Gene Therapy for Eye Disease

Abstract

Compositions and methods for treating ocular diseases in a subject are provided. In one aspect, an adeno-associated viral vector is provided comprising a nucleic acid molecule comprising a sequence encoding CNGB3. In another aspect, an adeno-associated viral vector is provided comprising a nucleic acid molecule comprising a sequence encoding CNGB3. In another aspect, an adeno-associated viral vector is provided comprising a nucleic acid molecule comprising a sequence encoding REP-1. In a desirable embodiment, the subject is a human, cat, dog, sheep, or non-human primate.

Description

[Technical Field]

[0001] Compositions and methods are provided for treating ocular diseases in a subject. In one aspect, an adeno-associated viral vector is provided comprising a nucleic acid molecule comprising a sequence encoding CNGB3. In another aspect, an adeno-associated viral vector is provided comprising a nucleic acid molecule comprising a sequence encoding CNGB3. In another aspect, an adeno-associated viral vector is provided comprising a nucleic acid molecule comprising a sequence encoding REP-1. In a desirable embodiment, the subject is a human, cat, dog, sheep, or non-human primate. [Background technology]

[0002] Incorporation of material submitted electronically Applicants incorporate by reference the attached sequence listing material, submitted in electronic form. This file is labeled "17-8318PCT_Seq_Listing_ST25.txt."

[0003] Background of the Invention Choroideremia (CHM) is an X-linked inherited retinal disease characterized by degeneration of photoreceptors, the retinal pigment epithelium (RPE), and the choriocapillaris. Symptoms manifest before the age of 10 or 20 years with complaints of poor night vision (night blindness) and progressive loss of peripheral vision. As the disease progresses, the visual field narrows, culminating in central vision loss and blindness as early as the third decade of life. Mutations in over 140 CHM genes have been found to cause choroideremia. Mutations can result in the production of abnormally small, nonfunctional, and / or unstable Rab escort protein-1 (REP-1) protein, a reduction in protein function, or the loss of REP-1 protein production. The lack of normal REP-1 disrupts the ability of Rab proteins to assist intracellular trafficking. Impaired intracellular protein and organelle movement impairs cellular function and leads to premature death.

[0004] The choroideremia gene, CHM, encodes Rab escort protein-1 (REP-1), a 653-amino acid protein involved in regulating membrane trafficking. Because the CHM locus is on the X-chromosome, choroideremia is typically diagnosed only in males. Female carriers of the disease are usually asymptomatic, but retinal examination often reveals patchy degeneration of the retina and RPE, and female subjects may be affected depending on the degree of X-inactivation (lyonization) of the normal X chromosome. See also J. J., et al., J. Med. Chem. Soc. 1999, 144:111-112, which is incorporated herein by reference. See also J. J. Med. Chem. Soc. 1999, 144:111-112, which is incorporated herein by reference.

[0005] Color vision deficiency is a heterogeneous group of autosomal recessive retinal disorders characterized by early-onset visual acuity loss, partial or complete color vision loss, nystagmus, photoaversion, and loss of cone photoreceptor function. Approximately 80% of color-blind subjects exhibit mutations in the alpha or beta subunits (A3 and B3) of the cGMP-regulated cation channel cyclic nucleotide-gated channel (CNG) of cone photoreceptors. Homologous to the human disease, Cnga3-deficient mice demonstrate a cone-specific loss of function, leading to dysfunction and degeneration of affected cone photoreceptors.

[0006] Therefore, there is a need for compositions useful for the expression of REP-1, CNGA3 or CNGB3 in human subjects. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Coussa, RG, Traboulsi, EI (2012) Choroideremia: a review of general findings and pathogenesis, Ophthalmic Genet 33(2):57-65 [Non-patent document 2] Vasireddy et al., AAV-mediated gene therapy for choroideremia:preclinical studies in personalized models.PLoS One.2013 May 7;8(5):e61396 Summary of the Invention

[0008] Summary of the invention Choroideremia (CHM) is an X-linked retinal degeneration that develops before the age of 10 or 20 years, causing night blindness and peripheral vision loss. The disease progresses through middle age, by which time most subjects become blind. CHM is a favorable target for gene augmentation therapy because the disease is caused by the loss of function of Rab escort protein 1 (REP1), a protein necessary for retinal cell health, encoded by the CHM gene. CHM cDNA can be packaged into recombinant adeno-associated viruses (rAAVs), which have an established track record in human gene therapy studies. Furthermore, sensitive and quantitative assays exist to document its activity, including the ability of REP1 to prenylate Rab proteins such as Rab27 and correct defects in Rab27 localization and trafficking due to the lack of prenylation in REP1-deficient cells.

