HK40101500A - Codon optimized rep1 genes and uses thereof - Google Patents

Description

Codon-optimized REP1 gene and its applications

Cross-references to related applications

This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/073,837, 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 (approximately 27KB in size) entitled “090400-5011-WO-Sequence-Listing”, created on or around July 28, 2021, contains the sequence list of this application and is hereby incorporated in its entirety by reference.

Background of the Invention

Achoroidemia is a rare, X-linked recessive form of inherited retinal degeneration that affects approximately 1 in 50,000 men. The disease causes progressive vision loss, beginning with night blindness in childhood, followed by peripheral vision loss, and progressing to central vision loss later in life. Progression continues throughout an individual's life, but the rate of change and the degree of vision loss can vary among affected individuals, even within the same family.

Achoroidal dysplasia is caused by a loss-of-function mutation in the CHM gene encoding Rab guard protein-1 (REP1), a protein involved in the lipid modification of Rab proteins. Although the full mechanism of the disease is not fully understood, the lack of functional proteins in the retina leads to cell death and the gradual degeneration of retinal pigment epithelial cells (RPE), photoreceptors, and the choroid.

Although there are currently no approved treatments for choroidal agenesis, several preclinical studies support the use of wild-type cDNA from CHM to salvage the disease phenotype. However, the suboptimal expression levels of wild-type sequences in human photoreceptors and RPE pose a challenge to gene therapy approaches for treating choroidal agenesis.

Invention Overview

Disclosed are codon-optimized nucleic acid molecules encoding the human Rab guardian protein-1 (REP1) 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 REP1 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 wild-type CHM nucleic acid sequences (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 CHM cDNA (GenBank accession number NM_000390.4; SEQ ID NO:3). In some embodiments, the codon-optimized nucleic acid molecules have a human codon fitness index of at least about 0.9, at least about 0.92, or at least about 0.94.

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 at least about 55%, at least about 57.5%, at least about 60%, or at least about 61%. In some aspects, the nucleic acid contains a G/C nucleotide percentage of about 55% to about 70%, about 57.5% to about 70%, or about 61% to about 70%.

In other embodiments, the nucleic acid relative to SEQ ID NO:3 includes one or more optimized parameters: frequency of the optimal codon; reduction of the maximum length of the direct repeat sequence; removal of restriction enzymes, including but not limited to removal of Bglll (AGATCT); removal of cis-acting elements, including but not limited to removal of destabilizing (ATTTA) elements.

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-specific or tissue-specific promoter that results in cell-specific expression of the nucleic acid, such as the vitrectomyces dystrophy 2 promoter, which selectively expresses the nucleic acid in RPE. In other aspects, the transcriptional control sequence is a constitutive promoter (e.g., CAG, CBA (chicken β-actin), CMV, or PGK promoter) that results in similar levels of nucleic acid expression in many cell types. In a preferred embodiment, the transcriptional control sequence comprises a CAG promoter containing (i) an early enhancer element of cytomegalovirus (CMV), (ii) a promoter, first exon, and first intron of the chicken β-actin gene, and (iii) a splice acceptor of the rabbit β-globin gene as described in Miyazaki et al., Gene 79(2):269-77 (1989). In a particularly preferred embodiment, the CAG 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.

ACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCC ACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCG AAGCGCGCGGCGGGCGGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGCCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTTCCTCCGGGCTGTAATTAGGCCTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGG CTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGGGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAA AGGCTGCGTGCGGGGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCG GGCCGGGGAGGGCTCGGGGGAGGGGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAG GAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTTCTTTTTCCTACAG (SEQ ID NO: 4).

The transcriptional control sequence may also include one or more elements downstream of the REP1 coding sequence, such as the marmot hepatitis virus posttranscriptional regulatory element (WPRE), which has been shown to enhance AAV transgene expression in the retina. In related embodiments, an expression cassette comprising a nucleic acid operatively linked to the expression control sequence 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 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 4, 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, 4, 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, or a vector comprising a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleotide sequence of SEQ ID NO:1 and encoding a polypeptide having the amino acid sequence of SEQ ID NO:2, 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 REP1 activity in human subjects, comprising administering to the subject a nucleic acid molecule or vector disclosed herein. In some embodiments, the retinal condition is choroidal agenesis.

Attached Figure Description

Figures 1A-D. Figures 1A and 1B show immunocytochemical analyses of iPSCs derived from CHM1 (Figure 1A) and CHM2 (Figure 1B) cells from patients with choroidal agenesis using antibodies against the pluripotent transcription factors NANOG, OCT4, and SOX2. Figures 1C and 1D show representative images of CHM1 (Figure 1C) and CHM2 (Figure 1D) cultures randomly differentiated into ectoderm, mesoderm, and endoderm cell lineages, indicated by the expression of TUJ1, α-smooth muscle actin (ASMA), and hepatocyte nuclear factor 4α (HNF4A).

Figures 2A-C and 2B illustrate immunocytochemical analysis of RPE cells derived from iPSCs derived from CHM1 (Figure 2A) and CHM2 (Figure 2B) patients with choroidal agenesis. RPE phenotypes after ICC differentiation and maturation for 45 days showed appropriate RPE transcription factor expression of melanin production-associated transcription factors (MITF) and orthodenticle homeobox 2 (OTX2), expression of the mature RPE cell marker RPE65, and expression of the tight junction marker occluder band 1 (ZO-1). For CHM1 images, the nuclei were counterstained with DAPI. Scale bar = 50 μm. Figure 2C is a diagram showing CHM1 and CHM2 RPEs phagocytizing at levels similar to wild-type (WT) RPEs. Furthermore, as seen from the reduced phagocytosis following αVβ5 inhibition, phagocytosis occurs via a known in vivo mechanism (αVβ5 integrin binding). For quantitative measurements, n=3; error bars ±S.D.; *P<0.05 compared to “ROS-free” condition; two-tailed t-test.

Figures 3A-B. Figure 3A: Shows Western blot images illustrating the levels of REP1 and housekeeping protein GADPH in RPE or normal iPSC-derived RPE cells after transduction with recombinant AAV viral particles carrying either a codon-optimized REP1 gene or an unmodified REP1 gene (driven by the CAG promoter in each case). Codon-optimized REP1 showed significantly higher protein expression levels. Figure 3B: Band intensity quantified and plotted as a ratio divided by GADPH. For quantitative measurements, n = 3; error bars ± S.D.; *p < 0.05 compared to WT-REP1; two-tailed t-test.

Figure 4 shows a schematic diagram of the functional isopreneation assay of the required components, including the biotinylated isoprene group that serves as the readout for the biochemical assay. Normal RPE cells with functional REP1 protein successfully promoted the isopreneation of the Rab27a GTPase, leading to the incorporation of the biotin group. In CHM RPE, cells lack the REP1 protein, which causes the accumulation of unsopreneated Rab27a GTPase protein.

Figures 5A-5D show the recovery of isoprenelation of Rab27a GTPase in CHM1 and CHM2 RPE cells transduced with rAAV viral particles containing codons optimized for REP1 (SEQ ID NO: 1) under the control of the CAG promoter. Figures 5A and 5B: Gel images showing REP1 protein levels and biotinylated isoprene donor incorporation as a measure of isoprenelation in cell lysates in transduced and untransduced CHM1 (Figure 5A) and CHM2 (Figure 5B) RPE cells (compared to RPE cells derived from normal iPSCs), as analyzed by Western blot. Figures 5C and 5D: Band intensity quantified and plotted as biotinylated Rab27a GTPase relative to the housekeeping protein GAPDH in CHM1 (Figure 5C) and CHM2 (Figure 5D) RPE cells. For quantitative measurements, n = 3; error bars ± S.D.; *p < 0.05 compared to untreated CHM RPE; two-tailed t-test.

