMFG

SNNWGQLGLGSKSAISKPTCVKALKPEKVKLAACGRNHTLVSTEGGNVYATGGNNEGQLG

LGDTEERNTFHVISFFTSEHKIKQLSAGSNTSAALTEDGRLFMWGDNSEGQIGLKNVSNVC

VPQQVTIGKPVSWISCGYYHSAFVTTDGELYVFGEPENGKLGLPNQLLGNHRTPQLVSEIPE

KVIQVACGGEHTVVLTENAVYTFGLGQFGQLGLGTFLFETSEPKVIENIRDQTISYISCGENH

TALITDIGLMYTFGDGRHGKLGLGLENFTNHFIPTLCSNFLRFIVKLVACGGCHMVVFAAPH

RGVAKEIEFDEINDTCLSVATFLPYSSLTSGNVLQRTLSARMRRRERERSPDSFSMRRTLPPIE

GTLGLSACFLPNSVFPRCSERNLQESVLSEQDLMQPEEPDYLLDEMTKEAEIDNSSTVESLG

ETTDILNMTHIMSLNSNEKSLKLSPVQKQKKQQTIGELTQDTALTENDDSDEYEEMSEMKE

GKACKQHVSQGIFMTQPATTIEAFSDEEVEIPEEKEGAEDSKGNGIEEQEVEANEENVKVHG

GRKEKTEILSDDLTDKAEVSEGKAKSVGEAEDGPEGRGDGTCEEGSSGAEHWQDEEREKG

EKDKGRGEMERPGEGEKELAEKEEWKKRDGEEQEQKEREQGHQKERNQEMEEGGEEEH

GEGEEEEGDREEEEEKEGEGKEEGEGEEVEGEREKEEGERKKEERAGKEEKGEEEGDQGE

GEEEETEGRGEEKEEGGEVEGGEVEEGKGEREEEEEEGEGEEEEGEGEEEEGEGEEEEGEG

KGEEEGEEGEGEEEGEEGEGEGEEEEGEGEGEEEGEGEGEEEEGEGEGEEEGEGEGEEEEG

EGKGEEEGEEGEGEGEEEEGEGEGEDGEGEGEEEEGEWEGEEEEGEGEGEEEGEGEGEEG

EGEGEEEEGEGEGEEEEGEEEGEEEGEGEEEGEGEGEEEEEEGEVEGEVEGEEGEGEGEEEE

GEEEGEEREKEGEGEENRRNREEEEEEEGKYQETGEEENERQDGEEYKKVSKIKGSVKYGKHKTYQKKSVTNTQGNGKEQRSKMPVQSKRLLKNGPSGSKKFWNNVLPHYLELK(SEQ ID NO:2).

The term "codon-optimized," when applied to the genes or coding regions of nucleic acid molecules used to transform various hosts, refers to altering the codons in the genes or coding regions of a nucleic acid molecule to reflect the typical codon usage of the host organism without changing the polypeptide encoded by DNA. Such optimization involves replacing at least one, more than one, or a large number of codons with one or more codons that are more frequently used in the organism's genes.

Deviations in the nucleotide sequence containing codons that encode amino acids in any polypeptide chain allow for variations in the sequence encoding genes. Since each codon consists of three nucleotides, and the nucleotides that make up DNA are limited to four specific bases, there are 64 possible nucleotide combinations, 61 of which encode amino acids (the remaining three codons encode signals that terminate translation). The “genetic code” showing which codons encode which amino acids is reproduced in Table 1 herein. Therefore, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are encoded by four triplets, serine and arginine by six, while tryptophan and methionine are encoded by only one triplet. This degeneracy allows for a wide range of variations in the base composition of DNA without altering the amino acid sequence of the protein encoded by the DNA.

Table US-00001 Table 1 Standard Genetic Code T C A G T TTT Phe(F) TCT Ser(S) TAT Tyr(Y) TTG TCys(C) TTC Phe(F) TCC Ser(S) TAC Tyr(Y) TGC TTA Leu(L) TCA Ser(S) TAA Stop TGA Stop TTG Leu(L) TCG Ser(S) TAG Stop p TGG Trp(W)C CTT Leu(L)CCT Pro(P)CAT His(H)CGTArg(R)CTC Leu(L)CCC Pro(P)CAC His(H )CGC Arg(R)CTALeu(L)CCA Pro(P)CAA Gln(Q)CGA Arg(R)CTG Leu(L)CCG Pro(P)CAG Gln(Q)CGG Arg(R)A ATT Ile(I)ACT Thr(T)AATAsn(N)AGT Ser(S)ATC Ile(I)ACC Thr(T)AAC Asn(N)AGC Ser(S)ATA Ile(I)ACA Thr(T)AAA Lys(K)AGA Arg(R)ATG Met(M)ACG Thr(T)AAG Lys(K)AGG Arg (R)G GTT Val(V)GCTAla(A)GAT Asp(D)GGT Gly(G)GTC Val(V)GCC Ala(A)GAC Asp(D)GGC Gly( G)GTA Val(V)GCA Ala(A)GAA Glu(E)GGA Gly(G)GTG Val(V)GCG Ala(A)GAG Glu(E)GGG Gly(G).

Many organisms exhibit a bias in using specific codons to encode the insertion of specific amino acids into the growing peptide chain. Codon bias, or codon skewness (the difference in codon use between organisms), is provided by the degeneracy of the genetic code and is well-documented in many organisms. Codon bias is generally associated with the translation efficiency of messenger RNA (mRNA), which is thought to depend in particular on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of the selected tRNA in a cell generally reflects the most frequently used codons in peptide synthesis. Therefore, genes can be tailored based on codon optimization to achieve optimal gene expression in a given organism.

Given the vast number of gene sequences available for use in a wide variety of animal, plant, and microbial species, the relative frequencies of codon usage have been calculated. Codon usage tables are available, for example, at the "Codon Usage Database," which can be found at www.kazusa.or.jp/codon/ (accessed June 18, 2012). See Nakamura, Y. et al., Nucl. Acids Res. 28:292 (2000).

Randomly assigning codons at optimized frequencies to encode a given polypeptide sequence can be done manually by calculating the codon frequency for each amino acid and then randomly assigning codons to the polypeptide sequence. Alternatively, various algorithms and computer software programs can be used to calculate the optimal sequence.

Non-viral vector

In some embodiments, a non-viral vector (e.g., an expression plasmid) comprising the modified nucleic acid described herein is provided. Preferably, the non-viral vector is a plasmid comprising the nucleic acid sequence of SEQ ID NO:1 or a sequence having at least 90% identity with it.

Viral vector

In a preferred embodiment, a viral vector comprising the modified (codon-optimized) nucleic acid described herein is provided. Preferably, the viral vector comprises a nucleic acid sequence operatively linked to, or having at least 90% identity with, the expression control sequence of SEQ ID NO:1. Examples of suitable viral vectors include, but are not limited to, adenovirus, retrovirus, lentivirus, herpesvirus, and adeno-associated virus (AAV) vectors.

In a preferred embodiment, the viral vector comprises a portion of the parvovirus genome, such as a deleted and/or modified RPGrf15 gene sequence and its associated expression control sequences replacing the AAV genome. The modified human RPGrf15 gene sequence typically has one or two adjacent (i.e., flanked) AAV TR or TR element insertions sufficient for viral replication (Xiao et al., 1997, J.Virol. 71(2):941-948), replacing the nucleic acids encoding the viral rep and cap proteins. Additional regulatory sequences suitable for promoting tissue-specific expression of the modified RPGrf15 gene sequence in target cells may also be included.

In some preferred embodiments, the AAV viral vector comprises nucleic acid comprising, from 5' to 3': (a) an AAV2 terminal repeat, (b) an hGRK promoter, (c) a codon-optimized RPGRorf15 gene as described herein, (d) a polyadenylated sequence, and (e) an AAV2 terminal repeat. In a particularly preferred embodiment, the AAV viral vector comprises nucleic acid (transgenic cassette) comprising the sequence of SEQ ID NO:5 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity with it.

(SEQ ID NO:5).

The components of the transgenic cassette of SEQ ID NO:5 and their corresponding locations are identified in Table 2 below:

Table 2

Location (bp) Components Length (bp) 1-145 5’ITR 145 170-368 GRK promoter 199 398-3856 RPGRorf15 cDNA 3459 3899-4143 SV40 Poly A 245 4159-4304 3’ITR 145

The 5'ITR has the following sequence:

TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO: 6)

The 3'ITR has the following sequence:

AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA(SEQ ID NO:7)

The SV40 polyadenylated sequence has the following sequence:

GGGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAAT AAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCA (SEQ ID NO: 8).

Those skilled in the art will understand that AAV vectors containing transgenes but lacking viral proteins (e.g., cap and rep) required for viral replication cannot replicate, as such proteins are essential for viral replication and packaging. Helper viruses typically include adenoviruses or herpes simplex viruses. Alternatively, as described below, helper functions (E1a, E1b, E2a, E4, and VA RNA) can be provided to packaging cells, including by transfecting the cells with one or more nucleic acids encoding various helper elements and/or by the cells containing nucleic acids encoding helper proteins. For example, HEK 293 is produced by transforming human cells with adenovirus 5 DNA and now expresses a large number of adenovirus genes, including but not limited to E1 and E3 (see, for example, Graham et al., 1977, J. Gen. Virol. 36:59-72). Thus, those helper functions can be provided by HEK 293 packaging cells without the need to supply them to the cells via, for example, plasmids encoding them.

The viral vector can be any suitable nucleic acid construct, such as DNA or RNA constructs, and can be single-stranded, double-stranded, or double-stranded (i.e., self-complementary as described in WO 2001/92551).

The viral capsid assembly of the packaged viral vector can be a small viral capsid. AAV caps and chimeric capsids are preferred. For example, the viral capsid can be an AAV capsid (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAVrh10, AAVrh74, RHM4-1, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAV-LK03, snake AAV, bird AAV, bovine AAV, canine AAV, horse AAV, sheep AAV, goat AAV, shrimp AAV, and any other AAV currently known or later discovered). See, for example, Fields et al., VIROLOGY, Vol. 2, Chapter 69 (4.sup.th ed., Lippincott-Raven Publishers).

In some embodiments, the viral capsid component of the packaged viral vector is a variant of the natural AAV capsid (i.e., containing one or more modifications relative to the natural AAV capsid). In some embodiments, the capsid is a variant of the AAV2, AAV5, or AAV8 capsid. In a preferred embodiment, the capsid is a variant of the AAV2 capsid, such as those described in U.S. Patent Application Publication No. 2019/0255192A1 (e.g., containing the amino acid sequence of any of SEQ ID NO: 42-59). In a particularly preferred embodiment, the capsid comprises a VP1 capsid protein having the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKAAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGC LPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRL QFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMIT DEEEIRTTNPVATEQYGSVSTNLQRGNLAISDQTKHARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIK NTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL(SEQ ID NO:9).

The variant AAV capsid protein of SEQ ID NO:9 contains the following modifications relative to the native AAV2 capsid: (i) a proline (P) to alanine (A) mutation at amino acid position 34 inside the assembled capsid (VP1 protein only), and (ii) an insertion of 10 amino acids (leucine-alanine-isoleucine-serine-aspartic acid-glutamine-threonine-lysine-histidine-alanine/LAISDQTKHA) at amino acid position 588 in VP1, VP2 and VP3.

The complete AAV Cap protein consists of VP1, VP2, and VP3. An ORF containing the nucleotide sequence encoding the AAV VP capsid protein may contain less than the complete AAV Cap protein or may provide the complete AAV Cap protein.

In another embodiment, the present invention provides the use of ancestral AAV vectors for therapeutic in vivo gene therapy. Specifically, computer-derived sequences were synthesized de novo and their biological activity was characterized. This effort resulted in the generation of nine functionally presumed ancestral AAVs and the identification of the predicted ancestor Anc80 for AAV serotypes 1, 2, 8, and 9 (Zinn et al., 2015, Cell Reports 12:1056-1068). In addition to assembly into viral particles, the prediction and synthesis of such ancestral sequences can also be achieved using the methods described in WO2015/054653, the contents of which are incorporated herein by reference. Notably, viral particles assembled from ancestral viral sequences can exhibit reduced sensitivity to pre-existing immunity in the current population compared to contemporary viruses or portions thereof.

This invention includes packaging cells encompassed by a “host cell”, which can be cultured to produce the packaging viral vector of this invention. The packaging cells of this invention typically comprise cells having heterologous (1) viral vector function, (2) packaging function, and (3) helper function. Each of these component functions is discussed in the following sections.

Initially, the vector can be manufactured using several methods known to those skilled in the art (see, for example, WO 2013/063379). A preferred method is described in Grieger et al., 2015, Molecular Therapy 24(2):287-297, the contents of which are incorporated herein by reference for all purposes. Briefly, efficient transfection of HEK293 cells is used as the starting point, wherein adherent HEK293 cell lines from a qualified clinical master cell bank are grown in shake flasks and in a WAVE bioreactor that allows for rapid and scalable production of rAAV under animal component-free suspension conditions. Using a triple transfection method (e.g., WO96/40240), suspension HEK293 cell lines yield more than ^10⁵ particles (vg)/cell containing the vector genome or more than ^10¹⁴ vg/L of cell culture at harvest 48 hours post-transfection. More specifically, triple transfection refers to the fact that packaging cells are transfected with three different plasmids: one plasmid encodes the AAV rep and cap genes, another plasmid encodes various auxiliary functions (such as adenovirus or HSV proteins, such as E1a, E1b, E2a, E4, and VA RNA), and a third plasmid encodes the transgene and its various control elements (such as the modified RPGRorf15 gene and the hGRK promoter).

To achieve the desired yield, numerous variables were optimized, such as the selection of a compatible serum-free suspension medium supporting both growth and transfection, transfection reagents, transfection conditions, and cell density. A universal purification strategy based on ion-exchange chromatography was also developed, yielding high-purity carriers for AAV serotypes 1–6, 8, 9, and various chimeric capsids. This user-friendly process can be completed within one week, producing a high full-particle to empty-particle ratio (>90% full-particle), providing purified yields (>1 × ^10¹³ vg/L) and purity suitable for clinical applications, and is universal for all serotypes and chimeric particles. This scalable manufacturing technology has been used to produce GMP Phase I clinical AAV carriers for retinal neovascularization (AAV2), hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), which have been administered to patients. Furthermore, by implementing a perfusion method that requires harvesting rAAV from the culture medium at multiple time points after transfection, the total vector production was increased by at least 5 times.