[0009] In one aspect, a codon-optimized cDNA sequence encoding Rab escort protein-1 (REP-1) is provided. In one embodiment, the codon-optimized cDNA sequence is a variant of SEQ ID NO: 3. In another embodiment, the codon-optimized cDNA sequence is SEQ ID NO: 1. In another embodiment, the cDNA sequence is codon-optimized for expression in humans.

[0010] In another embodiment, the expression cassette comprises a codon-optimized nucleic acid sequence encoding REP-1. In one embodiment, the expression cassette comprises the cDNA sequence of SEQ ID NO: 1. In yet another embodiment, the REP-1 coding sequence is located between the 5' and 3' AAV ITR sequences. In one embodiment, the vector genome comprises all of the nucleic acid sequences between and including the 5' and 3' ITRs.

[0011] In another embodiment, an adeno-associated virus (AAV) vector is provided. The AAV vector comprises an AAV capsid and a nucleic acid sequence comprising an AAV inverted terminal repeat, a nucleic acid sequence encoding human Rab escort protein-1 (REP-1), and an expression control sequence that directs expression of REP-1 in a host cell. In one embodiment, the REP-1 sequence encodes a full-length REP-1 protein. In one embodiment, the REP-1 sequence is the protein sequence of SEQ ID NO:2.

[0012] In one aspect, a codon-optimized cDNA sequence encoding cyclic nucleotide-gated channel alpha 3 (CNGA3) is provided. In one embodiment, the codon-optimized cDNA sequence is a mutant sequence of SEQ ID NO: 13 or SEQ ID NO: 15. In another embodiment, the codon-optimized cDNA sequence is SEQ ID NO: 9 or SEQ ID NO: 11. In another embodiment, the cDNA sequence is codon-optimized for expression in humans.

[0013] In another aspect, a codon-optimized cDNA sequence encoding CNGB3 is provided. In one embodiment, the codon-optimized cDNA sequence is a variant of SEQ ID NO: 19, or 21, or 23. In another embodiment, the codon-optimized cDNA sequence is SEQ ID NO: 45. In another embodiment, the cDNA sequence is codon-optimized for expression in humans.

[0014] In another aspect, the expression cassette comprises a codon-optimized nucleic acid sequence encoding cyclic nucleotide-gated channel alpha 3 (CNGA3). In one embodiment, the expression cassette comprises the cDNA sequence of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15. In yet another embodiment, the CNGA3 coding sequence is located between the 5' and 3' AAV ITR sequences.

[0015] In another aspect, the expression cassette comprises a codon-optimized nucleic acid sequence encoding cyclic nucleotide-gated channel beta 3 (CNGB3). In one embodiment, the expression cassette comprises the cDNA sequence of SEQ ID NO: 19 or SEQ ID NO: 21 or SEQ ID NO: 23 or SEQ ID NO: 45. In yet another embodiment, the CNGB3 coding sequence is located between the 5' and 3' AAV ITR sequences.

[0016] In another embodiment, an adeno-associated virus (AAV) vector is provided. The AAV vector comprises a nucleic acid sequence comprising an AAV capsid and an AAV inverted terminal repeat, a nucleic acid sequence encoding human CNGA3, and an expression control sequence that directs the expression of CNGA3 in a host cell. In one embodiment, the CNGA3 sequence encodes a full-length CNGA3 protein. In one embodiment, the CNGA3 sequence is the protein sequence of SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.

[0017] In another embodiment, an adeno-associated virus (AAV) vector is provided. The AAV vector comprises an AAV capsid and a nucleic acid sequence comprising an AAV inverted terminal repeat, a nucleic acid sequence encoding human CNGB3, and an expression control sequence that directs the expression of CNGB3 in a host cell. In one embodiment, the CNGB3 sequence encodes a full-length CNGB3 protein. In one embodiment, the CNGB3 sequence is the protein sequence of SEQ ID NO: 20.