Figures 6A-6C. Figure 6A: Immunostaining of CHM1 RPE with anti-REP1 and anti-RAB27A antibodies shows that CHM1 RPE lacks proper membrane localization of the Rab27a GTPase. Figures 6B and 6C: Delivery of codon-optimized REP1 of SEQ ID NO:1 via the rAAV vector corrected Rab27 GTPase membrane transport in CHM1 RPE (Figure 6B) and restored localization to the control RPE phenotype (Figure 6C).

Figure 7: Alignment of the optimized region of SEQ ID NO:1 with the DNA of the native REP1 of SEQ ID NO:3.

Figure 8 is a schematic diagram of the transgenic cassette contained in rAAV as described in Example 2 below. The transgenic cassette contains a 5'AAV2ITR, a CAG promoter, codon-optimized human CHM cDNA of SEQ ID NO:1, an SV40 polyadenylation signal, and a 3'AAV2ITR, and has the nucleotide sequence of SEQ ID NO:5.

Figure 9 illustrates the safety of 4D-110 (containing the transgenic cassette shown in Figure 8 and the capsid protein of SEQ ID NO: 9) after administration to the vitreous body of non-human primates, as assessed by aqueous flare, aqueous cells, and vitreous cells, based on quantitative observations of ocular inflammation. Fundus signs of transient, mild ocular inflammation were observed at high doses. These changes were in response to increased systemic steroid treatment. No adverse findings were considered to be associated with 4D-110. 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-110 NHP. LOD = Limit of Detection; for visualization purposes, all samples are plotted as LOD values.

Figure 11 shows the expression of REP1 transgenic mRNA in selected retinal, ocular, and non-ocular tissues at three autopsy time points in NHP after intravitreal administration of 4D-110. 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-110" refers to a recombinant AAV particle 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.

The term “having” as used in this document is equivalent to the term “containing” and is intended to be open-ended, allowing for additional elements.

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 has been 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 REP1) 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.

ATGGCTGATACACTGCCTTCTGAGTTTGATGTGATCGTGATTGGAACTGGACTGCCTGAGAGTATTATTGCTGCTGCTTGTAGTAGAAGCGGCCGGAGATGCTGCACGTGGACAGCAGATCC TACTATGGCGGCAACTGGGCCTCTTTCAGCTTTTCCGGCCTGCTGAGCTGGCTGAAGGAGTACCAGGAGAACTCCGACATCGTGTCTGATAGCCCCGTGTGGCAGGACCAGATCCTGGAGAATG AGGAGGCCATCGCCCTGTCCAGGAAGGATAAGACCATCCAGCACGTGGAGGTGTTCTGCTATGCCAGCCAGGACCTGCACGAGGATGTGGAGGAGGCAGGCGCCCTGCAGAAGAACCACGCCC TGGTGACCTCCGCCAATTCTACAGAGGCCGCCGACTCCGCCTTTCTGCCTACCGAGGATGAGTCCCTGTCTACAATGTCTTGTGAGATGCTGACCGAGCAGACACCTAGCTCCGATCCAGAGAA CGCCCTGGAGGTCAATGCGCCGAGGTGACCGGCGAGAAGGAGAACCACTGCGACGATAAGACCTGCGTGCCAAGCACATCCGCCGAGGACATGTCCGAGAACGTGCCTATCGCCGAGGATAC CACAGAGCAGCCAAAGAAGAATCGCATCACATACAGCCAGATCATCAAGGAGGGCAGGCGCTTCAATATCGACCTGGTGTCTAAGCTGCTGTACAGCCGGGGCCTGCTGATCGATCTGCTGATC AAGAGCAACGTGTCCCGCTATGCCGAGTTCAAGAATATCACCAGAATCCTGGCCTTTCGGGAGGGAAGAGTGGAGCAGGTGCCCTGCAGCAGAGCCGACGTGTTCAACTCCAAGCAGCTGACA ATGGTGGAGAAGAGGATGCTGATGAAGTTCCTGACATTTTGTATGGAGTACGAGAAGTATCCAGATGAGTACAAGGGCTATGAGGAGATCACCTTTTACGAGTATCTGAAGACCCAGAAGCTGA CACCCAATCTGCAGTACATCGTGATGCACTCCATCGCCATGACCTCTGAGACAGCCTCTAGCACCATCGACGGCCTGAAGGCCACAAAGAACTTCCTGCACTGCCTGGGCCGGTACGGCAATACACCCTTCCTGTTTCCTGTATGGCCAGGGCGAGCTGCCCCAGTGCTTCTGTAGAATGTGCGCCGTGTTTGGCGGCATCTATTGCCTGAGGCACTCTGTGCAGTGTCTGGTGGTGGACAAGGA GAGCCGCAAGTGTAAGGCCATCATCGATCAGTTTGGCCAGCGGATCATCTCTGAGCACTTCCTGGTGGAGGACAGCTACTTTCCTGAGAACATGTGCTCCAGGGTGCAGTATCGCCAGATCAG CCGGGCCGTGCTGATCACCGATAGATCCGTGCTGAAGACAGACAGCGATCAGCAGATCAGCATCCTGACCGTGCCAGCAGAGGAGCCAGGCACCTTCGCCGTGAGAGTGATCGAGCTGTGCTCC TCTACCATGACATGTATGAAGGGCACCTACCTGGTGCACCTGACCTGCACAAGCTCCAAGACAGCCCGCGAGGACCTGGAGAGCGTGGTGCAGAAGCTGTTCGTGCCCTACACCGAGATGGAG ATCGAGAACGAGCAGGTGGAGAAGCCTAGAATCCTGTGGGCCCTGTACTTCAACATGAGAGACTCTAGCGATATCTCTAGGAGCTGTTACAACGATCTGCCCTCTAACGTGTACGTGTGCAGCG GACCTGACTGTGGCCTGGGAAACGATAATGCCGTGAAGCAGGCCGAGACACTGTTCCAGGAGATTTGCCCTAACGAGGACTTTTGTCCCCCTCCACCCAATCCAGAGGATATCATCCTGGACGGCGATTCCCTGCAGCCAGAGGCCTCTGAGTCCTCTGCCATCCCCGAGGCCAATAGCGAAACATTCAAAGAAAGCACAAATCTGGGAAACCTGGAAGAAAGTAGTGAGTAA (SEQ ID NO: 1).