Packaging cells encompass viral vector functions, as well as packaging and vector functions. Viral vector functions typically include a portion of the parvovirus genome, such as the AAV genome, where the rep and cap sequences are deleted and replaced by a modified RPGrf15 sequence and its associated expression control sequences. Viral vector functions include sufficient expression control sequences to generate replication of the viral vector for packaging. Typically, a viral vector comprises a portion of the parvovirus genome, such as the AAV genome, where the rep and cap sequences are deleted and replaced by a transgene and its associated expression control sequences. The transgene typically side-mounts two AAV TRs, replacing the deleted viral rep and cap ORFs. Appropriate expression control sequences are included, such as tissue-specific promoters and other regulatory sequences suitable for promoting tissue-specific expression of the transgene in target cells. The transgene is typically a nucleic acid sequence that can be expressed to produce a therapeutic peptide or biomarker peptide.

The terminal repeats (TRs) used for the viral vector (both resolvable and non-resolvable) are preferably AAV sequences, with serotypes 1, 2, 3, 4, 5, and 6 being preferred. Resolvable AAV TRs do not need to have a wild-type TR sequence (e.g., the wild-type sequence can be altered by insertion, deletion, truncation, or missense mutations), as long as the TR mediates the desired function, such as viral packaging, integration, and/or provirus rescue. TRs can be synthetic sequences used as inverted terminal repeats of AAVs, such as the “double D sequence” described in U.S. Patent No. 5,478,745 to Samulski et al., the entire disclosure of which is incorporated herein by reference. Typically, but not necessarily, TRs originate from the same parvovirus; for example, both TR sequences may be from AAV2.

The packaging function includes a capsid assembly. The capsid assembly is preferably derived from a parvovirus capsid, such as an AAV capsid or a chimeric AAV capsid. Examples of suitable parvovirus capsid assemblies are those from the Parvoviridae family, such as autonomous parvoviruses or dependent parvoviruses. For example, the capsid assembly may be selected from AAV capsids, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10, AAVrh74, RHM4-1, RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6, AAV Hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAV2i8, AAV2G9, AAV2i8G9, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, and AAV-LK03, as well as other novel capsids that have not yet been identified or are derived from non-human primate sources. The shell assembly may include components from two or more AAV shells.

Packaged viral vectors typically include a modified RPGORf15 gene sequence flanked by TR elements and an expression control sequence, referred to herein as a “transgenic” or “transgenic expression cassette,” which is sufficient to result in the packaging of vector DNA and subsequent expression of the modified RPGORf15 gene sequence in transduced cells. Viral vector functions can be supplied to cells, for example, as components of plasmids or amplicones. Viral vector functions can be present extrachromosomally within cell lines and/or can be integrated into the chromosomal DNA of cells.

Any method can be used to introduce a nucleotide sequence carrying viral vector function into a cell host for replication and packaging, including but not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes combined with nuclear localization signals. In embodiments that provide viral vector function through transfection using a viral vector, standard methods for generating viral infection can be used.

Packaging functions include genes for viral vector replication and packaging. Thus, for example, packaging functions can include, as needed, functions necessary for viral gene expression, viral vector replication, rescuing the viral vector from its integrated state, viral gene expression, and packaging the viral vector into viral particles. Packaging functions can be supplied to packaging cells together or separately using genetic constructs such as plasmids or amplicones, baculoviruses, or HSV helper constructs. Packaging functions can exist extrachromosomally within packaging cells, but are preferably integrated into the cell's chromosomal DNA. Examples include genes encoding AAV Rep and Cap proteins.

Helper functions include auxiliary viral elements required for the establishment of active infection of packaging cells (which is necessary for the packaging of the initiating viral vector). Examples include functions derived from adenovirus, baculovirus, and/or herpesvirus sufficient to lead to viral vector packaging. For example, adenoviral helper functions would typically include adenoviral components E1a, E1b, E2a, E4, and VA RNA. Packaging functions can be supplied by infecting packaging cells with the desired virus. Packaging functions can be supplied to packaging cells together or separately using genetic constructs such as plasmids or amplicon. See, for example, the pXR helper plasmid described in Rabinowitz et al., 2002, J.Virol. 76:791, and the pDG plasmid described in Grimm et al., 1998, Human Gene Therapy 9:2745-2760. Packaging functions can be present extrachromosomally within packaging cells, but are preferably integrated into the cell's chromosomal DNA (e.g., E1 or E3 in HEK 293 cells).

Any suitable helper virus function can be employed. For example, baculoviruses can act as helper viruses when the packaging cells are insect cells. Herpesviruses can also be used as helper viruses in AAV packaging methods. Hybrid herpesviruses encoding one or more AAV Rep proteins can advantageously facilitate more scalable AAV vector production protocols.

Any method can be used to introduce a nucleotide sequence carrying an auxiliary function into the cell host for replication and packaging, including but not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes combined with nuclear localization signals. In embodiments that provide the auxiliary function by using transfection with a viral vector or infection with a helper virus, standard methods for generating viral infection can be used.

Any suitable sensory or packaging cells known in the art can be used to produce packaged viral vectors. Mammalian or insect cells are preferred. Examples of cells that can be used to produce packaging cells in the practice of this invention include, for example, human cell lines such as VERO, WI38, MRC5, A549, HEK293 cells (which express functional adenovirus E1 under the control of a constitutive promoter), B-50 or any other HeLa cells, HepG2, Saos-2, Huh7, and HT1080 cell lines. In one aspect, the packaging cells are capable of growth in suspension culture, more preferably, in serum-free culture. In one embodiment, the packaging cells are HEK293 cells grown in suspension in serum-free medium. In another embodiment, the packaging cells are HEK293 cells described in U.S. Patent No. 9,441,206 and deposited with ATCC No. PTA 13274. Many rAAV packaging cell lines are known in the art, including but not limited to those disclosed in WO 2002/46359. On the other hand, the packaging cells are cultured in the form of cell culture chambers (e.g., 10-layer cell culture chambers seeded with HEK293 cells).

Cell lines used as packaging cells include insect cell lines. Any insect cell that allows AAV replication and can be maintained in culture can be used according to the present invention. Examples include *Spodoptera frugiperda* cell lines such as Sf9 or Sf21, *Drosophila* spp. cell lines, or mosquito cell lines, such as those derived from *Aedes albopictus*. A preferred cell line is the *Spodoptera frugiperda* Sf9 cell line. The following references are incorporated herein by reference for their teachings concerning the use of insect cells for the expression of heterologous polypeptides, methods for introducing nucleic acids into such cells, and methods for maintaining such cells in culture: Methods in Molecular Biology, ed. Richard, Humana Press, N J (1995); O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, Oxford Univ. Press (1994); Sa Mullski et al., 1989, J. Virol. 63:3822-3828; Kajigaya et al., 1991, Proc. Nat'l. Acad. Sci. USA 88:4646-4650; Ruffing et al., 1992, J. Virol. 66:6922-6930; Kimbauer et al., 1996, Virol. 219:37-44; Zhao et al., 2000, Virol. 272:382-393; and Samulski et al., U.S. Patent No. 6,204,059.

The viral capsid according to the invention can be produced using any method known in the art, for example by expression from baculoviruses (Brown et al., (1994) Virology 198:477-488). As a further alternative, the viral vector of the invention can be produced in insect cells, for example, using a baculovirus vector to deliver the rep/cap gene and the rAAV template, as described by Urabe et al., 2002, Human Gene Therapy 13:1935-1943.

On the other hand, the present invention provides a method for producing rAAV in insect cells, wherein a baculovirus packaging system or vector carrying these gene coding regions can be constructed by engineering AAVRep and Cap into the polyhedral protein coding region of a baculovirus vector and generating a viral recombinant by transfection into a host cell. Notably, when using baculovirus to produce AAV, the preferred AAV DNA vector product is a self-complementary AAV-like molecule without mutations to the AAV ITR. This appears to be due to the lack of functional Rep enzyme activity, resulting in inefficient AAV rep cleavage byproducts in insect cells, which produce self-complementary DNA molecules. The host cell is a baculovirus-infected cell, or a cell in which additional nucleic acids encoding baculovirus helper functions have been introduced, or which include these baculovirus helper functions. These baculoviruses can express AAV components and subsequently promote capsid production.

During production, packaging cells typically include one or more viral vector functions sufficient to cause replication and packaging of viral vectors, as well as helper and packaging functions. These different functions can be supplied to packaging cells together or separately using genetic constructs such as plasmids or amplicones, and they can be present extrachromosomally within the cell line or integrated into the cell's chromosome.

Cells with any one or more of the aforementioned functions can be supplied, such as cell lines with one or more extrachromosomal incorporations or integrations into the chromosomal DNA of the cell as carriers, cell lines with one or more extrachromosomal incorporations or integrations into the chromosomal DNA of the cell as packaging cells, or cell lines with extrachromosomal incorporations or integrations into the chromosomal DNA of the cell as auxiliary cells.

The rAAV vector can be purified using methods standard in the art, such as column chromatography or cesium chloride gradient. Methods for purifying the rAAV vector are known in the art and include those described in Clark et al., 1999, Human Gene Therapy 10(6):1031-1039; Schenpp and Clark, 2002, Methods Mol. Med. 69:427-443; U.S. Patent Nos. 6,566,118 and WO 98/09657.

Treatment

In some embodiments, a method is provided for treating XLRP in a subject requiring such treatment, the method being carried out by administering to the subject a therapeutically effective amount of a nucleic acid having a nucleotide sequence having at least 90%, at least 95%, at least 98%, or 100% identity with the nucleotide sequence of SEQ ID NO:1, or a pharmaceutical composition comprising such nucleic acid and at least one pharmaceutically acceptable excipient.

In a related aspect, a nucleic acid comprising a nucleotide sequence having at least 90%, at least 95%, at least 98%, or 100% identity with the nucleotide sequence of SEQ ID NO:1 is provided for the treatment of XLRP.

In other related aspects, the use of nucleic acids comprising a nucleotide sequence having at least 90%, at least 95%, at least 98%, or 100% identity with the nucleotide sequence of SEQ ID NO:1 for the manufacture of a pharmaceutical is provided.

In other related aspects, the use of nucleic acids comprising a nucleotide sequence having at least 90%, at least 95%, at least 98%, or 100% identity with the nucleotide sequence of SEQ ID NO:1 for the manufacture of a medicament for the treatment of XLRP is provided.

In some aspects, a nucleotide sequence having at least 90%, at least 95%, at least 98%, or 100% identity with the nucleotide sequence of SEQ ID NO:1 is operatively linked to an expression control sequence. In some embodiments, the nucleotide sequence of SEQ ID NO:1 is operatively linked to a human G protein-coupled receptor rhodopsin kinase 1 (hGRK) promoter. In some preferred embodiments, the hGRK promoter has the sequence of SEQ ID NO:4.

In some embodiments, a nucleotide sequence having at least 90%, at least 95%, at least 98%, or 100% identity with the nucleotide sequence of SEQ ID NO:1 constitutes part of the expression cassette. In some aspects, the expression cassette from 5' to 3' comprises: (a) an AAV2 terminal repeat, (b) an hGRK promoter, (c) a codon-optimized RPGRorf15 gene of SEQ ID NO:1, (d) an SV40 polyadenylated sequence, and (e) an AAV2 terminal repeat. In a preferred embodiment, the 5' AAV2 terminal repeat has a nucleotide sequence as shown in SEQ ID NO:6 and/or the hGRK promoter has a nucleotide sequence as shown in SEQ ID NO:4 and/or the SV40 polyadenylated sequence has a nucleotide sequence as shown in SEQ ID NO:8 and/or the 3' AAV2 terminal repeat has a nucleotide sequence as shown in SEQ ID NO:7. In a particularly preferred embodiment, the expression cassette comprises a nucleic acid containing the nucleotide sequence of SEQ ID NO:5 or a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

In other embodiments, a method is provided for treating XLRP in a subject requiring such treatment, the method being carried out by administering to the subject a therapeutically effective amount of recombinant AAV (rAAV) viral particles, or a pharmaceutical composition comprising thereto, the rAAV viral particles comprising (i) a nucleic acid operatively linked to an expression control sequence having a nucleotide sequence having at least 90%, at least 95%, at least 98%, or 100% identity with the nucleotide sequence of SEQ ID NO:1, and (ii) an AAV capsid.

In related embodiments, the use of recombinant AAV (rAAV) viral particles for the treatment of XLRP is provided, the rAAV viral particles comprising (i) a nucleic acid operatively linked to an expression control sequence having a nucleotide sequence having at least 90%, at least 95%, at least 98%, or 100% identity with the nucleotide sequence of SEQ ID NO:1, and (ii) an AAV capsid.

In other related embodiments, the use of recombinant AAV (rAAV) viral particles for manufacturing a medicament for treating XLRP is provided, said rAAV viral particles comprising (i) a nucleic acid operatively linked to an expression control sequence having a nucleotide sequence having at least 90%, at least 95%, at least 98%, or 100% identity with the nucleotide sequence of SEQ ID NO:1, and (ii) an AAV capsid.

In some embodiments, the rAAV viral particle comprises a natural AAV2, AAV4, AAV5, or AAV8 capsid. In other embodiments, the rAAV viral particle comprises a variant AAV capsid that includes one or more modifications relative to AAV2, AAV4, AAV5, or AAV8. In a preferred embodiment, the AAV capsid comprises a capsid protein containing the sequence SEQ ID NO:9.

In some embodiments, the rAAV viral particle comprises (i) a natural AAV2 capsid or a variant thereof, and (ii) an expression cassette comprising, from 5' to 3',: (a) an AAV2 terminal repeat, (b) an hGRK promoter, (c) a codon-optimized RPGRorf15 gene of SEQ ID NO:1, (d) an SV40 polyadenylated sequence, and (e) an AAV2 terminal repeat. In a preferred embodiment, the rAAV comprises (i) a capsid containing the capsid protein of SEQ ID NO:9, and (ii) nucleic acid containing a 5' AAV2 terminal repeat of SEQ ID NO:6, an hGRK promoter of SEQ ID NO:4, an SV40 polyadenylated sequence of SEQ ID NO:8, and a 3' AAV2 terminal repeat of SEQ ID NO:7. In a particularly preferred embodiment, rAAV comprises (i) a capsid containing the capsid protein of SEQ ID NO: 9, and (ii) an expression cassette containing the nucleotide sequence of SEQ ID NO: 5.

In a particularly preferred embodiment, use of rAAV in the treatment of XLRP or in the manufacture of a medicament for the treatment of XLRP is provided, wherein rAAV comprises (i) a nucleic acid having the nucleotide sequence of SEQ ID NO:5, and (ii) a capsid having the amino acid sequence of SEQ ID NO:9. In some aspects, rAAV is administered by intravitreal injection.

In some other particularly preferred embodiments, a method for treating XLRP is provided, comprising administering to a subject an effective amount of rAAV, said rAAV comprising (i) a nucleic acid having the nucleotide sequence of SEQ ID NO:5, and (ii) a capsid having a capsid protein having the amino acid sequence of SEQ ID NO:9. In some aspects, rAAV is administered to the subject via intravitreal injection.