[0018] In another aspect, an adeno-associated virus (AAV) vector is provided comprising an AAV8 capsid and an expression cassette, wherein the expression cassette comprises a nucleic acid sequence encoding REP-1, an inverted terminal repeat, and an expression control sequence that directs expression of REP-1 in a host cell. In one embodiment, the expression control sequence comprises a chicken beta actin (CBA) promoter together with a cytomegalovirus (CMV) enhancer. In one embodiment, the nucleic acid sequence comprises SEQ ID NO:1.

[0019] In yet another aspect, an adeno-associated virus (AAV) vector is provided comprising an AAV8 capsid and an expression cassette, wherein the expression cassette comprises a nucleic acid sequence encoding CNGA3, an inverted terminal repeat, and an expression control sequence that directs expression of CNGA3 in a host cell. In one embodiment, the expression control sequence comprises a rhodopsin kinase promoter. In one embodiment, the expression control sequence comprises a human cone arrestin promoter. In one embodiment, the nucleic acid sequence comprises SEQ ID NO:9. In one embodiment, the nucleic acid sequence comprises SEQ ID NO:11.

[0020] In yet another aspect, an adeno-associated virus (AAV) vector is provided comprising an AAV8 capsid and an expression cassette, wherein the expression cassette comprises a nucleic acid sequence encoding CNGB3, an inverted terminal repeat sequence, and an expression control sequence that directs expression of CNGB3 in a host cell.

[0021] In another aspect, an adeno-associated virus (AAV) vector is provided comprising an AAV2 capsid and an expression cassette, wherein the expression cassette comprises a nucleic acid sequence encoding REP-1, an inverted terminal repeat, and an expression control sequence that directs expression of REP-1 in a host cell. In one embodiment, the expression control sequence comprises a CBA promoter together with a CMV enhancer. In one embodiment, the nucleic acid sequence comprises SEQ ID NO:1.

[0022] In another aspect, an adeno-associated virus (AAV) vector is provided comprising an AAV2 capsid and an expression cassette, wherein the expression cassette comprises a nucleic acid sequence encoding CNGA3, an inverted terminal repeat, and an expression control sequence that directs expression of CNGA3 in a host cell. In one embodiment, the expression control sequence comprises a rhodopsin kinase promoter. In one embodiment, the expression control sequence comprises a human cone arrestin promoter. In one embodiment, the nucleic acid sequence comprises SEQ ID NO:9. In one embodiment, the nucleic acid sequence comprises SEQ ID NO:11.

[0023] In yet another aspect, an adeno-associated virus (AAV) vector is provided comprising an AAV2 capsid and an expression cassette, wherein the expression cassette comprises a nucleic acid sequence encoding CNGB3, an inverted terminal repeat sequence, and an expression control sequence that directs expression of CNGB3 in a host cell.

[0024] In yet another aspect, an adeno-associated virus (AAV) vector is provided, comprising an AAV9 capsid and an expression cassette, the expression cassette comprising a nucleic acid sequence encoding CNGA3, an inverted terminal repeat, and an expression control sequence directing the expression of CNGA3 in a host cell. In one embodiment, the expression control sequence comprises a rhodopsin kinase promoter. In one embodiment, the expression control sequence comprises a human cone arrestin promoter. In one embodiment, the nucleic acid sequence comprises SEQ ID NO:9. In one embodiment, the nucleic acid sequence comprises SEQ ID NO:11.

[0025] In yet another aspect, an adeno-associated virus (AAV) vector is provided, comprising an AAV9 capsid and an expression cassette, the expression cassette comprising a nucleic acid sequence encoding CNGB3, an inverted terminal repeat sequence, and an expression control sequence that directs expression of CNGB3 in a host cell.

[0026] In another aspect, an adeno-associated virus (AAV) vector is provided, comprising an AAV9 capsid and an expression cassette, the expression cassette comprising a nucleic acid sequence encoding REP-1, an inverted terminal repeat, and expression control sequences that direct expression of REP-1 in a host cell. In one embodiment, the expression control sequences comprise a CBA promoter together with a CMV enhancer. In one embodiment, the nucleic acid sequence comprises SEQ ID NO:1.