In some implementations, a codon-optimized sequence encoding human REP1 is provided, which lacks the TAA stop codon of SEQ ID NO:1 (i.e., consists of nucleotides 1-1959 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 REP1 polypeptide having the amino acid sequence of SEQ ID NO:2:

MADTLPSEFDVIVIGTGLPESIIIAAACSRSGRRVLHVDSRSYYGGNWASFSFSGLLSWLKEY

QENSDIVSDSPVWQDQILENEEAIALSRKDKTIQHVEVFCYASQDLHEDVEEAGALQKNHA

LVTSANSTEAADSAFLPTEDESLSTMSCEMLTEQTPSSDPENALEVNGAEVTGEKENHCDD

KTCVPSTSAEDMSENVPIAEDTTEQPKKNRITYSQIIKEGRRFNIDLVSKLLYSRGLLIDLLIK

SNVSRYAEFKNITRILAFREGRVEQVPCSRADVFNSKQLTMVEKRMLMKFLTFCMEYEKYP

DEYKGYEEITFYEYLKTQKLTPNLQYIVMHSIAMTSETASSTIDGLKATKNFLHCLGRYGNT

PFLFPLYGQGELPQCFCRMCAVFGGIYCLRHSVQCLVVDKESRKCKAIIDQFGQRIISEHFLV

EDSYFPENMCSRVQYRQISRAVLITDRSVLKTDSDQQISILTVPAEEPGTFAVRVIELCSSTMT

CMKGTYLVHLTCTSSKTAREDLESVVQKLFVPYTEMEIENEQVEKPRILWALYFNMRDSSDI

SRSCYNDLPSNVYVCSGPDCGLGNDNAVKQAETLFQEICPNEDFCPPPPNPEDIILDGDSLQPEASESSAIPEANSETFKESTNLGNLEESSE(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 TTALeu(L) TCA Ser(S) TAA Stop TGA Stop TTG Leu(L) TCG Ser(S) TAG Stop 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)CTA Leu(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 the AAV genome with the rep and cap genes deleted and/or modified, and their associated expression control sequences replaced by the REP1 gene sequence. The modified human REP1 gene sequence is typically adjacent to one or two (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 REP1 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) a CAG promoter, (c) a codon-optimized REP1 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 145 170-1833 CAG promoter 1664 1863-3824 codon-optimized hREP1 cDNA 1962 3867-4111 SV40 Poly A 245 4127-4271 3'ITR 145

The 5'ITR has the following sequence:

TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO: 6)

The 3'ITR has the following sequence:

AGCCGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA(SEQ ID NO:7)

The SV40 polyadenylated sequence has the following sequence:

GAGCTCGGGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAA ACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCAATGCATCCT (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.

Viral vectors 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 capsid protein having the following amino acid sequence:

MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKAAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLG QPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLT LNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTT NPVATEQYGSVSTNLQRGNLAISDQTKHARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPV PANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL(SEQ IDNO: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, which are contained within “host cells”, and 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 will be discussed in subsequent 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 REP1 gene and the CAG 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 (> ^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 REP1 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 comprise a modified REP1 gene sequence flanked by TR elements and an expression control sequence, referred to herein as a “transgenic” or “transgenic expression cassette,” sufficient to result in the packaging of vector DNA and subsequent expression of the modified REP1 gene sequence in transduced cells. Viral vector functionality can be supplied to cells, for example, as a component of plasmids or amplicon. Viral vector functionality can be present extrachromosomally within cell lines and/or can integrate 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 choroidal agenesis 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 choroidal agenesis.

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 choroidal agenesis 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 CAG promoter. In some preferred embodiments, the CAG 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) a CAG promoter, (c) a codon-optimized REP1 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 CAG 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 choroidal agenesis 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 choroidal detachment 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 choroidal agenesis 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 particles comprise a natural AAV2, AAV4, AAV5, or AAV8 capsid. In other embodiments, the rAAV viral particles comprise 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 the sequence of 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) a CAG promoter, (c) a codon-optimized REP1 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) a nucleic acid containing a 5' AAV2 terminal repeat of SEQ ID NO:6, a CAG 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 choroidal agenesis or in the manufacture of a medicament for the treatment of choroidal agenesis 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 choroidal agenesis 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 containing 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 a CAG 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) a CAG promoter, (c) a codon-optimized REP1 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 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 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) nucleic acid containing a codon-optimized REP1 gene of SEQ ID NO:1, wherein said nucleic acid further comprises a 5' AAV2 terminal repeat of SEQ ID NO:6 and/or a CAG promoter of SEQ ID NO:4 and/or an SV40 polyadenylated sequence of SEQ ID NO:8 and/or an 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 REP1 in one or more retinal pigment epithelial cells and one or more rod photoreceptor cells of a human subject is provided, comprising administering an effective amount of infectious rAAV as described herein to the human subject, wherein REP1 is expressed in one or more retinal pigment epithelial cells and one or more rod photoreceptor cells. In some preferred embodiments, an effective amount of infectious rAAV is ^10⁹ vg/eye to ^10¹⁴ vg/eye and/or administering a single dose of rAAV (bilateral or unilateral) into the vitreous cavity of the 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 ^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, the nucleic acid or infectious rAAV described herein is administered to a person with achoroidal agenesis via periocular or intraocular injection (intravitreal, suprachoroidal, or subretinal), thereby treating the achoroidal agenesis in the subject. In other embodiments, the nucleic acid or infectious rAAV described herein is administered subretinally or intravitreal to a person with achoroidal agenesis, thereby treating the achoroidal agenesis in the subject. In a preferred embodiment, a single intravitreal injection (bilateral or unilateral) of the rAAV described herein is administered to a subject with achoroidal agenesis.

In related aspects, treatment of achoroidal dysplasia 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 achoroidal dysplasia 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 that is effective in treating choroidal agenesis 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 choroidal agenesis, thereby treating choroidal agenesis.

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 REP1 cDNA Sequence

The human REP1 open reading frame cDNA sequence (NCBI reference sequence NM_000390.4) was used for codon optimization for human expression. The optimization algorithm includes, but is not limited to, parameters related to codon usage bias, GC content, CpG dinucleotide content, negative CpG islands, mRNA secondary structure, RNA instability motifs, cryptic splicing sites, premature polyadenylation sites, internal chi sites and ribosome binding sites, as well as repetitive sequence parameters.

The natural human REP1 gene uses tandem rare codons, which can reduce translation efficiency or even disengage the translational machinery. Codon usage bias in humans was altered by increasing the codon fitness index (CAI) from 0.70 to 0.94. The average GC content was optimized from 54.24 in the natural sequence to 61.22 in the optimized sequence to extend the mRNA half-life. The stem-loop structure affecting ribosome binding and stability of the mRNA was disrupted in the optimized sequence. Furthermore, negative cis-acting sites, such as ATTTA (6 of which were deleted in the optimized sequence), were screened and deleted to optimize gene expression in human cells, and several restriction enzyme sites were deleted (two BglII sites (AGATCT), one EcoRI site (GAATTC), one XhoI site (CTCGAG), and one ARE site were deleted).

The resulting codon-optimized nucleotide sequence (as shown in SEQ ID NO:1 in this paper) contains improved codon usage, altered GC content, better mRNA stability, and modifications of a negative cis-acting element compared to the native sequence of SEQ ID NO:3.

Example 2 – Codon-optimized REP1 cDNA sequence expressed at higher levels in RPE cells from patients with choroidal asthenia to generate an in vitro human model system for evaluating the expression of codon-optimized human REP1 nucleic acid with the nucleotide sequence of SEQ ID NO:1 in diseased RPE cells derived from human choroidal asthenia patients and the functional correction for the CHM disease phenotype. This model system was chosen for in vitro pharmacology because a suitable non-human primate model of choroidal asthenia (CHM) was lacking for preclinical studies. Two CHM patient fibroblast samples were reprogrammed into iPSCs and subsequently differentiated into functionally mature RPE cells. It has been demonstrated that the deficiency of REP1 protein in CHM patients is associated with cellular defects in Rab27a transport and isopreneization (see, for example, Strunnikova, N.V. et al., PLoS Biol. 4, e8402 (2009); Sergeev, Y.V. et al., Mutat. Res. Fundam. Mol. Mech. Mutagen, 665, 44-50 (2009); Rak, A. et al., Cell 117, 749-760 (2004)).

Materials and Methods: Induced pluripotent stem cell lines were generated from cells of patients with choroidal agenesis.