In other aspects, pharmaceutical compositions are provided comprising a nucleic acid optionally operably linked to an expression control sequence, and at least one pharmaceutically acceptable excipient, said nucleic acid having a nucleotide sequence having at least 90%, at least 95%, at least 98%, or 100% identity with the nucleotide sequence of SEQ ID NO:1.

In some embodiments, the pharmaceutical composition comprises a nucleic acid operatively linked to a constitutive promoter (preferably an hGRK promoter having a sequence having at least 90%, at least 95%, at least 98%, or 100% identity with the nucleotide sequence of SEQ ID NO:4), said nucleic acid comprising the nucleotide sequence of SEQ ID NO:1.

In other aspects, pharmaceutical compositions are provided comprising at least one pharmaceutically acceptable excipient and infectious rAAV, said infectious rAAV comprising (i) an AAV capsid and (ii) a nucleic acid, said nucleic acid comprising, from 5' to 3': (a) an AAV 2-terminal repeat, (b) an hGRK promoter, (c) a codon-optimized RPGRorf15 gene of SEQ ID NO:1, (d) an SV40 polyadenylated sequence, and (e) an AAV 2-terminal repeat. In related embodiments, the pharmaceutical composition comprises ^10⁹ to ^10¹⁴ vg, preferably ^10¹⁰ to ^10¹³ vg of rAAV, more preferably 3 × ^10¹¹ vg or 1 × ^10¹² vg of rAAV.

In a preferred embodiment, the pharmaceutical composition comprises rAAV, said rAAV comprising (i) a capsid containing a capsid protein comprising or consisting of the sequence of SEQ ID NO:9, and (ii) a nucleic acid comprising the 5' AAV2 terminal repeat of SEQ ID NO:6 and/or the hGRK promoter of SEQ ID NO:4 and/or the SV40 polyadenylated sequence of SEQ ID NO:8 and/or the AAV2 terminal repeat of SEQ ID NO:7. In related embodiments, the pharmaceutical composition comprises ^10⁹ vg to ^10¹⁴ vg, preferably ^10¹⁰ vg to ^10¹³ vg of rAAV, more preferably approximately 3 × ^10¹¹ vg or approximately 1 × ^10¹² vg of rAAV.

In some embodiments, a method for expressing RPGR in one or more photoreceptor cells of a human subject is provided, comprising administering an effective amount of infectious rAAV as described herein to the human subject, wherein RPGR is expressed in one or more photoreceptor cells. In some preferred embodiments, an effective amount of infectious rAAV is ^10⁹ to ^10¹⁴ vg/eye and/or administering a single dose of rAAV (bilateral or unilateral) into the vitreous cavity of a human subject and/or the rAAV comprises a capsid of SEQ ID NO:9 and/or the rAAV comprises a heterologous nucleic acid containing the nucleotide sequence of SEQ ID NO:5.

In a particularly preferred embodiment, a pharmaceutical composition is provided comprising at least one pharmaceutically acceptable excipient and an infectious rAAV, said infectious rAAV comprising (i) a capsid containing a capsid protein comprising or consisting of the sequence of SEQ ID NO:9, and (ii) a nucleic acid containing or consisting of the nucleotide sequence of SEQ ID NO:5. In related embodiments, the pharmaceutical composition comprises 10 ^{× 10⁻¹⁰} vg to 10 ^{× 13} vg of rAAV, preferably approximately 3 × ^10¹¹ vg or approximately 1 × ^10¹² vg of rAAV.

In some embodiments, XLRP is treated in a subject by administering the nucleic acid or infectious rAAV described herein to a person with XLRP via periocular or intraocular injection (intravitreal, suprachoroidal, or subretinal). In other embodiments, XLRP is treated in a subject by administering the nucleic acid or infectious rAAV described herein subretinally or intravitreally. In a preferred embodiment, a single intravitreal injection (bilateral or unilateral) of the rAAV described herein is administered to a subject with XLRP.

In relevant respects, treatment of XLRP in treated subjects includes, for example, at 6, 12, or 24 months post-treatment, (i) improvement (i.e., gain) in visual function or functional vision relative to a control (e.g., relative to baseline measurements of the treated patient before treatment, or relative to an untreated eye if the nucleic acid or rAAV was administered unilaterally, or relative to a concurrent or historical control group of untreated XLRP patients) and/or (ii) reduction in visual function loss and/or retinal degeneration in the treated eye compared to a control (e.g., an untreated eye of the same patient or an untreated control group). These improvements can be assessed by appropriate ophthalmic tests, including but not limited to visual acuity tests, micro-field examinations and other visual field tests, anatomical tests such as optical coherence tomography and fundus autofluorescence imaging, retinal electrophysiology, and/or quality of life (QoL) assessments.

In some aspects, the effective amount of the nucleic acid or rAAV (or pharmaceutical composition comprising it) described herein is an amount effective in treating XLRP in human patients. In related aspects, the effective amount of rAAV described herein is ^10⁹ to ^10¹⁴ rAAV particles (or vector genome (vg))/eye, preferably ^10¹⁰ to ^10¹³ vg/eye, or 1 × ^10¹¹ vg/eye to 5 × ^10¹² vg/eye, more preferably about 3 × ^10¹¹ vg/eye or about 1 × ^10¹² vg/eye. In some preferred embodiments, a single dose of about 3 × ^10¹¹ vg/eye or about 1 × ^10¹² vg/eye is administered intravitreally to a human patient with XLRP, thereby treating XLRP.

Example

The following examples illustrate preferred embodiments of the invention and are not intended to limit the scope of the invention in any way. While the invention has been described with reference to its preferred embodiments, various modifications will be apparent to those skilled in the art upon reading this application.

Example 1 – Codon Optimization of the RPGORf15 cDNA Sequence with Improved Stability The open reading frame 15 (hRPGRorf15) sequence of the human retinitis pigmentosa GTPase regulator contains highly repetitive, purine-rich regions, leading to sequence instability during transgenic cassette cloning and plasmid amplification. The hRPGRorf15 cDNA sequence (NCBI reference sequence NM_001034853.1) was codon-optimized to generate an RPGGRorf15 cDNA sequence with improved expression in human cells and enhanced sequence stability.

The codon-optimized nucleotide sequence is shown below:

ATGAGAGAGCCTGAAGAGCTGATGCCTGATAGCGGAGCAGTGTTTACCTTTGGGAAGA

GCAAGTTCGCAGAGAATAACCCTGGGAAATTCTGGTTTAAGAACGACGTGCCCGTGCAC

CTGAGCTGTGGCGATGAGCACTCCGCCGTGGTGACAGGCAACAATAAGCTGTACATGTT

CGGCTCTAACAATTGGGGACAGCTGGGCCTGGGAAGCAAGTCCGCCATCAGCAAGCCA

ACCTGCGTGAAGGCCCTGAAGCCCGAGAAGGTGAAGCTGGCCGCCTGTGGCAGAAAC

CACACACTGGTGAGCACCGAGGGAGGAAACGTGTACGCAACAGGAGGCAACAATGAA

GGCCAGCTGGGCCTGGGCGACACAGAGGAGAGGAATACCTTTCACGTGATCAGCTTCTT

TACCTCCGAGCACAAGATCAAGCAGCTGTCCGCCGGCTCTAACACAAGCGCCGCCCTG

ACCGAGGACGGCCGCCTGTTCATGTGGGGCGATAATAGCGAGGGCCAGATCGGCCTGA

AGAACGTGTCCAACGTGTGCGTGCCTCAGCAGGTGACCATCGGCAAGCCAGTGTCCTG

GATCTCTTGTGGCTACTATCACAGCGCCTTCGTGACCACAGATGGCGAGCTGTACGTGTT

TGGAGAGCCAGAGAACGGCAAGCTGGGCCTGCCTAACCAGCTGCTGGGCAATCACCGG

ACACCCCAGCTGGTGTCCGAGATCCCTGAGAAAGTGATCCAGGTGGCATGCGGAGGAG

AGCACACAGTGGTGCTGACCGAGAATGCCGTGTATACCTTCGGCCTGGGACAGTTTGGA

CAGCTGGGCCTGGGCACATTCCTGTTTGAGACAAGCGAGCCAAAAGTGATCGAGAACA

TCCGCGACCAGACAATCAGCTACATCTCCTGCGGCGAGAATCACACAGCCCTGATCACC

GACATCGGCCTGATGTATACCTTTGGCGATGGCCGGCACGGCAAGCTGGGCCTGGGCCT

GGAGAACTTCACAAATCACTTTATCCCACCCTGTGCTCTAACTTCCTGCGGTTCATCGT

GAAGCTGGTGGCCTGCGGCGGCTGTCACATGGTGGTGTTCGCAGCACCTCACAGGGGA

GTGGCCAAGGAGATCGAGTTTGACGAGATCAACGATACATGCCTGTCCGTGGCCACCTT

CCTGCCATACAGCTCCCTGACATCCGGCAATGTGCTGCAGCGCACCCTGTCTGCCAGGA

TGCGGAGAAGGGAGAGGGAGCGGTCCCCTGACTCTTTCAGCATGAGGCGGACACTGCC

ACCTATCGAGGGCACCCTGGGCCTGTCTGCCTGCTTCCTGCCTAACAGCGTGTTCCCAA

GATGTAGCGAGAGGAATCTGCAGGAGTCTGTGCTGAGCGAGCAGGATCTGATGCAGCC

AGAGGAGCCCGACTACCTGCTGGATGAGATGACAAAGGAGGCCGAGATCGACAACTCT

AGCACCGTGGAGAGCCTGGGCGAGACAACAGATATCCTGAATATGACACACATCATGTC

CCTGAACTCTAATGAGAAGTCTCTGAAGCTGAGCCCAGTGCAGAAGCAGAAGAAGCAG

CAGACATCGGCGAGCTGACCCAGGACACAGCCCTGACCGAGAACGACGATTCTGATG

AGTATGAGGAGATGAGCGAGATGAAGGAGGGCAAGGCCTGTAAGCAGCACGTGTCCCA

GGGCATCTTCATGACCCAGCCAGCCACCACAATCGAGGCCTTTTCTGACGAAGAGGTGG

AGATCCCCGAGGAGAAGGAGGGCGCCGAGGATAGCAAGGGCAATGGCATCGAGGAGC

AGGAGGTGGAGGCCAACGAGGAGAATGTGAAGGTGCACGGCGGCAGAAAGGAGAAG

ACAGAGATCCTGTCCGACGATCTGACCGACAAGGCCGAGGTGTCCGAGGGCAAGGCCA

AGTCTGTGGGAGAGGCAGAGGACGGACCAGAGGGACGCGGCGATGGAACCTGCGAGG

AGGGATCCTCTGGAGCAGAGCACTGGCAGGACGAAGAAAGAGAGAAGGGCGAGAAGG

ATAAGGGCAGAGGAGAGATGGAGAGGCCTGGAGAGGGAGAGAAGGAGCTGGCAGAGA

AGGAGGAGTGGAAGAAGAGGGACGGCGAGGAGCAGGAGCAGAAGGAGAGAGAGCAG

GGCCACCAGAAGGAGAGGAACCAGGAGATGGAGGAGGGAGGAGAGGAGGAGCACGG

CGAGGGAGAGGAGGAGGAGGGCGATAGAGAGGAAGAAGAGGAGAAGGAGGGAGAGG

GCAAGGAGGAAGGCGAGGGAGAGGAGGTGGAGGGAGAAAGGGAGAAGGAGGAGGG

AGAGCGCAAGAAGGAAGAAAGAGCAGGCAAGGAAGAGAAGGGAGAGGAGGAGGGC

GATCAGGGCGAAGGAGAGGAGGAGGAGACAGAGGGAAGGGGAGAGGAGAAGGAGGA

GGGAGGAGAGGTCGAAGGAGGAGAAGTGGAGGAGGGCAAGGGCGAAAGAGAAGAGG

AGGAGGAGGAAGGCGAGGGCGAAGAAGAGGAGGGCGAGGGCGAGGAAGAAGAGGG

CGAGGGCGAAGAGGAAGAAGGCGAGGGCAAGGGCGAGGAGGAGGGCGAAGAAGGCG

AAGGGGAGGAGGAGGGCGAAGAGGGAGAGGGCGAGGGCGAGGAGGAAGAAGGCGA

AGGCGAAGGCGAAGAAGAAGGAGAAGGAGAGGGCGAAGAGGAGGAAGGCGAAGGA

GAAGGAGAGGAGGAAGGAGAAGGGGAGGGCGAAGAGGAGGAGGAAGAAGGCAAGG

GAGAAGAAGAAGGCGAAGAAGGCGAGGGAGAAGGCGAGGAAGAAGAAGGCGAGGG

AGAGGGAGAGGACGGCGAAGGCGAGGGCGAGGAAGAGGAAGGAGAGTGGGAGGGCG

AGGAAGAGGAGGGAGAAGGAGAAGGCGAAGAAGAAGGGGAAGGAGAGGGCGAGGA

AGGAGAAGGCGAAGGCGAAGAGGAGGAGGGGGAAGGGGAGGGCGAGGAGGAAGAG

GGAGAAGAGGAAGGCGAAGAAGAGGGAGAAGGCGAAGAGGAAGGAGAAGGCGAGG

GAGAAGAAGAGGAGGAGGGCGAGGTCGAAGGCGAGGTGGAGGGCGAAGAGGGGGAA

GGCGAAGGCGAGGAGGAGGAAGGGGAAGAAGAAGGCGAGGAGAGAGAGAAAGAAG

GCGAGGGCGAGGAGAACAGAAGGAATCGCGAAGAAGAAGAGGAAGAAGAGGGCAAG

TACCAGGAGACAGGCGAGGAGGAGAACGAGCGGCAGGATGGCGAGGAGTATAAGAAG

GTGTCCAAGATCAAGGGCTCTGTGAAGTACGGCAAGCACAAGACCTATCAGAAGAAGA

GCGTGACCAACACACAGGGCAATGGCAAGGAGCAGCGCAGCAAGATGCCTGTGCAGTC

CAAGCGGCTGCTGAAGAATGGCCCCTCTGGGAGCAAGAAGTTTTGGAATAATGTCCTGCCACACTACCTGGAGCTGAAATGA (SEQ ID NO: 10).

AAV plasmids containing the codon-optimized hRPGRorf15 gene (SEQ ID NO:10) under the control of the human G protein-coupled receptor kinase 1 promoter (also known as the human rhodopsin kinase promoter (hGRK)) or the ubiquitous 3-phosphoglycerate kinase (PGK) promoter were constructed using GenScript.