[0027] In another aspect, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, excipient and / or additive and at least one viral vector described herein.

[0028] In a further aspect, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, excipient and / or additive and a nucleic acid sequence, a plasmid, a vector, or a viral vector, such as an rAAV, as specifically described herein.

[0029] In another aspect, a method for treating choroideremia is provided. In one embodiment, the method comprises administering to a subject in need thereof a composition comprising an AAV vector encoding REP-1 as described herein.

[0030] In another aspect, a method for treating color vision deficiency is provided. In one embodiment, the method comprises administering to a subject in need thereof a composition comprising an AAV vector encoding CNGA3 as described herein.

[0031] In another aspect, a method for treating color vision deficiency is provided. In one embodiment, the method comprises administering to a subject in need thereof a composition comprising an AAV vector encoding CNGB3 as described herein.

[0032] In yet another aspect, a plasmid for generating an AAV vector is provided. In one embodiment, the plasmid comprises a codon-optimized cDNA sequence encoding REP-1 as described herein. In another embodiment, the plasmid comprises a codon-optimized cDNA sequence encoding CNGB3 as described herein. In another embodiment, the plasmid comprises a codon-optimized cDNA sequence encoding CNGB3, which is a sequence that shares at least 70% identity with SEQ ID NO: 19 or SEQ ID NO: 21. In yet another embodiment, the plasmid comprises a codon-optimized cDNA sequence encoding CNGB3 as described herein. In one embodiment, the plasmid is modular.

[0033] In another aspect, there is provided a method for producing a rAAV virus, comprising culturing packaging cells harboring a plasmid described herein in the presence of sufficient viral sequences to enable packaging of a gene expression cassette viral genome into an infectious AAV envelope or capsid. In another aspect, there is provided a recombinant AAV produced according to the method.

[0034] Other aspects and advantages of the present invention will become readily apparent from the following detailed description of the invention. [Brief explanation of the drawings]