Cell reprogramming was performed on fibroblasts from two patients with choroidal agenesis (referred to herein as CHM1 and CHM2). On day 10, approximately 5 × ^10⁴ –1 × ^10⁵ reprogrammed cells were replated on growth factor-depleted matrix gel (Corning) in mouse embryonic fibroblast (MEF) conditioned medium containing B18R protein (200 ng/ml), supplemented with human iPSC Reprogramming BoostSupplement II (EMD Millipore). On day 20, reprogrammed cells identified by altered morphology and the ability to form small colonies were transferred to mTeSR-1 medium (Stem Cell Technologies). Colonies of approximately 200 cells or larger were manually isolated and plated on growth factor-depleted matrix gel-coated plates in mTeSR-1 medium. The CHM-iPSC line was expanded from individual colonies. CHM-iPSC lines were cultured on telonexin XF (Stem Cell Technologies) in mTeSR-1 maintenance medium and passaged every 4–5 days at 70–80% confluence using Gentle Cell Dissociation Reagent (Stem Cell Technologies). To ensure random differentiation into all three germ layers, iPSC embryoids (EBs) were formed in suspension culture for one week and then redifferentiated for four weeks in mTeSR-1 basal medium with 20% Knockout serum substitute (Thermo Fisher Scientific) under adherent conditions.

Production of retinal pigment epithelial (RPE) cells in human choroidal agenesis

RPE cells were generated using the directed differentiation protocol described above (Leach et al., Investigative Ophthalmology & Visual Science 56(2):1002–13 (2015)). In short, iPSCs were directly passaged into Methylprednisolone (BD Biosciences) in DMEM/F12 containing 1×B27, 1×N2, and 1×NEAA (Invitrogen). From day 0 to day 2, 50 ng/ml Noggin, 10 ng/ml Dkk1, 10 ng/ml IGF1 (R&D Systems Inc.), and 10 mM nicotinamide were added to the basal medium. From day 2 to day 4, 10 ng/ml Noggin, 10 ng/ml Dkk1, 10 ng/ml IGF1, 5 ng/ml bFGF, and 10 mM nicotinamide were added to the basal medium. From day 4 to day 6, 10 ng/ml Dkk1, 10 ng/ml IGF1, and 100 ng/ml activator A (R&D Systems) were added to the basal medium. From day 6 to day 14, 100 ng/ml activator A, 10 μM SU5402 (EMD Millipore), and 1 mM VIP (Sigma-Aldrich) were added to the basal medium. On day 14, cells were mechanically enriched by scraping off cells with non-RPE morphology. Subsequently, the remaining RPE was digested at 37°C for approximately 5 minutes using TrypLE Express (Invitrogen). Cells were then passed through a 30-μm single-cell filter and seeded onto CC2-treated chamber slides in Matrigel-coated tissue culture plastic, transwell membranes (Corning Enterprises), or XVIVO-10 medium (Lonza).

Functional characterization of RPE cells in human choroidal dysplasia

CHM RPE cells were cultured using a prepared culture medium to analyze phagocytosis of the outer rod segment (ROS) (Maminishkis, et al., Investigative Ophthalmology and Visual Science, 47(8):3612-24 (2006)). Cells were seeded in quadruplicates at 1 × ^10⁵ cells/ ^cm² on 0.1% gelatin-coated black-walled transparent 96-well plates and cultured for 30 days. Photoreceptor ROS were isolated from bovine eyes (Sierra for Medical Science) and fluorescently labeled with fluorescein isothiocyanate (FITC) protein (Thermo Fisher Scientific) as previously described (Molday RS and Molday LL, Journal of Cell Biology, 105(6Pt 1):2589-601 (1987)). Under certain conditions, cultured cells were treated with 62.5 μg/ml αvβ5 integrin function blocking antibody (Abcam) or IgG isotype control (Abcam) at 37°C for 30 minutes. After initial antibody incubation, cells were excited for five hours at 37°C and 5% _CO2 with 1 × ^10⁶ FITC-ROS cells per well (Buchholz et al., STEM CELLS TRANSLATIONAL MEDICINE 2(5):384–93(2013))(Rowland et al., Journal of Tissue Engineering and Regenerative Medicine, 7(8):642-53(2013)). After ROS incubation, the wells were washed six times with PBS, followed by the addition of 0.4% trypan blue for 20 minutes to quench fluorescence from extracellular ROS. Each well was imaged using epifluorescence microscopy, and the integrated pixel density of the micrographs was determined using ImageJ software (National Institutes of Health) with a rolling pixel radius of 50.

Immunocytochemistry

Cells were fixed with 4% paraformaldehyde (PFA) (Santa Cruz Biotechnologies) 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 (Calbiochem), and 5% goat serum (Thermo Fisher). 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 Zeiss Axio Observer.D1. Image processing was performed using Zeiss Zen 2 software and FIJI. A list of primary and secondary antibodies is provided in Table 3 below.

Table 3:

SDS-PAGE and Western blot

CHM RPE cell lysates were harvested using standard RIPA buffer (Thermo Fisher) containing Complete protease inhibitor tablets (Millipore Sigma) and incubated on ice for 15 min. Samples were then centrifuged at 21×g for 15 min. The supernatant was collected, and protein concentrations were determined using BCA protein assay (Thermo Fisher Scientific), normalized, and adjusted to 2 μM DTT. Biorad 4× sample buffer was added, and the samples were heated at 70 °C for 10 min. An XT Criterion gel was run, and the gel was then transferred to a membrane. The membrane was subsequently blocked and probed with REP1 and GADPH antibodies. The membrane was incubated with HRP-conjugated secondary antibody, and the bands were visualized using ECL.

Isopreneation determination

As described in As et al., PLoS ONE 8(12):1–11 (2013), isopreneation assays were performed using RPE cell lysates. Cell lysates were prepared after washing with PBS in cold isopreneation buffer (500 μM HEPES pH 7.0, 50 μM NaCl, ₂ μM MgCl₂, 0.1 μM GDP, 0.5% NP-40, and Complete protease inhibitor tablets) and incubated on ice for 10–15 min. Protein concentrations were determined using a BCA protein assay (Thermo Fisher Scientific). Protein concentrations were normalized, and the lysates were adjusted to 2 μM DTT. Isopreneation reactions were performed using 20 μL of lysate corresponding to 50–200 μg of protein. The reaction of the functional complex consisted of 2 μM RabGGT enzyme, 4 μM Rab27a, and 4 μM biotin-geranyl-PPi (Jena Bioscience). The reaction was incubated at 25 °C for 5 h and terminated by adding 4× sample buffer (BioRad), DTT to 40 mM, and heating at 70 °C for 10 min. Western blotting was performed on XTCriterion gels according to the manufacturer's protocol. The isoprenelation reaction was analyzed using streptavidin-HRP (Abcam).

Rab27a transport measurement

RPE cells were seeded at 25,000 cells/ ^cm² in XVIVO-10 medium onto porphyrin-coated eight-chamber slides. Two days post-inoculation, CHM RPE cells were transduced with recombinant AAV virus particles at a multiplicity of infection (MOI) of 5000 vg/cell: (i) a transgenic expression cassette having the sequence of SEQ ID NO:5 and (ii) a modified AAV2 capsid protein having the amino acid sequence of SEQ ID NO:9. Fourteen days post-infection, cells were fixed and stained as described above.