20 ng of AAV plasmid DNA was used to transform competent *E. coli* (Cat. #C3040H, New England BioLabs, Ipswich, MA), and the cells were plated on kanamycin 50 μg/ml plates (#L1025, Teknova, Hollister, CA). Microcultures were prepared from the resulting colonies, and DNA was prepared using the GeneJET plasmid micropreparation kit (Cat. #0503, Thermo Fisher, Waltham, MA) and restriction digested to identify positive clones.

Despite codon optimization, sequence instability of the codon-optimized hRPGRorf15 (SEQ ID NO:10) was still detected after restriction digestion during plasmid production.

A second codon-optimized hRPGRorf15 sequence was developed using different optimization algorithms, including but not limited to parameters related to codon use bias, GC content, AT-rich or GC-rich regions, mRNA secondary structure, RNA instability motifs, cryptic splicing sites, internal chi sites and ribosome binding sites, and repetitive sequences. Codon use bias in humans was altered by increasing the codon fitness index (CAI) to 0.89. The average GC content was optimized from 59.16 in the native sequence to 57 in the optimized sequence to extend the mRNA half-life. The resulting codon-optimized nucleotide sequence (as shown in SEQ ID NO:1 in this paper) contains improved codon use, altered GC content, better mRNA stability, and modifications with negative cis-acting elements.

Construct an AAV plasmid (pAAV-GRK promoter-cohRPGRorf15-SV40) containing the nucleotide sequence of SEQ ID NO:5 (SEQ ID NO:5 contains (i) 5'AAV2 ITR (SEQ ID NO:6); (ii) codon-optimized hRPGRorf15 cDNA (SEQ ID NO:1) under the control of the hGRK promoter (SEQ ID NO:4); (iii) SV40 late poly-A element (SEQ ID NO:8) and (iv) 3'AAV2 ITR (SEQ ID NO:7)).

pAAV-GRK promoter-cohRPGRorf15-SV40 DNA was prepared as follows. 20 ng of plasmid DNA from GenScript was used to transform competent *E. coli* (Cat. #C3040H, New England BioLabs, Ipswich, MA), and the cells were plated on kanamycin 50 μg/ml plates (#L1025, Teknova, Hollister, CA). Microcultures were grown from the resulting colonies, and DNA was prepared using the GeneJET Plasmid Microculture Kit (Cat. #0503, Thermo Fisher, Waltham, MA) and subjected to restriction digestion to identify positive clones. A culture was grown from a positive clone in 50 mL of Terrific Broth, and DNA was prepared using the Qiagen EndoFree Plasmid Maxi Kit (Cat. #12362, Qiagen, Hilden, Germany). Large quantities of pAAV-GRK-cohRPGRorf15-SV40 were digested with multiple restriction enzymes to verify the plasmid's identity. Gel electrophoresis of the restriction digests and the expected fragments is shown in Figure 1. All actual fragments matched the expected fragments. The expression cassette sequence was verified by Sanger DNA sequencing.

Conclusion: A large number of preparations of pAAV-GRK-cohRPGRorf15-SV40 were correctly mapped by restriction digestion, and their integrity was verified by Sanger DNA sequencing. Thus, the codon-optimized hRPGRorf15 sequence shown in SEQ ID NO:1 exhibits excellent stability compared to the native sequence in SEQ ID NO:3 and the codon-optimized sequence in SEQ ID NO:10.

Example 2 – Expression and activity of human RPGrf15 protein expressed by hRPGRorf15 codon optimized by SEQ ID NO:1

The expression and activity of human RPGRorf15 protein expressed by pAAV-GRK-cohRPGRorf15-SV40 were evaluated in transfected HEK293T cells.

In summary, HEK293T cells were seeded at 2.0 x 10^5 cells/well in 1.0 mL of DMEM/10% FBS medium in 12-well plates. This medium was used due to the high transfectivity and protein expression of HEK293T cells. The next day, 1.0 μg of AAV plasmid DNA, compounded with 3.0 μl of FuGene6 (Cat.#E2691, Promega, Madison, WI), was added to the cells in duplicate wells. Two days post-transfection, cells were washed with PBS and lysed in 0.25 mL of 1× passive lysis buffer (Promega) containing 1× Halt protease inhibitor (ThermoFisher), with shaking for 15 min at room temperature. Cell debris was pelleted by centrifugation at 12,000 g for 10 min at 4 °C. The supernatant was collected and stored at -20 °C. Transfection included samples without plasmid and pAAV-PGK promoter-cohRPGRorf15-SV40 as negative and positive controls, respectively. The pAAV-PGK promoter-cohRPGRorf15-SV40 is identical to the AAV vector described above, except that the codon-optimized hRPGRorf15 is operatively linked to the ubiquitous 3-phosphoglycerate kinase (PGK) promoter instead of the hGRK promoter.

Cell lysate (20 μl) was mixed with 10 μl 4×LDS, 4 μl 10× reducing agent, and 6 μl water (final volume = 40 μl) and denatured at 70 °C for 10 min. The sample was loaded onto a 12-well Bolt 4-12% Bis-Tris Plus polyacrylamide gel (Invitrogen, NW04122BOX) and run at 200 V for 32 min in 1×MOPS buffer. The isolated proteins were transferred to a nitrocellulose membrane using an iBlot 2 device (ThermoFisher) for 10 minutes and detected using an iBind Flex device (ThermoFisher) with primary antibodies against RPGR (Sigma HPA001593 1:2000 and GenScript CT-15U1729DC260_16 1:500) and anti-polyglutamyl GT335 (AG-20B-0020 1:500, Adipogen, San Diego, CA). Secondary antibodies were HRP-conjugated goat anti-rabbit (ThermoFisher 31460) (for the anti-RPGR primary antibody) and HRP-conjugated goat anti-mouse (ThermoFisher 31430) (for the anti-polyglutamyl GT335 primary antibody). Proteins were visualized using the SuperSignal WestDura chemiluminescent substrate (ThermoFisher 34076) and imaged on a ChemiDoc MP (BioRad, Hercules, CA). All antibodies used are listed in Table 3 below.

Table 3: Western blot antibodies

Antibody host species Salesperson Table of contents# Dilution Anti-RPGR polyclonal rabbit Sigma HPA001593 1:2,000 Anti-CT-15 rabbit GenScript U1729DC260_16 1:500 Anti-polyglutamylation GT335 mice Adipogen AG-20B-0020 1:500 HRP anti-rabbit IgG (H+L) goat Thermo 31460 1:5,000 HRP anti-mouse IgG (H+L) goat Thermo 31430 1:5,000

Figure 2 shows a representative Western blot image of lysates from transfected HEK293T cells. CT-15 and Sigma antibodies detected what appeared to be the same 135-140 kDa species as RPGRorf15, present in RPGR-transfected lysates but not in untransfected lysates, exhibiting the correct size and being recognized by the polyglutamylation detection antibody GT335. Expression was higher when driven by the ubiquitous PGK promoter (which is not preferentially active in photoreceptor cells).

Conclusion—Western blot analysis of lysates from transfected HEK293T cells demonstrated the expression and polyglutamylation of hRPGRorf15 protein of the correct size, optimized by the codon of SEQ ID NO:1.

Example 3 – Functional Expression of hRPGRorf15 in an In Vitro Human XLRP Model: A human in vitro model system was generated to evaluate the correction of the X-linked retinitis pigmentosa (XLRP) phenotype to codons optimized with the nucleotide sequence of SEQ ID NO:1. For this purpose, an AAV vector was constructed comprising the nucleotide sequence of SEQ ID NO:1 (i.e., the AAV vector backbone described in Examples 1 and 2, having the sequence of SEQ ID NO:5) driven by the human G protein-coupled receptor rhodopsin kinase 1 (hGRK) promoter and a variant capsid protein having the amino acid sequence of SEQ ID NO:9. The hGRK promoter was selected to restrict the expression of RPGrrf15 to photoreceptors.

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood drawn from individuals with XLRP and reprogrammed into induced pluripotent stem cells (iPSCs) using the CytoTuneiPS 2.0 Sendai reprogramming kit (Thermo Fisher Scientific, Waltham, MA). The pluripotency of the iPSCs was confirmed by immunocytochemistry for the detection of iPSC markers, including Sox2, Oct4, and Nanog. The induced pluripotent stem cells were then differentiated into photoreceptors using methods described in Gonzalez-Cordero et al., Stem Cell Report, 9, 820:837 (2017); Gonzalez-Cordero et al., Human Gene Therapy, 29(1) (2018); and Meyer et al., Stem Cells, 29(8):1206-1218 (2011). Photoreceptor differentiation was confirmed by immunocytochemistry for the detection of specific markers, namely, recovery protein and rhodopsin. This confirmed the absence of hRPGorf15 protein expression in photoreceptors and the known functionalization of hRPGorf15 protein through glutamylation.

Immunocytochemistry was performed as follows: Cells were fixed with 4% paraformaldehyde (PFA) (Santa Cruz Biotechnologies, Dallas, TX) at 4°C for 15 minutes. All antibody staining was performed in PBS blocking solution containing 0.2% Triton-X100 (Sigma-Aldrich), 2% bovine serum albumin (Millipore Sigma, Burlington, MA), and 5% goat serum (Thermo Fisher Scientific). Primary antibodies were incubated overnight at 4°C. Cells were then incubated with secondary antibodies at room temperature for one hour, followed by counterstaining with DAPI (Sigma-Aldrich) in PBS at room temperature for five minutes. Cells were imaged using a Zeiss Axio Observer.D1 fluorescence microscope. Image processing was performed using Zeiss Zen 2 software (Carl Zeiss Microscopy LLC, White Plains, NY). A list of primary and secondary antibodies is provided in Table 4 below.

Table 4

To assess the transcript levels of the codon-optimized RPGrrf15 transgene in diseased photoreceptors transduced into XLRP-iPSC-derived cells, XLRP photoreceptors (PRs) were transduced with the aforementioned AAV vector at a multiplicity of infection (MOI, viral genome per cell) of 50,000 to ensure levels above the detection limit. RNA was isolated and cDNA synthesized 30 days post-transduction. Droplet digital PCR was run on the prepared samples, and transcript levels were analyzed as copies/mL. Quantification of the number of droplets containing the primer/probe set (above a set threshold) was examined. Two primer/probe sets were created to specifically distinguish the codon-optimized human RPGrrf15 transgene from the endogenous human RPGR1-19 constitutive isoform (hRPGR1-19).

As expected, untransduced XLRP-infected cells expressed low, background levels of the cohRPGRorf15 transcript. After transduction with the AAV vector, cells showed a more than 400-fold increase in cohRPGRorf15 transcript levels compared to hRPGR1-19. Transduced cells showed a more than 1000-fold increase in cohRPGRorf15 transcript levels compared to untransduced cells. Untransduced cells had higher hRPGR1-19 levels than cohRPGRorf15. See Figure 3. Analyses were performed in triplicate, and levels were averaged. Transduction with an AAV vector containing hRPGRorf15 codons optimized for SEQ ID NO:1 significantly increased cohRPGRorf15 transcript levels in photoreceptor cultures.

To assess the protein levels of codon-optimized human RPGrf15 transgenes generated by transduction of XLRP-iPSC-derived photoreceptors with an AAV vector, diseased photoreceptors derived from XLRP-iPSCs were transduced at an MOI of 50,000 vg/cell. Cell lysates were collected 30 days post-transduction, and SDS-PAGE and Western blot analysis were performed to assess hRPGRorf15 protein levels. Band intensities were quantified and plotted as a histogram in Figure 4. Transduction with the AAV vector resulted in a significant increase in human RPGrf15 protein expression compared to untransduced cells.

To determine whether the exogenously introduced cohRPGRorf15 protein in the photoreceptor was functional, gamma-glutamylation (a representative of function) was detected. According to published literature, hRPGRorf15 gamma-glutamylation is strongly correlated with protein function (Fischer et al., 2017; Rao et al., 2016; Sun et al., 2016). Diseased PR cells derived from XLRP-iPSCs were transduced at an MOI of 50,000 vg/cell. Cell lysates were collected 30 days post-transduction, and SDS-PAGE and Western blot analysis were performed to assess gamma-glutamylation of expressed hRPGRorf15 protein. Gamma-glutamylation was determined by probing the membrane with the gamma-glutamylation-specific antibody GT335 and examining the positive band pattern at the hRPGRorf15 size of 127 kDa. Band intensity was quantified and plotted as a histogram in Figure 5. In diseased photoreceptors derived from two XLRP patients, transduction of PR cells with an AAV vector containing a codon-optimized hRPGRorf15 nucleotide sequence resulted in a significant increase in glutamylation of the human RPGRorf15 protein compared to untransduced cells.

The dose-response of the codon-optimized transgene (cohRPGRorf15) was validated because low hRPGRorf15 protein levels were detected in Western blots at high MOIs. To this end, an AAV vector was constructed containing the codon-optimized RPGRorf15 sequence of SEQ ID NO:1 operably linked to the ubiquitous promoter 3-phosphoglycerate kinase (PGK) and the capsid of SEQ ID NO:9 (this AAV vector is identical to the AAV vector described above, except for the promoter). Diseased photoreceptors were transduced at three MOIs (5,000, 10,000, and 20,000). Cell lysates were collected 30 days post-transduction and subjected to SDS-PAGE and Western blot analysis to assess hRPGRorf15 protein levels and glutamylation (GT335 = anti-glutamylation antibody). Band intensities were quantified and plotted as histograms (Figure 6). Despite high variability due to the heterogeneity of the cultures, hRPGRorf15 protein and hRPGRorf15 glutamylation were observed at low MOIs when constitutive promoters were used to drive cohRPGRorf15 expression.

Conclusion—In vitro studies using iPSC-derived photoreceptors have demonstrated that AAV-mediated delivery of codon-optimized hRPGRorf15 (SEQ ID NO:1) restores the human RPGORf15 transcript and transgene expression in human XLRP-affected photoreceptors. Furthermore, the RPGORf15 protein expressed after 4D-125 transduction was post-translational glutonylated. Based on published literature, glutonylation confers functionality to RPGORf15.

Example 4 – Safety and biodistribution assessment of intravitreal administration of a codon-optimized RPGrf15 cDNA sequence delivered by R100 in non-human primates

Materials and methods

GLP toxicology and biodistribution studies

Male cynomolgus macaques (macaca fascicularis) aged 2–14 years were administered the drug to each eye via scleral injection twice, with a total dose volume of 100 μL/eye. Doses of 1 × ^10¹¹ vg/eye and 1 × ^10¹² vg/eye were evaluated. Animals were anesthetized with ketamine IM and given a topical ophthalmic solution to relieve pain. Methylprednisolone was administered weekly via IM at 20–80 mg. Euthanasia was performed by trained veterinarians at weeks 3, 13, and 26 post-administration.