[0035] [Figure 1]Figures 1A and 1B are gels showing in vitro REP-1 protein expression after transfection of cultured 84-31 HEK cells. The first lane of each gel shows expression of the codon-optimized REP-1 described herein expressed from plasmid p944. The second lane shows expression of native REP-1 from plasmid p742. The third lane shows endogenous expression of REP-1 by 84-31 cells that were not transfected with the plasmid. The last lane is blank. The gels demonstrate that the codon-optimized REP-1 sequence described herein results in higher levels of protein expression than the native REP-1 sequence, and that the level of expression from the exogenously transfected plasmid is many-fold higher than endogenous REP-1 expression. [Figure 2] 2A through 2F are alignments of the native REP-1 coding sequence of SEQ ID NO:1 versus the codon-optimized REP-1 coding sequence of SEQ ID NO:3. [Figure 3] 3A through 3F are alignments of the native CNGA3 coding sequence of SEQ ID NO:13 versus the codon-optimized CNGA3 coding sequence of SEQ ID NO:9. [Figure 4] Figures 4A through 4F are alignments of the CNGB3 native ORF (SEQ ID NO: 19) versus the CNGB3 modified ORF (SEQ ID NO: 21) versus the CNGB3 modified orf with modified ends (SEQ ID NO: 23). Point mutations are highlighted. [Figure 5] 5 is a plasmid map of p584 described herein. The sequence of p584 is shown in SEQ ID NO:7. [Figure 6] 6 is a plasmid map of AAV.hCHMco.version 2a described herein. The sequence of version 2a is set forth in SEQ ID NO:25. [Figure 7] 7 is a plasmid map of AAV.hCHMco.version 2b described herein. The sequence of version 2b is set forth in SEQ ID NO:26. [Figure 8]8 is a plasmid map of AAV.hCHMco.version 3a described herein. The sequence of version 3a is set forth in SEQ ID NO:27. [Figure 9] 9 is a plasmid map of AAV.hCHMco.version 3b described herein. The sequence of version 3b is set forth in SEQ ID NO:28. [Figure 10] 10 is a plasmid map of AAV.hCHM.version 1 described herein. The sequence of version 1 is set forth in SEQ ID NO:29. [Figure 11] Figure 11 is a graph of the effect of lambda inserts on AAV product impurities. From qPCR testing, all a-version (lambda-containing) vectors have a much reduced Kan+ signal. [Figure 12] Figure 12A is a Western blot showing human anti-REP-1 antibody detection of a ∼75-80 kDa protein in the ocular tissues of CD-1 mice injected with AAV8.2b at 5 × 10 (5E9) vector genome copies (high dose). Animals injected with AAV8.2b at 5 × 10 (low dose) showed a very weak protein band at ∼75-80 kDa. Figure 12B is a Western blot analysis of ocular tissues from AAV8.3b-injected CD1 mice (two mice per group) detected with an anti-REP-1 antibody, which revealed the presence of a ∼75-80 kDa protein in one eye injected with the low dose and in both eyes injected with the high dose of AAV8.3b. No REP-1 expression was detected in the ocular tissues of uninjected mice. [Figure 13] 13A and 13B provide a plasmid map of pAAV-RK1-native CNGA3 described herein. The sequence is shown in SEQ ID NO: 30. [Figure 14] 14A and 14B provide a plasmid map of pAAV-RK1-codon-optimized CNGA3 described herein. The sequence is set forth in SEQ ID NO:31. [Figure 15]15A and 15B provide a plasmid map of pAAV-RK1-codon-optimized CNGA3 variant 3 described herein. The sequence is shown in SEQ ID NO: 32. [Figure 16] Figures 16A and 16B provide a plasmid map of pAAV-hCAR-native CNGA3 described herein. The sequence is shown in SEQ ID NO: 33. [Figure 17] Figures 17A and 17B provide the plasmid map of pAAV-hCAR-codon-optimized CNGA3 described herein. The sequence is shown in SEQ ID NO: 34. [Figure 18] Figures 18A and 18B provide the plasmid map of pAAV-hCAR-codon-optimized CNGA3 variant 3 described herein. The sequence is shown in SEQ ID NO: 35. [Figure 19] 19A and 19B provide a plasmid map of pAAV-CMV-CBA-native CNGA3 described herein. The sequence is set forth in SEQ ID NO: 36. [Figure 20] 20A and 20B provide the plasmid map of pAAV-CMV-CBA-codon-optimized CNGA3 described herein. The sequence is set forth in SEQ ID NO: 37. [Figure 21] 21A and 21B provide the plasmid map of pAAV-CMV-CBA-codon-optimized CNGA3 variant 3 described herein. The sequence is shown in SEQ ID NO: 38. [Figure 22] 22A and 22B provide a plasmid map of pAAV-RK1-native CNGB3 described herein. The sequence is shown in SEQ ID NO:39. [Figure 23] 23A and 23B provide a plasmid map of pAAV-RK1-codon-optimized CNGB3 described herein. The sequence is shown in SEQ ID NO:40. [Figure 24] Figures 24A and 24B provide a plasmid map of pAAV-hCAR-native CNGB3 described herein. The sequence is shown in SEQ ID NO: 41. [Figure 25]Figures 25A and 25B provide the plasmid map of pAAV-hCAR-codon-optimized CNGB3 described herein. The sequence is shown in SEQ ID NO: 42. [Figure 26] 26A and 26B provide a plasmid map of pAAV-CMV-CBA-native CNGB3 described herein. The sequence is shown in SEQ ID NO:43. [Figure 27] 27A and 27B provide a plasmid map of pAAV-CMV-CBA-codon-optimized CNGB3 described herein. The sequence is set forth in SEQ ID NO:44. [Figure 28] Figure 28 is a Western blot showing hCNGA3 protein expression in 84-31 cells transduced with the indicated vectors. 