Experimental data

Two types of CHM patient fibroblast samples were obtained as described above and reprogrammed into iPSCs, which were then differentiated into RPE cells. More specifically, fibroblast (CHM1 and CHM2) cells were reprogrammed via Simplicon RNA reprogramming using synthetic in vitro transcribed RNA expressing four reprogramming factors (Oct4, Klf4, Sox2, and Glis1) in polycistronic transcripts that have undergone a limited number of self-replicating cell divisions. Immunocytochemical analysis of human PSC markers NANOG, SOX2, and OCT4 was performed to confirm the pluripotency of the CHM1 and CHM2 iPSC lines in choroidal agenesis (Figs. 1a and 1b). To confirm the pluripotency of the generated iPSC lines, cells were randomly differentiated into EBs in suspension culture and subsequently differentiated under adherent conditions for four weeks, and their ability to differentiate into ectodermal, mesodermal, and endoderm lineages was assessed. At this point, markers associated with neurons (TUJ1+), smooth muscle cells (ASMA+), and hepatocytes (HNF4A+) belonging to the ectoderm, mesoderm, and endoderm germ layers, respectively, were detected (Fig. 1c and 1d). After confirming pluripotency, iPSC lineages derived from CHM1 and CHM2 patient cells differentiated into RPE cells. RPE cells were allowed to mature for 30 days, followed by analysis of appropriate RPE cell marker expression and function. Protein expression and localization of melanin-associated transcription factor (MITF) and adjacent dentate homeobox 2 (OTX2), RPE65, and occluder band (ZO-1) (Fig. 2a and 2b) were normal. No changes were observed in extrasegmental phagocytosis of photoreceptors (a known function of RPE) (Fig. 2c), confirming that CHM iPSC-derived RPE cells exhibited key physiological characteristics similar to native RPE.

The expression levels of codon-optimized REP1 transgenes relative to unmodified REP1 were evaluated. For this purpose, recombinant AAV (rAAV) viral particles were isolated, comprising (i) a modified AAV2 capsid protein having the amino acid sequence of SEQ ID NO:9 and (ii) a transgene expression cassette containing either codon-optimized REP1 of SEQ ID NO:1 or native REP1 of SEQ ID NO:3, each under the control of the CAG promoter of SEQ ID NO:4. Briefly, CHM RPE cells were transduced with rAAV viral particles at two different MOIs (500 or 5000 vg/cell). Cell lysates were collected 14 days post-transduction and subjected to SDS-PAGE and Western blot analysis to assess REP1 expression levels. As shown in Figures 3A-B, codon-optimized REP1 resulted in higher expression levels than unmodified REP1. Furthermore, at lower doses (MOI 500 vg/cell), REP1 protein levels reached those found in normal RPE.

Functional assays were developed to evaluate the ability of delivered REP1 protein to isoprene-modify Rab27a GTPase (Figure 4). CHM RPE cells were transduced with rAAV containing (i) a transgenic expression cassette having the sequence of SEQ ID NO:5 and (ii) a modified AAV2 capsid protein having the amino acid sequence of SEQ ID NO:9. Cell lysates from transduced or control CHM1 and CHM2 RPE cells were collected 14 days post-infection. Wild-type RPE cells were used as a positive control. Isoprene modification of Rab27a GTPase would occur only in the presence of REP1 and the isoprene donor, RabGGT enzyme. To visualize the transfer of isoprene from RabGGT enzyme to Rab27a GTPase, the isoprene group was biotinylated. After transduction, cell lysates were combined with Rab27a GTPase, RabGGT enzyme, and the biotinylated isoprene group. The in vitro reaction was incubated for 5 hours to optimize the transfer of isoprenyl groups. After the reaction, the lysates were analyzed by SDS-PAGE and Western blotting. The SA-HRP conjugates revealed the level of isoprenylation in each reaction (Figures 5a-d). RPE cells derived from normal fibroblast-derived iPSC lines were used as a positive control in this experiment.

A second functional assay was performed to confirm that isoprenelation of the Rab27a GTPase following rAAV delivery led to the appropriate transport of Rab27a to the target membrane. CHM RPE cells were cultured at a low density (2.5 × ^10⁴ cells/ ^cm² ) and subsequently transduced with rAAV at an MOI of 5000 vg/cell, the rAAV comprising (i) a transgenic expression cassette having the sequence of SEQ ID NO:5 and (ii) a modified AAV2 capsid protein having the amino acid sequence of SEQ ID NO:9. After 14 days, the cultures were immunostained and imaged with anti-REP1 and anti-RAB27A antibodies to visualize the subcellular localization of RAB27A in the transduced cultures compared to untreated cultures. Treatment of CHM RPE cells with rAAV (Fig. 6a) induced the transport of RAB27A from the cytoplasmic region to the target membrane (Fig. 6b), similar to that in normal FB-iPSC-derived RPE cells (Fig. 6c). These data indicate that two weeks after delivery of codon-optimized REP1 of SEQ ID NO:1, the transport of RAB27A from the cytoplasmic region to the target membrane is normalized, and this correction is associated with the restoration of the normal cell RPE phenotype.

in conclusion

The above studies demonstrate that codon-optimized REP1 of SEQ ID NO:1 is expressed at significantly higher levels in disease-associated (REP1-deficient) human RPE cells compared to the native (unmodified) REP1 gene. The studies also show that REP1 expressed from codon-optimized REP1 of SEQ ID NO:1 is functional, rescuing isoprenoidation defects (Rab27) and correcting intracellular transport defects in the Rab protein, thereby restoring the normal cellular RPE phenotype in diseased RPE cells. In vitro pharmacology shows that cohREP1 exhibits superior correction for REP1 protein defects in retinal pigment epithelial (RPE) cells derived from patients with choroidal agenesis compared to the normal gene.

Example 3 – Safety and biodistribution assessment of intravitreal administration of a codon-optimized REP1 cDNA sequence delivered by R100 in nonhuman 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 (unilateral administration), 3 × ^10¹¹ vg/eye (bilateral administration only), and 1 × ^10¹² vg/eye (unilateral and bilateral administration) were evaluated. Animals were anesthetized with ketamine IM and given topical ophthalmic solutions to relieve pain. Methylprednisolone 20–80 mg was administered weekly via IM following injection. Euthanasia was performed by trained veterinarians at weeks 3, 13, and 26 post-administration.

The biodistribution of the 4D-110 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 capsid (containing the variant capsid protein of SEQ ID NO:9) and the 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-110 delivery is safe and results in the expression of therapeutic transgenes in NHP.

4D-110 (R100.CAG-cohRep1) has entered Phase 1-2 clinical trials. The Investigational New Drug (IND) data for this product includes evaluations in two separate 6-month Good Laboratory Practice (GLP) toxicology and biodistribution studies (Table 4). A total of 61 eyes with 44 NHPs were injected intravitreally via unilateral, sequential bilateral, or simultaneous bilateral administration.

Table 4. 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 resolved by week 3 (Figure 9); in some cases, the systemic steroid dose was instantaneously increased. In both the low-dose and high-dose groups, bilateral administration of 4D-110 resulted in transient minimal to moderate anterior uveitis; this finding typically resolved within two weeks, consistent with the increase in systemic steroid treatment.

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 in 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). R100 vector-derived transgene expression 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 in any study (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-110 developed anti-capsid antibody responses following administration (data not shown).

Summarize

4D-110 (R100.CAG-cohRep1) has recently been converted to a clinical trial for the hereditary retinal disease choroidal agenesis (NCT04483440). This therapeutic product has been evaluated in two separate GLP toxicology and biodistribution studies (Table 4). A total of 44 NHPs were injected via unilateral, sequential bilateral, or simultaneous bilateral administration; a total of 61 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. Gene 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 4 – Safety assessment of intravitreal administration of a codon-optimized REP1 cDNA sequence delivered by R100 in human patients with choroidal agenesis.