The biodistribution of the 4D-125 genome (rAAV containing the capsid protein of SEQ ID NO:9 and a heterologous nucleic acid containing the nucleotide sequence of SEQ ID NO:5) in all major ocular compartments (retina, optic nerve, ciliary body, iris, trabecular meshwork) and major systemic organs (including the testes) was assessed using validated, GLP-compliant qPCR assays. Transgenic expression was assessed in tissues where the genome was detected using qualified, GLP-compliant RT-qPCR assays.

The series of toxicological assessments performed in the study included: clinical ophthalmic assessment (complete ophthalmic examination, including SD-OCT imaging and ERG), systemic assessment, clinicopathology, gross pathology, and micropathology. Assays were validated to determine anti-capsid and anti-GMO antibody responses. The ELISpot assay was validated to detect cellular responses to the R100 (a variant capsid protein containing SEQ ID NO:9) capsid and expressed proteins.

Neutralizing antibody assay

24 hours prior to infection, 2v6.11 cells were plated at a density of 3 × ^10⁴ cells/well. Prior to infection, the rAAV vector encoding firefly luciferase driven by the CAG promoter was incubated with individual serum samples at 37°C for 1 hour, followed by cell infection with a genomic MOI of 1,000. 48 hours post-infection, luciferase activity was assessed using either the Luc-Screen Extended-Glow luciferase reporter assay system (Invitrogen) or the ONE-Glo luciferase assay system (Promega), and quantified using a BioTek Cytation 3 multi-mode microplate reader and Gen5 software.

Prior to inclusion in the study, the presence of neutralizing antibodies against R100 in non-human primate (NHP) serum was screened. NHPs were included in the study when samples resulted in less than 50% neutralization of AAV transduction at a 1:10 serum dilution.

AAV Manufacturing

Recombinant R100 viral vectors were generated by transient transfection into HEK293 cells. Cells were cultured in DMEM supplemented with FBS and maintained at 37°C and 5% _CO2 . Cells were transfected three times (load, capsid, and helper plasmid) using polyethyleneimine (PEI). Viral particles were harvested from cells and/or supernatant 48–96 h post-transfection, and cells were lysed via microfluidic lysis. Cell lysates and/or supernatant were enzymatically treated to degrade plasmids and host cell DNA, followed by clarification and concentration by tangential flow filtration (TFF). The TFF residue was then loaded onto an affinity resin column for purification. After elution with a pH gradient, the post-affinity material was buffer-exchanged and then further purified by anion exchange chromatography (if desired). The purified rAAV was then reconstituted in DPBS containing 0.001% polysorbate-20, aseptically filtered, and filled to produce the rAAV drug product.

result

4D-125 delivery is safe and results in the expression of therapeutic transgenes in NHP.

4D-125 (R100.GRK-cohRPGRorf15) has entered Phase 1-2 clinical trials. The Investigational New Drug (IND) data for this product includes evaluations in a 6-month Good Laboratory Practice (GLP) toxicology and biodistribution study (Table 5). A total of 30 eyes were injected intravitreally via unilateral administration to 30 NHPs.

Table 5: Good Laboratory Practice (GLP) Toxicological and Biodistribution Studies

As determined by clinical observation, histopathology, OCT, or ERG, no significant toxicity was observed with 4D-110 at any dose level. Administration of 4D-110 to a single eye resulted in only minimal to mild anterior uveitis, which was limited to the immediate period after administration and subsided by week 3 (Figure 9); in some cases, the systemic steroid dose was instantaneously increased.

Very high levels of the vector genome were present in the retina of the treated eyes at all time points (week 3, left panel; week 13, middle panel; week 26, right panel), indicating the persistence of the vector in ocular tissues (Fig. 10). In addition to the retina, the vector genome was detected in samples from the aqueous humor, vitreous humor, iris/ciliary body, and optic nerve of the treated eyes at all time points. The vector genome was generally not detectable in non-ocular tissues, except for low levels in the liver, spleen, and lymph nodes (Fig. 10). Transgenic expression derived from the R100 vector was detected in the treated retina and iris/ciliary body from both the low-dose and high-dose groups (Fig. 11). Gene expression was dose-dependent and increased from week 3 to week 13, remaining stable at week 26 (Fig. 11, left, middle, and right panels, respectively). No non-ocular vector expression was detected at week 26 (Fig. 11).

Cellular immune responses were assessed using the ELISpot assay; no animals showed a significant response to the R100 capsid peptide or the transgenic peptide (data not shown). Most animals administered 4D-125 developed anti-capsid antibody responses following administration (data not shown).

Summarize

4D-125 (R100.GRK-cohRPGRorf15) has recently been converted to a clinical trial for the hereditary retinal disease X-linked retinitis pigmentosa (NCT04517149). This therapeutic product has been evaluated in GLP toxicology and biodistribution studies (Table 5). A total of 30 NHPs were treated with unilateral injection; a total of 30 NHP eyes were injected. No significant test-product-related adverse events or T-cell responses were reported. Mild to moderate transient corticosteroid-responsive anterior uveitis was observed. Transgenic expression was localized to the retina and was not detected in any of the systemic organs evaluated. Human clinical trials are ongoing to determine the safety, pharmacodynamics, and efficacy of this product via intravitreal injection (including by continuous visual field testing and optical coherence tomography).

Example 5 – Safety assessment of intravitreal administration of a codon-optimized RPGrf15 cDNA sequence delivered by R100 in patients with human X-linked retinitis pigmentosa.

Summary of safety and tolerability data for initial phase 1 dose escalation

Clinical trial design and recruitment

The clinical trial employed a standard “3+3” dose escalation design to evaluate the safety, tolerability, and bioactivity of a single intravitreal injection of 4D-125 at two dose levels (3E11 or 1E12 vg/eye). A total of six patients were recruited in the dose escalation cohort, three at each dose level. Patients received a standard immunosuppressive regimen with gradual dose reduction; adjustments were determined by the investigator. Results were based on data cutoffs between 1 and 9 months post-administration.

Initial Tolerability and Adverse Events Overview

As outlined in the summary table of adverse events (AEs) that occurred during treatment (Table 6), 4D-125 was well tolerated throughout the evaluation period:

Table 6. Summary of Adverse Events

Recruited patients# 6 dose 3E11 or 1E12 vg/eye Follow-up (months) at the data cutoff point 4-9 months Dose-limiting toxicities (DLT) 0 (0%) Severe AE 0 (0%) Any CTCAE of level 3 or higher 0 (0%) Retinal AE (any level) 0 (0%) Uveitis, CTCAE grade 2 (moderate) 1/6 (17%) Uveitis, CTCAE grade 1 (mild) 2/6 (33%)

Clinical assessment

Preliminary bioactivity was assessed using micro-field examination (MP) to measure retinal sensitivity and ellipsoidal zone area (EZA) to measure using SD-OCT. Seven subjects (median age 42.5 years; range 27–56 years) received 4D-125 (3 × ^10¹¹ vg/eye (n=3) and 1 × ^10¹² vg/eye (n=4)) and were followed up for 4.2–12.5 months. Intraocular inflammation (4/7 subjects) was mild to moderate, transient (duration 0.9–1.6 months), and steroid-responsive. Most subjects had advanced disease, and only 2 subjects had measurable EZA and mean MP retinal sensitivity (mMPRS) in both eyes at baseline (BL) and at least 4 months of follow-up. Both subjects showed greater improvements in mMPRS (+1.65 dB vs. +0.25 dB at 9 months and +0.50 dB vs. +0.10 dB at 4 months; BL values 1.5–3.2 dB) and the number of loci achieving ≥7 dB sensitivity (6 vs. 1 at 9 months and 3 vs. 0 at 4 months) compared to BL in the treated eye versus the untreated eye. For both subjects, EZA showed a smaller relative decrease compared to BL in the treated eye versus the untreated eye (-12.4% vs. -16.2% at 9 months and -20.2% vs. -28.7% at 6 months).

Throughout the Phase 1/2 study, patients' ocular and systemic conditions were closely monitored, including detailed ophthalmic evaluations and retinal imaging, as well as blood tests and systemic examinations where necessary. Various visual functional and anatomical assessments were performed to detect any preliminary efficacy signals. These assessments included, but were not limited to, measurement of the ellipsoidal zone (EZ) area, fundus autofluorescence, micro-field examination, static automated visual field testing, and best-corrected visual acuity (BCVA).

in conclusion

Intravitreal administration of 4D-125 was well tolerated, with mild to moderate, transient, and steroid-responsive intraocular inflammation. Preliminary indications of biological activity were observed in two evaluable dose-escalation subjects based on micro-field examination and SD-OCT. These findings support dose escalation at 1 × ^10¹² vg/eye in XLRP subjects with less advanced disease in an ongoing phase 1/2 study.

While the materials and methods of the invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that changes may be made to the methods described herein without departing from the concept, spirit, and scope of the invention. All such similar alternatives and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention.

<110> 4D Molecular Therapeutics, Inc.