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-110 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 5), 4D-110 was well tolerated throughout the evaluation period:

Table 5. Summary of Adverse Events

Clinical assessment

Closely monitor the patient's ocular and general condition, including a detailed ophthalmic evaluation and retinal imaging, as well as blood tests and general physical examinations where necessary. Perform a variety of visual functional and anatomical assessments to detect any preliminary efficacy signals. These assessments include, but are not limited to, measurement of the ellipsoidal zone (EZ) area, fundus autofluorescence, micro-field examination, static automatic visual field testing, and best-corrected visual acuity (BCVA).

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 REP1 gene and its applications

<130> 090400-5011 WO

<150> US 63/073,837

<151> 2020-09-02

<160> 9

<170> PatentIn version 3.5

<210>  1

<211> 1962

<212> DNA

<213> Artificial sequence

<220>

<223> Codon Optimizer REP1

<400>  1

atggctgata cactgccttc tgagtttgat gtgatcgtga ttggaactgg actgcctgag 60

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tcctactatg gcggcaactg ggcctctttc agcttttccg gcctgctgag ctggctgaag 180

gagtaccagg agaactccga catcgtgtct gatagccccg tgtggcagga ccagatcctg 240

gagaatgagg aggccatcgc cctgtccagg aaggataaga ccatccagca cgtggaggtg 300

ttctgctatg ccagccagga cctgcacgag gatgtggagg aggcaggcgc cctgcagaag 360

aaccacgccc tggtgacctc cgccaattct agaggccg ccgactccgc ctttctgcct 420

accgaggatg agtccctgtc tacaatgtct tgtgagatgc tgaccgagca gacacctagc 480

tccgatccag agaacgccct ggaggtcaat ggcgccgagg tgaccggcga gaaggagaac 540

cactgcgacg atagacctg cgtgccaagc acatccgccg aggacatgtc cgagaacgtg 600

cctatcgccg aggataccac agcagcca aagaagaatc gcatcacata cagccagatc 660

atcaaggagg gcaggcgctt caatatcgac ctggtgtcta agctgctgta cagccggggc 720

ctgctgatcg atctgctgat caagagcaac gtgtcccgct atgccgagtt caagaatatc 780

accagaatcc tggcctttcg ggagggaaga gtggagcagg tgccctgcag cagagccgac 840

gtgttcaact ccaagcagct gacaatggtg gagaagagga tgctgatgaa gttcctgaca 900

ttttgtatgg agtacgagaa gtatccagat gagtacaagg gctatgagga gatcaccttt 960

tacgagtatc tgaagaccca gaagctgaca cccaatctgc agtacatcgt gatgcactcc 1020

atcgccatga cctctgagac agcctctagc accatcgacg gcctgaaggc cacaaagaac 1080

ttcctgcact gcctgggccg gtacggcaat acacccttcc tgtttcctct gtatggccag 1140

ggcgagctgc cccagtgctt ctgtagaatg tgcgccgtgt ttggcggcat ctattgcctg 1200

aggcactctg tgcagtgtct ggtggtggac aaggagagcc gcaagtgtaa ggccatcatc 1260

gatcagtttg gccagcggat catctctgag cacttcctgg tggaggacag ctactttcct 1320

gagaacatgt gctccagggt gcagtatcgc cagatcagcc gggccgtgct gatcaccgat 1380

agatccgtgc tgaagacaga cagcgatcag cagatcagca tcctgaccgt gccagcagag 1440

gagccaggca ccttcgccgt gagagtgatc gagctgtgct cctctaccat gacatgtatg 1500

aagggcacct acctggtgca cctgacctgc acaagctcca agacagcccg cgaggacctg 1560

gagagcgtgg tgcagaagct gttcgtgccc tacaccgaga tggagatcga gaacgagcag 1620

gtggagaagc ctagaatcct gtgggccctg tacttcaaca tgagagactc tagcgatatc 1680

tctaggagct gttacaacga tctgccctct aacgtgtacg tgtgcagcgg acctgactgt 1740

ggcctgggaa acgataatgc cgtgaagcag gccgagacac tgttccagga gatttgccct 1800

aacgaggact tttgtccccc tccacccaat ccagaggata tcatcctgga cggcgattcc 1860

ctgcagccag aggcctctga gtcctctgcc atccccgagg ccaatagcga aacattcaaa 1920

gaaagcacaa atctgggaaa cctggaagaa agtagtgagt aa 1962

<210>  2

<211> 653

<212> PRT

<213> Homo sapiens

<400>  2

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

1 5 10 15

Gly Leu Pro Glu Ser Ile Ile Ala Ala Ala Cys Ser Arg Ser Gly Arg

20 25 30

Arg Val Leu His Val Asp Ser Arg Ser Tyr Tyr Gly Gly Asn Trp Ala

35 40 45

Ser Phe Ser Phe Ser Gly Leu Leu Ser Trp Leu Lys Glu Tyr Gln Glu

50 55 60

Asn Ser Asp Ile Val Ser Asp Ser Pro Val Trp Gln Asp Gln Ile Leu

65 70 75 80

Glu Asn Glu Glu Ala Ile Ala Leu Ser Arg Lys Asp Lys Thr Ile Gln

85 90 95

His Val Glu Val Phe Cys Tyr Ala Ser Gln Asp Leu His Glu Asp Val

100 105 110

Glu Glu Ala Gly Ala Leu Gln Lys Asn His Ala Leu Val Thr Ser Ala

115 120 125

Asn Ser Thr Glu Ala Ala Asp Ser Ala Phe Leu Pro Thr Glu Asp Glu

130 135 140

Ser Leu Ser Thr Met Ser Cys Glu Met Leu Thr Glu Gln Thr Pro Ser

145 150 155 160

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

165 170 175

Glu Lys Glu Asn His Cys Asp Asp Lys Thr Cys Val Pro Ser Thr Ser

180 185 190

Ala Glu Asp Met Ser Glu Asn Val Pro Ile Ala Glu Asp Thr Thr Thr Glu

195 200 205

Gln Pro Lys Lys Asn Arg Ile Thr Tyr Tyr Ser Gln Ile Ile Lys Glu Gly

210 215 220

Arg Arg Phe Asn Ile Asp Leu Val Ser Lys Leu Leu Tyr Ser Arg Gly

225 230 235 240

Leu Leu Ile Asp Leu Leu Ile Lys Ser Asn Val Ser Arg Tyr Ala Glu

245 250 255

Phe Lys Asn Ile Thr Arg Ile Leu Ala Phe Arg Glu Gly Arg Val Glu

260 265 270

Gln Val Pro Cys Ser Arg Ala Asp Val Phe Asn Ser Lys Gln Leu Thr

275 280 285

Met Val Glu Lys Arg Met Leu Met Lys Phe Leu Thr Phe Cys Met Glu

290 295 300

Tyr Glu Lys Tyr Pro Asp Glu Tyr Lys Gly Tyr Glu Glu Ile Thr Phe

305 310 315 320

Tyr Glu Tyr Leu Lys Thr Gln Lys Leu Thr Pro Asn Leu Gln Tyr Ile