<120> Codon-optimized RPGRORF15 gene and its applications

<130> 090400-5012 WO

<150> US 63/073,843

<151> 2020-09-02

<160> 10

<170> PatentIn version 3.5

<210>  1

<211> 3459

<212> DNA

<213> Artificial sequence

<220>

<223> Codon optimization RPGRorf15

<400>  1

atgagagaac ccgaggaact gatgcccgac tctggcgccg tgtttacctt cggcaagagc 60

aagttcgccg agaacaaccc cggcaagttc tggttcaaga acgacgtgcc agtgcacctg 120

agctgcggag atgaacactc tgccgtggtc accggcaaca acaagctgta catgttcggc 180

agcaacaact ggggccagct cggcctggga tctaagtctg ccatcagcaa gcctacctgc 240

gtgaaggccc tgaagcctga gaaagtgaaa ctggccgcct gcggcagaaa tcacaccctg 300

gtttctaccg aaggcggcaa tgtgtatgcc accggcggaa acaatgaggg acaagcttgga 360

ctgggcgaca ccgaggaaag aaacaccttc cacgtgatca gctttttcac cagcgagcac 420

aagatcaagc agctgagcgc cggctctaat acctctgccg ctctgacaga ggacggcaga 480

ctgtttatgt ggggcgacaa ttctgagggc cagatcggac tgaagaacgt gtccaatgtg 540

tgcgtgcccc agcaagtgac aatcggcaag cctgtgtctt ggatcagctg cggctactac 600

cacagcgcct ttgtgacaac cgatggcgag ctgtatgtgt tcggcgagcc agagaatggc 660

aagctgggac tgcctaacca gctgctgggc aatcacagaa cccctcagct ggtgtctgag 720

atccccgaaa aagtgatcca ggtggcctgt ggcggagagc acaagtggt gctgacagag 780

aatgccgtgt aaccctttgg cctgggccag tttggacaac tcggactggg aaccttcctg 840

ttcgagacaa gcgagcccaa agtgatcgag aacatccggg accagaccat cagctacatc 900

agctgtggcg agaaccacac agccctgatc acagacatcg gcctgatgta cacattcggc 960

gacggaaggc atggaaagct cggacttggc ctggaaaact tcaccaacca cttcatccct 1020

acgctgtgca gcaacttcct gcggttcatt gtgaagctgg tggcctgcgg aggatgccac 1080

atggtggttt ttgctgcccc tcacagaggc gtggccaaag agattgagtt cgacgagatc 1140

aacgatacct gcctgagcgt ggccaccttc ctgccttaca gcagcctgac atctggcaac 1200

gtgctgcaga ggacactgag cgccagaatg cgcagacggg aaagagagag aagccccgac 1260

agcttcagca tgagaagaac cctgcctcca atcgagggca cactgggcct gtctgcctgc 1320

tttctgccta acagcgtgtt ccccagatgc agcgagagaa acctgcaaga gagcgtgctg 1380

agcgagcagg atctgatgca gcctgaggaa cccgactacc tgctggacga gatgaccaaa 1440

gaggccgaga tcgacaacag cagcacagtg gaaagcctgg gcgagacaac cgacatcctg 1500

aacatgaccc acatcatgag cctgaacagc aacgagaagt ctctgaagct gagccccgtg 1560

cagaagcaga agaagcagca gaccatcggc gagctgacac aggatactgc cctgaccgag 1620

aacgacgaca gcgacgagta cgaagagatg agcgagatga aggaaggcaa ggcctgcaag 1680

cagcacgtgt cccagggcat ctttatgacc cagcctgcca ccaccatcga ggccttttcc 1740

gacgaggaag tggaaatccc cgaggaaaaa gagggcgccg aggacagcaa aggcaacggc 1800

attgaggaac aagaggtgga agccaacgaa gagaacgtga aggtgcacgg cggacggaaa 1860

gaaaagaccg agatcctgag cgacgacctg accgataagg ccgaggtttc cgagggcaaa 1920

gccaagtctg tgggagaagc cgaggatgga cctgaaggcc gcggagatgg aacctgtgaa 1980

gaaggatcta gcggagccga gcactggcag gatgaggaac gcgagaaggg cgagaaagac 2040

aaaggcagag gcgagatgga aagacccggc gaggcgaaa aagagctggc cgagaaagag 2100

gaatggaaga aacgcgacgg cgaagaacaa gagcagaaag aaagagagca gggccaccag 2160

aaagaacgga atcaagagat ggaagaaggc ggcgaggaag aacacggcga agggggaagaa 2220

gaggaaggcg accgagagga agaagaagag aaagaaggcg aaggcaaaga agaaggcgag 2280

ggcgaagagg tggaaggcga gcgtgaaaaa gaagagggcg aacgcaagaa agaagaacgc 2340

gccggaaaag aggaaaaagg cgaggaagag ggcgaccaag gcgaaggcga ggaagaagaa 2400

actgaaggca gaggggaaga gaaagaggaa ggcggcgaag tcgaaggcgg agaggttgaa 2460

gaaggcaaag gcgagcgaga agaggaagaa gaagaaggcg aaggcgagga agaggaaggc 2520

gaaggcgaag aggaagaagg cgaaggggaa gaagaagaag gcgaaggcaa gggcgaagag 2580

gagggcgaag aaggcgaggg cgaagaggag ggcgaagaag gcgaaggcga gggcgaagaa 2640

gaagaaggcg aaggcgaagg cgaggaagaa ggcgaaggcg aaggggaaga agggaaggc 2700

gaaggcgaag gcgaagaaga aggcgaaggc gagggcgaag aggaagaagg cgaaggcaaa 2760

ggggaagaag aaggcgagga aggcgaaggc gaaggcgagg aagaagaagg cgaaggcgag 2820

ggcgaagatg gcgaaggcga aggcgaagag gaagagggcg agtgggaggg cgaagaagag 2880

gaaggcgaag gcgagggcga agaggaaggc gaaggcgagg gcgaagaagg cgaaggcgaa 2940

ggcgaggaag aggaaggcga aggcgaaggg gaagaagaag agggcgaaga agaaggcgaa 3000

gaggaaggcg aaggggaaga agaaggcgaa ggcgaaggcg aagaagagga agagggcgaa 3060

gttgaaggcg aggttgaggg cgaagaaggc gaaggcgaag gggaagaaga agaaggcgag 3120

gaagaagggg aagagaga aaaagaaggc gagggcgaag aaaaccgccg gaaccgcgaa 3180

gaggaagagg aagaagaggg caagtaccaa gagactggcg aggaagagaa cgagcggcag 3240

gatggcgaag agtacaagaa ggtgtccaag atcaagggca gcgtgaagta cggcaagcac 3300

aagacctacc agaagaagtc cgtcaccaac acgcaaggca atggaaaaga acagcggagc 3360

aagatgcccg tgcagtccaa gaggctgctg aagaatggcc ctagcggcag caagaaattc 3420

tggaacaatg tgctgcccca ctacctcgag ctgaagtga 3459

<210>  2

<211>  1152

<212> PRT

<213> Homo sapiens

<400>  2

Met Arg Glu Pro Glu Glu Leu Met Pro Asp Ser Gly Ala Val Phe Thr

1 5 10 15

Phe Gly Lys Ser Lys Phe Ala Glu Asn Asn Pro Gly Lys Phe Trp Phe

20 25 30

Lys Asn Asp Val Pro Val His Leu Ser Cys Gly Asp Glu His Ser Ala

35 40 45

Val Val Thr Gly Asn Asn Lys Leu Tyr Met Phe Gly Ser Asn Asn Trp

50 55 60

Gly Gln Leu Gly Leu Gly Ser Lys Ser Ala Ile Ser Lys Pro Thr Cys

65 70 75 80

Val Lys Ala Leu Lys Pro Glu Lys Val Lys Leu Ala Ala Cys Gly Arg

85 90 95

Asn His Thr Leu Val Ser Thr Glu Gly Gly Asn Val Tyr Ala Thr Gly

100 105 110

Gly Asn Asn Glu Gly Gln Leu Gly Leu Gly Asp Thr Glu Glu Arg Asn

115 120 125

Thr Phe His Val Ile Ser Phe Phe Thr Ser Glu His Lys Ile Lys Gln

130 135 140

Leu Ser Ala Gly Ser Asn Thr Ser Ala Ala Leu Thr Glu Asp Gly Arg

145 150 155 160

Leu Phe Met Trp Gly Asp Asn Ser Glu Gly Gln Ile Gly Leu Lys Asn

165 170 175

Val Ser Asn Val Cys Val Pro Gln Gln Val Thr Ile Gly Lys Pro Val

180 185 190

Ser Trp Ile Ser Cys Gly Tyr Tyr His His Ser Ala Phe Val Thr Thr Thr Asp

195 200 205

Gly Glu Leu Tyr Val Phe Gly Glu Pro Glu Asn Gly Lys Leu Gly Leu

210 215 220

Pro Asn Gln Leu Leu Gly Asn His Arg Thr Pro Gln Leu Val Ser Glu

225 230 235 240

Ile Pro Glu Lys Val Ile Gln Val Ala Cys Gly Gly Glu His Thr Val

245 250 255

Val Leu Thr Glu Asn Ala Val Tyr Thr Phe Gly Leu Gly Gln Phe Gly

260 265 270

Gln Leu Gly Leu Gly Thr Phe Leu Phe Glu Thr Ser Glu Pro Lys Val

275 280 285

Ile Glu Asn Ile Arg Asp Gln Thr Ile Ser Tyr Ile Ser Cys Gly Glu

290 295 300

Asn His Thr Ala Leu Ile Thr Asp Ile Gly Leu Met Tyr Thr Phe Gly

305 310 315 320

Asp Gly Arg His Gly Lys Leu Gly Leu Gly Leu Glu Asn Phe Thr Asn

325 330 335

His Phe Ile Pro Thr Leu Cys Ser Asn Phe Leu Arg Phe Ile Val Lys

340 345 350

Leu Val Ala Cys Gly Gly Cys His Met Val Val Phe Ala Ala Pro His

355 360 365

Arg Gly Val Ala Lys Glu Ile Glu Phe Asp Glu Ile Asn Asp Thr Cys

370 375 380

Leu Ser Val Ala Thr Phe Leu Pro Tyr Ser Ser Leu Thr Ser Gly Asn

385 390 395 400

Val Leu Gln Arg Thr Leu Ser Ala Arg Met Arg Arg Arg Glu Arg Glu

405 410 415

Arg Ser Pro Asp Ser Phe Ser Met Arg Arg Thr Leu Pro Pro Ile Glu

420 425 430

Gly Thr Leu Gly Leu Ser Ala Cys Phe Leu Pro Asn Ser Val Phe Pro

435 440 445

Arg Cys Ser Glu Arg Asn Leu Gln Glu Ser Val Leu Ser Glu Gln Asp

450 455 460

Leu Met Gln Pro Glu Glu Pro Asp Tyr Leu Leu Asp Glu Met Thr Lys

465 470 475 480

Glu Ala Glu Ile Asp Asn Ser Ser Thr Val Glu Ser Leu Gly Glu Thr

485 490 495

Thr Asp Ile Leu Asn Met Thr His Ile Met Ser Leu Asn Ser Asn Glu

500 505 510

Lys Ser Leu Lys Leu Ser Pro Val Gln Lys Gln Lys Lys Gln Gln Thr

515 520 525

Ile Gly Glu Leu Thr Gln Asp Thr Ala Leu Thr Glu Asn Asp Asp Ser

530 535 540

Asp Glu Tyr Glu Glu Met Ser Glu Met Lys Glu Gly Lys Ala Cys Lys

545 550 555 560

Gln His Val Ser Gln Gly Ile Phe Met Thr Gln Pro Ala Thr Thr Ile

565 570 575

Glu Ala Phe Ser Asp Glu Glu Val Glu Ile Pro Glu Glu Lys Glu Gly

580 585 590

Ala Glu Asp Ser Lys Gly Asn Gly Ile Glu Glu Gln Glu Val Glu Ala

595 600 605

Asn Glu Glu Asn Val Lys Val His Gly Gly Arg Lys Glu Lys Thr Glu

610 615 620

Ile Leu Ser Asp Asp Leu Thr Asp Lys Ala Glu Val Ser Glu Gly Lys

625 630 635 640

Ala Lys Ser Val Gly Glu Ala Glu Asp Gly Pro Glu Gly Arg Gly Asp

645 650 655

Gly Thr Cys Glu Glu Gly Ser Ser Gly Ala Glu His Trp Gln Asp Glu

660 665 670

Glu Arg Glu Lys Gly Glu Lys Asp Lys Gly Arg Gly Glu Met Glu Arg

675 680 685

Pro Gly Glu Gly Glu Lys Glu Leu Ala Glu Lys Glu Glu Trp Lys Lys

690 695 700

Arg Asp Gly Glu Glu Gln Glu Gln Lys Glu Arg Glu Gln Gly His Gln

705 710 715 720

Lys Glu Arg Asn Gln Glu Met Glu Glu Gly Gly Glu Glu Glu His Gly

725 730 735

Glu Gly Glu Glu Glu Glu Glu Gly Asp Arg Glu Glu Glu Glu Glu Lys Glu

740 745 750

Gly Glu Gly Lys Glu Glu Gly Glu Gly Glu Glu Val Glu Gly Glu Arg

755 760 765

Glu Lys Glu Glu Gly Glu Arg Lys Lys Glu Glu Arg Ala Gly Lys Glu

770 775 780

Glu Lys Gly Glu Glu Glu Glu Gly Asp Gln Gly Glu Gly Glu Glu Glu Glu

785 790 795 800

Thr Glu Gly Arg Gly Glu Glu Lys Glu Glu Gly Gly Glu Val Glu Gly

805 810 815

Gly Glu Val Glu Glu Gly Lys Gly Glu Arg Glu Glu Glu Glu Glu

820 825 830

Gly Glu Gly Glu Glu Glu Glu Gly Glu Gly Glu Glu Glu Glu Glu Gly Glu

835 840 845

Gly Glu Glu Glu Glu Gly Glu Gly Lys Gly Glu Glu Glu Gly Glu Glu

850 855 860

Gly Glu Gly Glu Glu Glu Gly Glu Glu Gly Glu Gly Glu Gly Glu Glu

865 870 875 880

Glu Glu Gly Glu Gly Glu Gly Glu Glu Glu Gly Glu Gly Glu Gly Glu

885 890 895

Glu Glu Glu Gly Glu Gly Glu Gly Glu Glu Glu Gly Glu Gly Glu Gly

900 905 910

Glu Glu Glu Glu Gly Glu Gly Lys Gly Glu Glu Glu Gly Glu Glu Glu Gly

915 920 925

Glu Gly Glu Gly Glu Glu Glu Glu Gly Glu Gly Glu Gly Glu Asp Gly

930 935 940

Glu Gly Glu Gly Glu Glu Glu Glu Gly Glu Trp Glu Gly Glu Glu Glu

945 950 955 960

Glu Gly Glu Gly Glu Gly Glu Glu Glu Gly Glu Gly Glu Gly Glu Glu

965 970 975

Gly Glu Gly Glu Gly Glu Glu Glu Glu Gly Glu Gly Glu Gly Glu Glu

980 985 990

Glu Glu Gly Glu Glu Glu Glu Gly Glu Glu Glu Gly Glu Gly Glu Glu Glu

995 1000 1005

Gly Glu Gly Glu Gly Glu Glu Glu Glu Glu Gly Glu Val Glu Gly

1010 1015 1020

Glu Val Glu Gly Glu Glu Gly Glu Gly Glu Gly Glu Glu Glu Glu

1025 1030 1035

Gly Glu Glu Glu Gly Glu Glu Arg Glu Lys Glu Gly Glu Gly Glu

1040 1045 1050

Glu Asn Arg Arg Asn Arg Glu Glu Glu Glu Glu Glu Glu Gly Lys

1055 1060 1065

Tyr Gln Glu Thr Gly Glu Glu Glu Asn Glu Arg Gln Asp Gly Glu

1070 1075 1080

Glu Tyr Lys Lys Val Ser Lys Ile Lys Gly Ser Val Lys Tyr Gly

1085 1090 1095

Lys His Lys Thr Tyr Tyr Gln Lys Lys Ser Val Thr Asn Thr Gln Gly

1100 1105 1110

Asn Gly Lys Glu Gln Arg Ser Lys Met Pro Val Gln Ser Lys Arg

1115 1120 1125

Leu Leu Lys Asn Gly Pro Ser Gly Ser Lys Lys Phe Trp Asn Asn

1130 1135 1140

Val Leu Pro His Tyr Leu Glu Leu Lys

1145 1150

<210> 3

<211> 3459

<212> DNA

<213> Homo sapiens

<400> 3

atgagggagc cggaagagct gatgcccgat tcgggtgctg tgtttacatt tgggaaaagt 60

aaatttgctg aaaataatcc cggtaaattc tggtttaaaa atgatgtccc tgtacatctt 120

tcatgtggag atgaacattc tgctgttgtt accggaaata ataaacttta catgtttggc 180

agtaacaact ggggtcagtt aggattagga tcaaagtcag ccatcagcaa gccaacatgt 240

gtcaaagctc taaaacctga aaaagtgaaa ttagctgcct gtggaaggaa ccacaccctg 300

gtgtcaacag aaggaggcaa tgtatatgca actggtggaa ataatgaagg acagttgggg 360

cttggtgaca ccgaagaaag aaacactttt catgtaatta gcttttttac atccgagcat 420

aagattaagc agctgtctgc tggatctaat acttcagctg ccctaactga ggatggaaga 480

ctttttatgt ggggtgacaa ttccgaaggg caaattggtt taaaaaatgt aagtaatgtc 540

tgtgtccctc agcaagtgac cattgggaaa cctgtctcct ggatctcttg tggatattac 600

cattcagctt ttgtaacaac agatggtgag ctatatgtgt ttggagaacc tgagaatggg 660

aagttaggtc ttcccaatca gctcctgggc aatcacagaa caccccagct ggtgtctgaa 720

attccggaga aggtgatcca agtagcctgt ggtggagagc atactgtggt tctcacggag 780

aatgctgtgt atacctttgg gctgggacaa tttggtcagc tgggtcttgg cacttttctt 840

tttgaaactt cagaacccaa agtcattgag aatattaggg atcaaacaat aagttatatt 900

tcttgtggag aaaatcacac agctttgata acagatatcg gccttatgta tacttttgga 960

gatggtcgcc acggaaaatt aggacttgga ctggagaatt ttaccaatca cttcattcct 1020

actttgtgct ctaatttttt gaggtttata gttaaattgg ttgcttgtgg tggatgtcac 1080

atggtagttt ttgctgctcc tcatcgtggt gtggcaaaag aaattgaatt cgatgaaata 1140

aatgatactt gcttatctgt ggcgactttt ctgccgtata gcagtttaac ctcaggaaat 1200

gtactgcaga ggactctatc agcacgtatg cggcgaagag agagggag gtctccagat 1260

tctttttcaa tgaggagaac actacctcca atagaaggga ctcttggcct ttctgcttgt 1320

tttctcccca attcagtctt tccacgatgt tctgagagaa acctccaaga gagtgtctta 1380

tctgaacagg acctcatgca gccagaggaa ccagattatt tgctagatga aatgaccaaa 1440

gaagcagaga tagataattc ttcaactgta gaaagccttg gagaaactac tgatatctta 1500

aacatgacac acatcatgag cctgaattcc aatgaaaagt cattaaaatt atcaccagtt 1560

cagaaacaaa agaaacaaca aacaattggg gaactgacgc aggatacagc tcttactgaa 1620

aacgatgata gtgatgaata tgaagaaatg tcagaaatga aagaagggaa agcatgtaaa 1680

caacatgtgt cacaagggat tttcatgacg cagccagcta cgactatcga agcattttca 1740

gatgaggaag tagagatccc agaggagaag gaaggagcag aggattcaaa aggaaatgga 1800

atagaggagc aagaggtaga agcaaatgag gaaaatgtga aggtgcatgg aggaagaaag 1860

gagaaaacag agatcctatc agatgacctt acagacaaag cagaggtgag tgaaggcaag 1920

gcaaaatcag tgggagaagc agaggatggg cctgaaggta gaggggatgg aacctgtgag 1980

gaaggtagtt caggagcaga acactggcaa gatgaggaga gggagaaggg ggagaaagac 2040

aagggtagag gagaaatgga gaggccagga gagggagaga aggaactagc agagaaggaa 2100

gaatggaaga agagggatgg ggaagagcag gagcaaaagg agagggagca gggccatcag 2160

aaggaaagaa accaagagat ggaggaggga ggggaggagg agcatggaga aggagaagaa 2220

gaggagggag acagagaaga ggaagaagag aaggagggag aagggaaaga ggaaggagaa 2280

ggggaagaag tggagggaga acgtgaaaag gaggaaggag agaggaaaaa ggaggaaaga 2340

gcggggaagg aggagaaagg agggaagaa ggagaccaag gagaggggga agaggaggaa 2400

acagagggga gaggggagga aaaagaggag ggaggggaag tagagggagg ggaagtagag 2460

gaggggaaag gagagaggga agaggaagag gagagggtg agggggaaga ggaggaaggg 2520

gagggggaag aggaggaagg ggagggggaa gagggaag gagaagggaa aggggaggaa 2580

gaaggggaag aaggagaagg ggaggaagaa ggggaggaag gagaagggga gggggaagag 2640

gaggaaggag aaggggaggg agaagaggaa ggagaagggg agggaagaaga ggaggaagga 2700

gaaggggagg gagaagagga aggagaaggg gagggagaag aggaggaagg agaagggaaa 2760

ggggaggagg aaggagagga aggagaaggg gagggggaag aggagggaagg agaaggggaa 2820

ggggaggatg gagaagggga gggggaagag gaggaaggag aatgggaggg ggaagaggag 2880

gaaggagaag gggaggggga agaggaagga gaaggggaag gggaggaagg agaaggggag 2940

ggggaagagg aggaaggaga agggggagggg gaagaggagg aaggggaaga agaaggggag 3000

gaagaaggag agggagga agaaggggag ggagaagggg aggaagaaga ggaaggggaa 3060

gtggaagggg aggtggaagg ggaggaagga gagggggaag gagaggaaga ggaaggagag 3120

gaggaaggag aagaaaggga aaaggagggg gaaggagaag aaaacaggag gaacagagaa 3180

gaggaggagg aagaagaggg gaagtatcag gagacaggcg aagaagagaa tgaaaggcag 3240

gatggagagg agtacaaaaa agtgagcaaa ataaaggat ctgtgaaata tggcaaacat 3300

aaaacatatc aaaaaaagtc agttactaac acacagggaa atgggaaaga gcagaggtcc 3360

aaaatgccag tccagtcaaa acgactttta aaaaacgggc catcaggttc caaaaagttc 3420

tggaataatg tattaccaca ttacttggaa ttgaagtaa 3459

<210> 4

<211> 199

<212> DNA

<213> Artificial sequence

<220>

<223> hGRK bootloader

<400>  4

gggccccaga agcctggtgg ttgtttgtcc ttctcagggg aaaagtgagg cggccccttg 60

gaggaagggg ccgggcagaa tgatctaatc ggattccaag cagctcaggg gattgtcttt 120

ttctagcacc ttcttgccac tcctaagcgt cctccgtgac cccggctggg atttagcctg 180

gtgctgtgtc agccccggg                               199

<210> 5

<211> 4303

<212> DNA

<213> Artificial sequence

<220>

<223> cohRPGR expression cassette

<400> 5

ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60

cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120

gccaactcca tcactagggg ttcctatcga ttgaattccc cggggatccg ggccccagaa 180

gcctggtggt tgtttgtcct tctcagggga aaagtgaggc ggccccttgg aggaaggggc 240

cgggcagaat gatctaatcg gattccaagc agctcagggg attgtctttt tctagcacct 300

tcttgccact cctaagcgtc ctccgtgacc ccggctggga tttagcctgg tgctgtgtca 360

gccccgggtc tagagtcgac ctgcagaagc ttccaccatg agagaacccg aggaactgat 420

gcccgactct ggcgccgtgt ttaccttcgg caagagcaag ttcgccgaga acaaccccgg 480

caagttctgg ttcaagaacg acgtgccagt gcacctgagc tgcggagatg aacactctgc 540

cgtggtcacc ggcaacaaca agctgtacat gttcggcagc aacaactggg gccagctcgg 600

cctggggatct aagtctgcca tcagcaagcc tacctgcgtg aaggccctga agcctgagaa 660

agtgaaactg gccgcctgcg gcagaaatca caccctggtt tctaccgaag gcggcaatgt 720

gtatgccacc ggcggaaaca atgagggaca gcttggactg ggcgacaccg aggaaagaaa 780

caccttccac gtgatcagct ttttcaccag cgagcacaag atcaagcagc tgagcgccgg 840

ctctaatacc tctgccgctc tgacagagga cggcagactg tttatgtggg gcgacaattc 900

tgagggccag atcggactga agaacgtgtc caatgtgtgc gtgccccagc aagtgacaat 960

cggcaagcct gtgtcttgga tcagctgcgg ctactaccac agcgcctttg tgacaaccga 1020

tggcgagctg tatgtgttcg gcgagccaga gaatggcaag ctgggactgc ctaaccagct 1080

gctgggcaat cacagaaccc ctcagctggt gtctgagatc cccgaaaaag tgatccaggt 1140

ggcctgtggc ggagagcaca cagtggtgct gacagagaat gccgtgtaca cctttggcct 1200

gggccagttt ggacaactcg gactgggaac cttcctgttc gagacaagcg agcccaaagt 1260

gatcgagaac atccgggacc agaccatcag ctacatcagc tgtggcgaga accacacagc 1320

cctgatcaca gacatcggcc tgatgtacac attcggcgac ggaaggcatg gaaagctcgg 1380

acttggcctg gaaaacttca ccaaccactt catccctacg ctgtgcagca acttcctgcg 1440

gttcattgtg aagctggtgg cctgcggagg atgccacatg gtggtttttg ctgcccctca 1500

cagaggcgtg gccaaagaga ttgagttcga cgagatcaac gatacctgcc tgagcgtggc 1560

caccttcctg ccttacagca gcctgacatc tggcaacgtg ctgcagagga cactgagcgc 1620

cagaatgcgc agacgggaaa gagagagaag ccccgacagc ttcagcatga gaagaaccct 1680

gcctccaatc gaggcacac tgggcctgtc tgcctgcttt ctgcctaaca gcgtgttccc 1740

cagatgcagc gagagaaacc tgcaagagag cgtgctgagc gagcaggatc tgatgcagcc 1800

tgaggaaccc gactacctgc tggacgagat gaccaaagag gccgagatcg acaacagcag 1860

cacagtggaa agcctgggcg agacaaccga catcctgaac atgacccaca tcatgagcct 1920

gaacagcaac gagaagtctc tgaagctgag ccccgtgcag aagcagaaga agcagcagac 1980

catcggcgag ctgacacagg atactgcct gaccgagaac gacgacagcg acgagtacga 2040

agagatgagc gagatgaagg aaggcaaggc ctgcaagcag cacgtgtccc agggcatctt 2100

tatgacccag cctgccacca ccatcgaggc cttttccgac gaggaagtgg aaatccccga 2160

ggaaaaagag ggcgccgagg acagcaaagg caacggcatt gaggaacaag aggtggaagc 2220

caacgaagag aacgtgaagg tgcacggcgg acggaaagaa aagaccgaga tcctgagcga 2280

cgacctgacc gataaggccg aggtttccga gggcaaagcc aagtctgtgg gagaagccga 2340

ggatggacct gaaggccgcg gagatggaac ctgtgaagaa ggatctagcg gagccgagca 2400

ctggcaggat gaggaacgcg agaagggcga gaaagacaaa ggcagaggcg agatggaaag 2460

acccggcgag ggcgaaaaag agctggccga gaaagaggaa tggaagaaac gcgacggcga 2520

agaacaagag cagaaagaaa gagagcaggg ccaccagaaa gaacggaatc aagagatgga 2580

agaaggcggc gaggaagaac acggcgaagg ggaagaagag gaaggcgacc gagaggaaga 2640

agaagagaaa gaaggcgaag gcaaagaaga aggcgagggc gaagaggtgg aaggcgagcg 2700

tgaaaaagaa gagggcgaac gcaagaaaga agaacgcgcc ggaaaagagg aaaaaggcga 2760

ggaagagggc gaccaaggcg aaggcgagga agaagaaact gaaggcagag gggaagagaa 2820

agaggaaggc ggcgaagtcg aaggcggaga ggttgaagaa ggcaaaggcg agcgagaaga 2880

ggaagaagaa gaaggcgaag gcgaggaaga ggaaggcgaa ggcgaagagg aagaaggcga 2940

aggggaagaa gaagaaggcg aaggcaaggg cgaagaggag ggcgaagaag gcgagggcga 3000

agaggagggc gaagaaggcg aaggcgaggg cgaagaagaa gaaggcgaag gcgaaggcga 3060

ggaagaaggc gaaggcgaag gggaagaaga ggaaggcgaa ggcgaaggcg aagaagaagg 3120

cgaaggcgag ggcgaagagg aagaaggcga aggcaaaggg gaagaagaag gcgaggaagg 3180

cgaaggcgaa ggcgaggaag aagaaggcga aggcgagggc gaagatggcg aaggcgaagg 3240

cgaagaggaa gagggcgagt gggagggcga agaagaggaa ggcgaaggcg agggcgaaga 3300

ggaaggcgaa ggcgagggcg aagaaggcga aggcgaaggc gaggaagagg aaggcgaagg 3360

cgaaggggaa gaagaagagg gcgaagaaga aggcgaagag gaaggcgaag gggaagaaga 3420

aggcgaaggc gaaggcgaag aagaggaaga gggcgaagtt gaaggcgagg ttgagggcga 3480

agaaggcgaa ggcgaagggg aagaagaaga aggcgaggaa gaaggggaag agagaaaa 3540

agaaggcgag ggcgaagaaa accgccggaa ccgcgaagag gaagaggaag aagagggcaa 3600

gtaccaagag actggcgagg aagagaacga gcggcaggat ggcgaagagt acaagaaggt 3660

gtccaagatc aagggcagcg tgaagtacgg caagcacaag acctaccaga agaagtccgt 3720

caccaacacg caaggcaatg gaaaagaaca gcggagcaag atgcccgtgc agtccaagag 3780

gctgctgaag aatggcccta gcggcagcaa gaaattctgg aacaatgtgc tgccccacta 3840

cctcgagctg aagtgagcct cgagcagcgc tgctcgagag atctgcggcc gcgagctcgg 3900

ggatccagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg 3960

aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag 4020

ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga 4080

ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga 4140

tcaatgcatc ctagccggag gaacccctag tgatggagtt ggccactccc tctctgcgcg 4200

ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc 4260

cggcctcagt gagcgagcga gcgcgcagag aggagtggc caa 4303

<210> 6

<211> 145

<212> DNA

<213> Adeno-associated virus 2

<400> 6

ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60

cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120

gccaactcca tcactagggg ttcct 145

<210> 7

<211> 145

<212> DNA

<213> Adeno-associated virus 2

<400> 7

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc 120

gagcgcgcag agagggagtg gccaa                           145

<210> 8

<211>  245

<212> DNA

<213> Artificial sequence

<220>

<223> SV40 polyadenylation sequence

<400> 8

ggggatccag acatgataag atatacattgat gagtttggac aaaccacaac tagaatgcag 60

tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata 120

agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg 180

gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat 240

gatca 245

<210> 9

<211> 745

<212> PRT

<213> Artificial sequence

<220>

<223> rAAV variant capsid protein

<400>  9

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Ala Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Ala Ile Ser Asp