325 330 335

Val Met His Ser Ile Ala Met Thr Ser Glu Thr Ala Ser Ser Thr Ile

340 345 350

Asp Gly Leu Lys Ala Thr Lys Asn Phe Leu His Cys Leu Gly Arg Tyr

355 360 365

Gly Asn Thr Pro Phe Leu Phe Pro Leu Tyr Gly Gln Gly Glu Leu Pro

370 375 380

Gln Cys Phe Cys Arg Met Cys Ala Val Phe Gly Gly Ile Tyr Cys Leu

385 390 395 400

Arg His Ser Val Gln Cys Leu Val Val Asp Lys Glu Ser Arg Lys Cys

405 410 415

Lys Ala Ile Ile Asp Gln Phe Gly Gln Arg Ile Ile Ser Glu His Phe

420 425 430

Leu Val Glu Asp Ser Tyr Phe Pro Glu Asn Met Cys Ser Arg Val Gln

435 440 445

Tyr Arg Gln Ile Ser Arg Ala Val Leu Ile Thr Asp Arg Ser Val Leu

450 455 460

Lys Thr Asp Ser Asp Gln Gln Ile Ser Ile Leu Thr Val Pro Ala Glu

465 470 475 480

Glu Pro Gly Thr Phe Ala Val Arg Val Ile Glu Leu Cys Ser Ser Thr

485 490 495

Met Thr Cys Met Lys Gly Thr Tyr Tyr Leu Val His Leu Thr Cys Thr Ser

500 505 510

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

515 520 525

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

530 535 540

Arg Ile Leu Trp Ala Leu Tyr Phe Asn Met Arg Asp Ser Ser Asp Ile

545 550 555 560

Ser Arg Ser Cys Tyr Asn Asp Leu Pro Ser Asn Val Tyr Val Cys Ser

565 570 575

Gly Pro Asp Cys Gly Leu Gly Asn Asp Asn Ala Val Lys Gln Ala Glu

580 585 590

Thr Leu Phe Gln Glu Ile Cys Pro Asn Glu Asp Phe Cys Pro Pro Pro

595 600 605

Pro Asn Pro Glu Asp Ile Ile Leu Asp Gly Asp Ser Leu Gln Pro Glu

610 615 620

Ala Ser Glu Ser Ser Ala Ile Pro Glu Ala Asn Ser Glu Thr Phe Lys

625 630 635 640

Glu Ser Thr Asn Leu Gly Asn Leu Glu Glu Ser Ser Glu

645 650

<210> 3

<211> 1962

<212> DNA

<213> Homo sapiens

<400> 3

atggcggata ctctcccttc ggagtttgat gtgatcgtaa tagggacggg tttgcctgaa 60

tccatcattg cagctgcatg ttcaagaagt ggccggagag ttctgcatgt tgattcaaga 120

agctactatg gaggaaactg ggccagtttt agcttttcag gactattgtc ctggctaaag 180

gaataccagg aaaacagtga cattgtaagt gacagtccag tgtggcaaga ccagatcctt 240

gaaaatgaag aagccattgc tcttagcagg aaggacaaaa ctattcaaca tgtggaagta 300

ttttgttatg ccagtcagga tttgcatgaa gatgtcgaag aagctggtgc actgcagaaa 360

aatcatgctc ttgtgacatc tgcaaactcc agaagctg cagattctgc cttcctgcct 420

acggaggatg agtcattaag cactatgagc tgtgaaatgc tcacagaaca aactccaagc 480

agcgatccag agaatgcgct agaagtaaat ggtgctgaag tgacagggga aaaagaaaac 540

cattgtgatg ataaaacttg tgtgccatca acttcagcag aagacatgag tgaaaatgtg 600

cctatagcag aagataccac agagcaacca aagaaaaaca gaattactta ctcacaaatt 660

attaaagaag gcaggagatt taatattgat ttagtatcaa agctgctgta ttctcgagga 720

ttaattg atcttctaat caaatctaat gttagtcgat atgcagagtt taaaaatatt 780

accaggattc ttgcatttcg agaaggacga gtggaacagg ttccgtgttc cagagcagat 840

gtctttaata gcaaacaact tactatggta gaaaagcgaa tgctaatgaa atttcttaca 900

ttttgtatgg aatatgagaa atatcctgat gaatataaag gatatgaaga gatcacattt 960

tatgaatatt taaagactca aaaattaacc cccaacctcc aatatattgt catgcattca 1020

attgcaatga catcagagac agccagcagc accatagatg gtctcaaagc taccaaaaac 1080

tttcttcact gtcttgggcg gtatggcaac actccatttt tgtttccttt atatggccaa 1140

ggagaactcc cccagtgttt ctgcaggatg tgtgctgtgt ttggtggaat ttattgtctt 1200

cgccattcag tacagtgcct tgtagtggac aaagaatcca gaaaatgtaa agcaattata 1260

gatcagtttg gtcagagaat aatctctgag catttcctcg tggaggacag ttactttcct 1320

gagaacatgt gctcacgtgt gcaatacagg cagatctcca gggcagtgct gattacagat 1380

agatctgtcc taaaaacaga ttcagatcaa cagatttcca ttttgacagt gccagcagag 1440

gaaccaggaa cttttgctgt tcgggtcatt gagttatgtt cttcaacgat gacatgcatg 1500

aaaggcacct atttggttca tttgacttgc acatcttcta aaacagcaag agaagattta 1560

gaatcagttg tgcagaaatt gtttgttcca tatactgaaa tggagataga aaatgaacaa 1620

gtagaaaagc caagaattct gtgggctctt tacttcaata tgagagattc gtcagacatc 1680

agcaggagct gttataatga tttaccatcc aacgtttatg tctgctctgg cccagattgt 1740

ggtttaggaa atgataatgc agtcaaacag gctgaaacac ttttccagga aatctgcccc 1800

aatgaagatt tctgtccccc tccaccaaat cctgaagaca ttatccttga tggagacagt 1860

ttacagccag aggcttcaga atccagtgcc ataccagg ctaactcgga gactttcaag 1920

gaaagcacaa accttggaaa cctagaggag tcctctgaat aa 1962

<210> 4

<211> 1664

<212> DNA

<213> Artificial sequence

<220>

<223> CAG promoter

<400>  4

actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc 60

cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 120

ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 180

caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 240

ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 300

tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 360

accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca 420

cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg 480

gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg 540

agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg 600

cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcggggag tcgctgcgac 660

gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac 720

tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt 780

agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc 840

tccgggaggg ccctttgtgc gggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg 900

tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg 960

cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc 1020

ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt 1080

gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc cctccccgag 1140

ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg 1200

ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg 1260

ccggggaggg ctcggggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg 1320

cggcgagccg cagccattgc cttttatggt aatcgtgcga gaggcgcag ggacttcctt 1380

tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc 1440

gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt 1500

cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct 1560

gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta 1620

gagcctctgc taaccatgtt catgccttct tctttttcct acag 1664

<210> 5

<211>  4271

<212> DNA

<213> Artificial sequence

<220>

<223> REP1 transgenic box

<400> 5

ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60

cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120

gccaactcca tcactagggg ttcctatcga ttgaattccc cggggatcca ctagttatta 180

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 240

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 300

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 360

gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 420

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 480

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga 540

ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt 600

gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggggcg 660

cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc 720

ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg 780

gcggccctat aaaaagcgaa gcgcgcggcg ggcggggagt cgctgcgacg ctgccttcgc 840

cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta 900

ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt 960

taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc 1020

cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt gggagcgcc 1080

gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt 1140

gcgctccgca gtgtgcgcga gggagcgcg gccgggggcg gtgccccgcg gtgcgggggg 1200

ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tggggggggtg agcagggggt 1260

gtgggcgcgt cggtcgggct gcaaccccc ctgcaccccc ctccccgagt tgctgagcac 1320

ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc 1380

ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc cggggagggc 1440

tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc ggcgagccgc 1500

agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt gtcccaaatc 1560

tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag 1620

cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc 1680

cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg ccttcggggg 1740

ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct 1800

aaccatgttc atgccttctt ctttttccta cagtctagag tcgacctgca gaagcttcca 1860

ccatggctga tacactgcct tctgagtttg atgtgatcgt gattggaact ggactgcctg 1920

agagtattat tgctgctgct tgtagtagaa gcggccggag agtgctgcac gtggacagca 1980

gatcctacta tggcggcaac tgggcctctt tcagcttttc cggcctgctg agctggctga 2040

aggagtacca ggagaactcc gacatcgtgt ctgatagccc cgtgtggcag gaccagatcc 2100

tggagaatga ggaggccatc gccctgtcca ggaaggataa gaccatccag cacgtggagg 2160

tgttctgcta tgccagccag gacctgcacg aggatgtgga ggaggcaggc gccctgcaga 2220

agaaccacgc cctggtgacc tccgccaatt ctacagaggc cgccgactcc gcctttctgc 2280

ctaccgagga tgagtccctg tctacaatgt cttgtgagat gctgaccgag cagacaccta 2340

gctccgatcc agagaacgcc ctggaggtca atggcgccga ggtgaccggc gagaaggaga 2400

accactgcga cgataagacc tgcgtgccaa gcacatccgc cgaggacatg tccgagaacg 2460

tgcctatcgc cgaggatacc acaggcagc caaagaagaa tcgcatcaca tacagccaga 2520

tcatcaagga gggcaggcgc ttcaatatcg acctggtgtc taagctgctg tacagccggg 2580

gcctgctgat cgatctgctg atcaagagca acgtgtcccg ctatgccgag ttcaagaata 2640

tcaccagaat cctggccttt cgggagggaa gagtggagca ggtgccctgc agcagagccg 2700

acgtgttcaa ctccaagcag ctgacaatgg tggagaagag gatgctgatg aagttcctga 2760

cattttgtat ggagtacgag aagtatccag atgagtacaa gggctatgag gagatcacct 2820

tttacgagta tctgaagacc cagaagctga cacccaatct gcagtacatc gtgatgcact 2880

ccatcgccat gacctctgag acagcctcta gcaccatcga cggcctgaag gccacaaaga 2940

acttcctgca ctgcctgggc cggtacggca atacacctt cctgtttcct ctgtatggcc 3000

agggcgagct gccccagtgc ttctgtagaa tgtgcgccgt gtttggcggc atctattgcc 3060

tgaggcactc tgtgcagtgt ctggtggtgg acaaggagag ccgcaagtgt aaggccatca 3120

tcgatcagtt tggccagcgg atcatctctg agcacttcct ggtggaggac agctactttc 3180

ctgagaacat gtgctccagg gtgcagtatc gccagatcag ccgggccgtg ctgatcaccg 3240

atagatccgt gctgaagaca gacagcgatc agcagatcag catcctgacc gtgccagcag 3300

aggagccagg caccttcgcc gtgagagtga tcgagctgtg ctcctctacc atgacatgta 3360

tgaagggcac ctacctggtg cacctgacct gcacaagctc caagacagcc cgcgaggacc 3420

tggagagcgt ggtgcagaag ctgttcgtgc cctacaccga gatggagatc gagaacgagc 3480

aggtggagaa gcctagaatc ctgtgggccc tgtacttcaa catgagagac tctagcgata 3540

tctctaggag ctgttacaac gatctgcct ctaacgtgta cgtgtgcagc ggacctgact 3600

gtggcctggg aaacgataat gccgtgaagc aggccgagac actgttccag gagatttgcc 3660

ctaacgagga cttttgtccc cctccaccca atccagagga tatcatcctg gacggcgatt 3720

ccctgcagcc agaggcctct gagtcctctg ccatccccga ggccaatagc gaaacattca 3780

aagaaagcac aaatctggga aacctggaag aaagtagtga gtaagcctcg agcagcgctg 3840

ctcgagagat ctgcggccgc gagctcgggg atccagacat gataagatac attgatgagt 3900

ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg 3960

ctattgcttt atttgtaacc attaagct gcaataaaca agttaacaac aacaattgca 4020

ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc 4080

tctacaaatg tggtatggct gattatgatc aatgcatcct agccggagga acccctagtg 4140

atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag 4200

cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag 4260

ggagtggcca a 4271

<210> 6

<211> 145

<212> DNA

<213> Artificial sequence

<220>

<223>  5' ITR

<400> 6

ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60

cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120

gccaactcca tcactagggg ttcct 145

<210> 7

<211> 151

<212> DNA

<213> Artificial sequence

<220>

<223>  3' ITR

<400> 7

agccggagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca 60

ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga 120

gcgagcgagc gcgcagagag ggagtggcca a 151

<210> 8

<211>  260

<212> DNA

<213> Artificial sequence

<220>

<223> Polyadenylation signaling

<400> 8

gagctcgggg atccagacat gataagatac attgatgagt ttggacaaac cacaactaga 60

atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc 120

attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt 180

cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct 240

gattatgatc aatgcatcct 260

<210> 9

<211> 745

<212> PRT

<213> Artificial sequence

<220>

<223> Variant AAV 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

Claims (41)

1. A nucleic acid encoding the human Rab guardian protein-1 (REP1) 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, wherein said nucleic acid is expressed at a higher level than the expression level in cells otherwise identical to that of the wild-type REP1 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 0.94. 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 cassette of claim 4, wherein the expression control sequence is a constitutive promoter, preferably comprising a CAG promoter having at least 90%, at least 95%, or at least 98% identity with the nucleotide sequence shown in SEQ ID NO:4. 6. The expression cassette of claim 5, comprising from 5' to 3': (a) an AAV2 terminal repeat, (b) a CAG promoter, (c) a codon-optimized REP1 gene of SEQ ID NO:1, (d) an SV40 polyadenylated sequence, and (e) an AAV2 terminal repeat. 7. The expression cassette of claim 6, wherein the 5'AAV2 terminal repeat has a nucleotide sequence as shown in SEQ ID NO:6 and/or wherein the CAG 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. 8. The expression cassette of claim 7, 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. 9. The expression cassette of claim 3, wherein the expression control sequence is a promoter that guides the preferential expression of the nucleic acid in rods and cones. 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, 4, 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) a CAG promoter, (c) a codon-optimized REP1 gene of SEQ ID NO:1, (d) an SV40 polyadenylated sequence, and (e) 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 CAG 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 comprises a 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 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 of treating choroidal agenesis in subjects 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 of treating choroidal agenesis in a subject in need, comprising administering 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) a CAG promoter, (c) a codon-optimized REP1 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 CAG 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 intravitreal 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 choroidal agenesis. 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 choroidal agenesis. 29. A nucleic acid, expression cassette, or vector for use according to claim 27 or 28, wherein the nucleic acid or vector is administered via intravitreal or subretinal injection 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 choroidal agenesis, comprising (i) an AAV capsid and (ii) a nucleic acid, said nucleic acid comprising from 5' to 3': (a) an AAV2 terminal repeat, (b) a CAG promoter, (c) a codon-optimized REP1 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 choroidal agenesis, comprising (i) an AAV capsid and (ii) a nucleic acid, said nucleic acid comprising from 5' to 3': (a) an AAV2 terminal repeat, (b) a CAG promoter, (c) a codon-optimized REP1 gene of SEQ ID NO:1, (d) an SV40 polyadenylated sequence, and (e) an AAV2 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 CAG 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 via 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 REP1 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 REP1 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) a CAG promoter, (c) a codon-optimized REP1 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 CAG 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.