580 585 590

Gln Thr Lys His Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 10

<211> 3459

<212> DNA

<213> Artificial sequence

<220>

<223> RPGR, the codon optimizer

<400> 10

atgagagagc ctgaagagct gatgcctgat agcggagcag tgtttacctt tgggaagagc 60

aagttcgcag agaataaccc tgggaaattc tggtttaaga acgacgtgcc cgtgcacctg 120

agctgtggcg atgagcactc cgccgtggtg acaggcaaca ataagctgta catgttcggc 180

tctaacaatt ggggacagct gggcctggga agcaagtccg ccatcagcaa gccaacctgc 240

gtgaaggccc tgaagcccga gaaggtgaag ctggccgcct gtggcagaaa ccacacactg 300

gtgagcaccg agggaggaaa cgtgtacgca acaggaggca acaatgaagg ccagctgggc 360

ctgggcgaca cagaggagag gaataccttt cacgtgatca gcttctttac ctccgagcac 420

aagatcaagc agctgtccgc cggctctaac acaagcgccg ccctgaccga ggacggccgc 480

ctgttcatgt ggggcgataa tagcgagggc cagatcggcc tgaagaacgt gtccaacgtg 540

tgcgtgcctc agcaggtgac catcggcaag ccagtgtcct ggatctcttg tggctactat 600

cacagcgcct tcgtgaccac agatggcgag ctgtacgtgt ttggagagcc agagaacggc 660

aagctgggcc tgcctaacca gctgctgggc aatcaccgga caccccagct ggtgtccgag 720

atccctgaga aagtgatcca ggtggcatgc ggaggagagc acaagtggt gctgaccgag 780

aatgccgtgt ataccttcgg cctggggacag tttggacagc tgggcctggg cacattcctg 840

tttgagacaa gcgagccaaa agtgatcgag aacatccgcg accagacaat cagctacatc 900

tcctgcggcg agaatcacac agccctgatc accgacatcg gcctgatgta tacctttggc 960

gatggccggc acggcaagct gggcctgggc ctggagaact tcacaaatca ctttatcccc 1020

accctgtgct ctaacttcct gcggttcatc gtgaagctgg tggcctgcgg cggctgtcac 1080

atggtggtgt tcgcagcacc tcacagggga gtggccaagg agatcgagtt tgacgagatc 1140

aacgatacat gcctgtccgt ggccaccttc ctgccataca gctccctgac atccggcaat 1200

gtgctgcagc gcaccctgtc tgccaggatg cggagaaggg agagggagcg gtcccctgac 1260

tctttcagca tgaggcggac actgccacct atcgagggca ccctgggcct gtctgcctgc 1320

ttcctgccta acagcgtgtt cccaagatgt agcgagagga atctgcagga gtctgtgctg 1380

agcgagcagg atctgatgca gccagaggag cccgactacc tgctggatga gatgacaaag 1440

gaggccgaga tcgacaactc tagcaccgtg gagagcctgg gcgagacaac agatatcctg 1500

aatatgacac acatcatgtc cctgaactct aatgagaagt ctctgaagct gagcccagtg 1560

cagaagcaga agaagcagca gaccatcggc gagctgaccc aggacacagc cctgaccgag 1620

aacgacgatt ctgatgagta tgaggagatg agcgagatga aggagggcaa ggcctgtaag 1680

cagcacgtgt cccagggcat cttcatgacc cagccagcca ccacaatcga ggccttttct 1740

gacgaagagg tggagatccc cgaggagaag gagggcgccg aggatagcaa gggcaatggc 1800

atcgaggagc aggaggtgga ggccaacgag gagaatgtga aggtgcacgg cggcagaaag 1860

gagaagacag agatcctgtc cgacgatctg accgacaagg ccgaggtgtc cgagggcaag 1920

gccaagtctg tgggagaggc agaggacgga ccagagggac gcggcgatgg aacctgcgag 1980

gagggatcct ctggagcaga gcactggcag gacgaagaaa gagagaaggg cgagaaggat 2040

aagggcagag gagagatgga gagcctgga gagggagaga aggagctggc agagaaggag 2100

gagtggaaga agagggacgg cgaggagcag gagcagaagg agagagagca gggccaccag 2160

aaggagagga acccaggagat ggaggaggga ggagaggagg agcacggcga gggagaggag 2220

gaggagggcg atagagagga agaagaggag aaggagggag agggcaagga ggaaggcgag 2280

ggagaggagg tggagggaga aagggagaag gaggagggag agcgcaagaa ggaagaaaga 2340

gcaggcaagg aagagaaggg agaggagggag ggcgatcagg gcgaaggaga ggaggaggag 2400

acagagggaa ggggagagga gaaggaggag ggaggagagg tcgaaggagg agaagtggag 2460

gagggcaagg gcgaaagaga agaggaggag gaggaaggcg agggcgaaga agaggagggc 2520

gagggcgagg aagaagaggg cgagggcgaa gaggaagaag gcgagggcaa gggcgaggag 2580

gagggcgaag aaggcgaagg ggaggaggag ggcgaagagg gagagggcga gggcgaggag 2640

gaagaaggcg aaggcgaagg cgaagaagaa ggagaaggag agggcgaaga ggaggaaggc 2700

gaaggagaag gagaggagga aggagaaggg gagggcgaag aggaggaggg agaaggcaag 2760

ggagaagaag aaggcgaaga aggcgaggga gaaggcgagg aagaagaagg cgagggag 2820

ggagaggacg gcgaaggcga gggcgaggaa gaggaaggag agtgggaggg cgaggaagag 2880

gagggagaag gagaaggcga agaagaaggg gaaggagagg gcgaggaagg agaaggcgaa 2940

ggcgaagagg aggaggggga aggggagggc gaggaggaag agggaaga ggaaggcgaa 3000

gaagaggggag aaggcgaaga ggaaggagaa ggcgagggag aagaagagga ggagggcgag 3060

gtcgaaggcg aggtggaggg cgaagagggg gaaggcgaag gcgaggagga ggaaggggaa 3120

gaagaaggcg aggagagaga gaaagaaggc gaggcgagg agaacagaag gaatcgcgaa 3180

gaagaagagg aagaagaggg caagtaccag gagacaggcg aggaggagaa cgagcggcag 3240

gatggcgagg agtataagaa ggtgtccaag atcaagggct ctgtgaagta cggcaagcac 3300

aagacctatc agaagaagag cgtgaccaac acaagggca atggcaagga gcagcgcagc 3360

aagatgcctg tgcagtccaa gcggctgctg aagaatggcc cctctgggag caagaagttt 3420

tggaataatg tcctgccaca ctacctggag ctgaaatga 3459

Claims (41)

1. A nucleic acid encoding the human retinitis pigmentosa GTPase regulatory factor (RPGR) protein of SEQ ID NO:2 and codon-optimized for expression in humans, said nucleic acid comprising a nucleotide sequence as shown in SEQ ID NO:1 or comprising a nucleotide sequence having at least 95% identity with it, said nucleic acid being expressed at a higher level than the expression level in cells otherwise identical to the wild-type RPGR nucleotide sequence of SEQ ID NO:3. 2. The nucleic acid according to claim 1, wherein the nucleotide sequence has a codon fitness index of at least 0.89. 3. The nucleic acid according to claim 1, comprising the nucleotide sequence shown in SEQ ID NO:1. 4. An expression cassette comprising a nucleic acid according to any one of claims 1 to 3 and an expression control sequence operatively linked to and heterologous to the nucleic acid sequence. 5. The expression box of claim 4, wherein the expression control sequence is a constitutive promoter. 6. The expression cassette of claim 4, wherein the expression control sequence is a promoter that directs the preferential expression of the nucleic acid in rods and cones, preferably a human G protein-coupled receptor rhodopsin kinase 1 (hGRK) promoter, comprising a nucleotide sequence as shown in SEQ ID NO:4 or a sequence having at least 90%, at least 95%, or at least 98% identity with it. 7. The expression cassette of claim 6, comprising from 5' to 3': (a) an AAV2 terminal repeat, (b) an hGRK promoter, (c) a codon-optimized RPGRorf15 gene of SEQ ID NO:1, (d) an SV40 polyadenylated sequence, and (e) an AAV2 terminal repeat. 8. The expression cassette of claim 7, wherein the 5'AAV2 terminal repeat has a nucleotide sequence as shown in SEQ ID NO:6 and/or wherein the hGRK promoter has a nucleotide sequence as shown in SEQ ID NO:4 and/or wherein the SV40 polyadenylated sequence has a nucleotide sequence as shown in SEQ ID NO:8 and/or wherein the 3'AAV2 terminal repeat has a nucleotide sequence as shown in SEQ ID NO:7. 9. The expression cassette of claim 8, comprising or consisting of the nucleotide sequence of SEQ ID NO:5 or a sequence having at least 90%, at least 95%, or at least 98% identity with it. 10. A vector comprising the nucleic acid according to any one of claims 1 to 3 or the expression cassette according to any one of claims 4 to 9. 11. The vector of claim 10, wherein the vector is a recombinant gland-associated (rAAV) vector. 12. The vector of claim 11, wherein the rAAV vector comprises an AAV capsid of serotype 2, 5 or 8 or a variant thereof. 13. The vector of claim 12, wherein the rAAV vector comprises an AAV2 capsid or a variant thereof. 14. The vector of claim 13, wherein the rAAV vector comprises an AAV2 capsid variant containing a capsid protein comprising or consisting of the sequence of SEQ ID NO:9. 15. The vector according to any one of claims 11-14, wherein the rAAV vector comprises nucleic acid, the nucleic acid comprising from 5' to 3': (a) an AAV2 terminal repeat, (b) an hGRK promoter, (c) a codon-optimized RPGRorf15 gene of SEQ ID NO:1, and (d) an AAV2 terminal repeat. 16. The vector of claim 15, wherein the 5'AAV2 terminal repeat has a nucleotide sequence as shown in SEQ ID NO:6 and/or wherein the hGRK promoter has a nucleotide sequence as shown in SEQ ID NO:4 and/or wherein the SV40 polyadenylated sequence has a nucleotide sequence as shown in SEQ ID NO:8 and/or wherein the 3'AAV2 terminal repeat has a nucleotide sequence as shown in SEQ ID NO:7. 17. The vector of claim 16, wherein the rAAV vector comprises nucleic acid, the nucleic acid comprising the nucleotide sequence of SEQ ID NO:5 or a sequence having at least 90%, at least 95%, or at least 98% identity with it. 18. The vector of claim 17, wherein the rAAV vector comprises (i) a capsid containing a capsid protein comprising or consisting of the sequence of SEQ ID NO:9, and (ii) a nucleic acid comprising or consisting of the nucleotide sequence of SEQ ID NO:5. 19. A host cell comprising the nucleic acid according to any one of claims 1 to 3 or the expression cassette according to any one of claims 4 to 9. 20. The host cell of claim 19, wherein the host cell is a mammalian cell. 21. The host cell of claim 19 or 20, wherein the host cell is a CHO cell, HEK293 cell, HEK293T cell, HeLa cell, BHK21 cell, or Vero cell and/or wherein the host cell is grown in suspension culture or cell culture chamber culture and/or wherein the host cell is a photoreceptor cell, retinal ganglion cell, glial cell, bipolar cell, amacrine cell, horizontal cell, or retinal pigment epithelial cell. 22. A method for treating XLRP in a subject in need, comprising administering to the subject a therapeutically effective amount of a nucleic acid according to any one of claims 1-3, an expression cassette according to any one of claims 4-9, or a vector according to any one of claims 10-18. 23. A method for treating XLRP in a subject in need, comprising administering an infectious rAAV to the subject, the infectious rAAV comprising (i) an AAV capsid and (ii) a nucleic acid, the nucleic acid comprising from 5' to 3': (a) an AAV2 terminal repeat, (b) an hGRK promoter, (c) a codon-optimized RPGRorf15 gene of SEQ ID NO:1, (d) an SV40 polyadenylated sequence, and (e) an AAV2 terminal repeat. 24. The method of claim 23, wherein the 5'AAV2 terminal repeat has a nucleotide sequence as shown in SEQ ID NO:6 and/or wherein the hGRK promoter has a nucleotide sequence as shown in SEQ ID NO:4 and/or wherein the SV40 polyadenylated sequence has a nucleotide sequence as shown in SEQ ID NO:8 and/or wherein the 3'AAV2 terminal repeat has a nucleotide sequence as shown in SEQ ID NO:7. 25. The method according to claim 23 or 24, wherein the rAAV comprises (i) a capsid containing a capsid protein comprising or consisting of the sequence of SEQ ID NO:9, and (ii) a nucleic acid comprising or consisting of the nucleotide sequence of SEQ ID NO:5. 26. The method according to any one of claims 22-25, wherein the nucleic acid or vector is administered to the subject via periocular, intravitreal, choroidal, or subretinal injection and/or wherein the vector is administered to the subject at a dose of approximately ^10¹⁰ vector genomes (vg)/eye to approximately ^10¹³ vg/eye, preferably approximately 1 × ^10¹¹ vg/eye to approximately 5 × ^10¹² vg/eye, more preferably at a dose of approximately 3 × ^10¹¹ vg/eye or at a dose of approximately 1 × ^10¹² vg/eye. 27. A nucleic acid according to any one of claims 1-3, an expression cassette according to any one of claims 4-9, or a vector according to any one of claims 10-18 for the treatment of XLRP. 28. A nucleic acid according to any one of claims 1-3, an expression cassette according to any one of claims 4-9, or a vector according to any one of claims 10-18 for manufacturing a medicament for treating XLRP. 29. A nucleic acid, expression cassette, or vector for use according to claim 27 or 28, wherein the nucleic acid or vector is administered by injection periocularly, intravitreally, choroidally, or subretinal and/or wherein the vector is administered at a dose of approximately ^10¹⁰ vector genomes (vg)/eye to approximately ^10¹³ vg/eye, preferably approximately 1 × ^10¹¹ vg/eye to approximately 5 × ^10¹² vg/eye, more preferably at a dose of approximately 3 × ^10¹¹ vg/eye or at a dose of approximately 1 × ^10¹² vg/eye. 30. An infectious rAAV for treating XLRP, comprising (i) an AAV capsid and (ii) a nucleic acid, said nucleic acid comprising from 5' to 3': (a) an AAV2 terminal repeat, (b) an hGRK promoter, (c) a codon-optimized RPGRorf125 gene of SEQ ID NO:1, (d) an SV40 polyadenylated sequence, and (e) an AAV2 terminal repeat. 31. An infectious rAAV for manufacturing a medicament for treating XLRP, comprising (i) an AAV capsid and (ii) a nucleic acid, said nucleic acid comprising from 5' to 3': (a) an AAV 2-terminal repeat, (b) an hGRK promoter, (c) a codon-optimized RPGRorf125 gene of SEQ ID NO:1, (d) an SV40 polyadenylated sequence, and (e) an AAV 2-terminal repeat. 32. The infectious rAAV according to claim 30 or 31, wherein the 5'AAV2 terminal repeat has a nucleotide sequence as shown in SEQ ID NO:6 and/or wherein the hGRK promoter has a nucleotide sequence as shown in SEQ ID NO:4 and/or wherein the SV40 polyadenylated sequence has a nucleotide sequence as shown in SEQ ID NO:8 and/or wherein the 3'AAV2 terminal repeat has a nucleotide sequence as shown in SEQ ID NO:7. 33. The infectious rAAV of claim 32, wherein the rAAV comprises (i) a capsid containing a capsid protein comprising or consisting of the sequence of SEQ ID NO:9, and (ii) a nucleic acid comprising or consisting of the nucleotide sequence of SEQ ID NO:5. 34. An infectious rAAV for use according to any one of claims 30-33, wherein the rAAV is administered by intravitreal injection and/or wherein the vector is administered at a dose of about ^10¹⁰ vector genomes (vg)/eye to about ^10¹³ vg/eye, preferably about 1 × ^10¹¹ vg/eye to about 5 × ^10¹² vg/eye, more preferably at a dose of about 3 × ^10¹¹ vg/eye or at a dose of about 1 × ^10¹² vg/eye. 35. A method for treating a disease or condition mediated by reduced RPGrf15 levels in mammals, the method comprising administering a therapeutically effective amount of a nucleic acid according to any one of claims 1-3, an expression cassette according to any one of claims 4-9, or a vector according to any one of claims 10-18. 36. A method for increasing RPGrf15 levels in a mammal, the method comprising administering to the mammal a nucleic acid according to any one of claims 1-3, an expression cassette according to any one of claims 4-9, or a vector according to any one of claims 10-18. 37. A pharmaceutical composition comprising a nucleic acid according to any one of claims 1-3, an expression cassette according to any one of claims 4-9, or a carrier according to any one of claims 10-18, and at least one pharmaceutically acceptable excipient. 38. A pharmaceutical composition comprising infectious rAAV, said infectious rAAV comprising (i) an AAV capsid and (ii) a nucleic acid, said nucleic acid comprising from 5' to 3': (a) an AAV2 terminal repeat, (b) an hGRK promoter, (c) a codon-optimized RPGRorf gene of SEQ ID NO:1, (d) an SV40 polyadenylated sequence, and (e) an AAV2 terminal repeat. 39. The pharmaceutical composition of claim 38, wherein the 5'AAV2 terminal repeat has a nucleotide sequence as shown in SEQ ID NO:6 and/or wherein the hGRK promoter has a nucleotide sequence as shown in SEQ ID NO:4 and/or wherein the SV40 polyadenylated sequence has a nucleotide sequence as shown in SEQ ID NO:8 and/or wherein the 3'AAV2 terminal repeat has a nucleotide sequence as shown in SEQ ID NO:7. 40. The pharmaceutical composition of claim 39, wherein the rAAV comprises (i) a capsid containing a capsid protein comprising or consisting of the sequence of SEQ ID NO:9, and (ii) a nucleic acid comprising or consisting of the nucleotide sequence of SEQ ID NO:5. 41. The pharmaceutical composition according to any one of claims 38-40, wherein the pharmaceutical composition comprises ^10⁹ vg to ^10¹⁴ vg of rAAV, preferably ^10¹⁰ vg to ^10¹³ vg of rAAV, more preferably comprising about 3 × ^10¹¹ vg or about 1 × ^10¹² vg of rAAV.