48 hours after transduction, proteins were harvested and Western blotting was performed. The native and codon-optimized (hopt) proteins are predicted to be 79 kDa, and V3-hopt is predicted to be 85 kDa. [Figure 29] Figure 29 is a Western blot showing hCNGA3 protein expression in 84-31 cells transduced with the indicated vectors. 48 hours after transduction, proteins were harvested and Western blotted. The native and codon-optimized (hopt) proteins are predicted to be 79 kDa, and V3-hopt is predicted to be 85 kDa. [Figure 30] FIG. 30 is a three bar graph showing vector / plasmid expression as measured by RT-PCR for the three indicated vectors. [Figure 31] Figure 31 is a bar graph showing cone ERG results for CNGA3 null mice treated with the indicated vectors, as described in Example 8. n>5 for all groups, error bars represent standard deviation. [Figure 32] Figures 32A and 32B are two line graphs showing the fluorescence intensity of non-transduced (Figure 32A) and transduced (Figure 32B) cells. These results show calcium uptake in transduced versus non-transduced 84-31 cells after addition of cGMP. Each series represents one cell. [Figure 33] FIG. 33 is a bar graph showing cone ERG results for CNGB3 null mice treated with the vectors described in Example 9. [Figure 34] Figure 34 is a graph of the effect of lambda inserts on AAV product impurities. [Figure 35] Figure 35 is a Western blot showing human anti-REP-1 antibody detection of an approximately 75-80 kDa protein in ocular tissues of CD-1 mice injected with the indicated rAAV at 5E7 vector genome copies per eye. A1, A2, and A3 represent animals 1, 2, and 3, respectively. [Figure 36] Figure 36 is a Western blot showing human anti-REP-1 antibody detection of an approximately 75-80 kDa protein in ocular tissues of CD-1 mice injected with the indicated rAAV at 1E8 vector genome copies per eye. A1, A2, and A3 represent animals 1, 2, and 3, respectively. [Figure 37] Figure 37 is a Western blot showing human anti-REP-1 antibody detection of an approximately 75-80 kDa protein in ocular tissues of CD-1 mice injected with the indicated rAAV at 5E8 vector genome copies per eye. A1 and A2 represent animals 1 and 2, respectively. [Figure 38] Figure 38 is a Western blot showing human anti-REP-1 antibody detection of an approximately 75-80 kDa protein in ocular tissues of CD-1 mice injected with the indicated rAAV at 5E9 vector genome copies per eye. A1 and A2 represent animals 1 and 2, respectively. [Figure 39] Figures 39A and 39B are Western blots showing human anti-REP-1 antibody detection of an approximately 75-80 kDa protein in ocular tissues of CD-1 mice injected with the indicated rAAV at 5E9 vector genome copies per eye (Figure 39A) or 1E10 vector genome copies per eye (Figure 39B). A3, A4, A5, and A6 represent animals 3, 4, 5, and 6, respectively. [Figure 40]Figure 40 is a graph of the transduction efficiency of AAV8.CMV / CbA-GFP (designated article 6) and AAV2.CMV / CbA-GFP (designated article 5) at various doses in PBWT3.1 or BMC1 cells. The fold change in GFP above background is plotted on the y-axis, and the dose in units of vector genome copies is shown on the x-axis. [Figure 41] Figure 41 provides a bar graph showing the percentage of caspase-3 positive cells among the cells illustrated in Figure 44. Stauro indicates staurosporine-treated cells, and UNT indicates untreated cells. Substance 1 is AAV2.V2a; substance 2 is AAV8.V3a; and substance 3 is AAV8.V1. [Figure 42] Figure 42 provides a bar graph showing the prenylation of target RAB proteins in CHM patient-derived iPSCs after transduction with AAV8.V2a, AAV8.V3a, and AAV8.V1. The CHM patient-derived iPSC cell line JB588 displays a CHM mutation of Arg555 stop (AGA to TGA). The CHM patient-derived iPSC cell line JB527 displays a CHM deletion of Ex2-4. The CHM patient-derived iPSC cell line JB415 displays a CHM mutation of Ex10c.1327-1328delAT. In the left panel, incorporated 3H-GGPP normalized to untransduced iPSC cells is plotted on the y-axis. In the right panel, the amount of incorporated 3H-GGPP in pmol is plotted on the y-axis. [Figure 43]Figure 43A shows the results of cone responses 5 to 7 weeks after injection for the test substances described in Example 16. WT = wild type, Un = uninjected Cnga3 null mice, Ex = vehicle-injected Cnga3 null mice; OPT = codon optimized, NAT = native, V3 = mutant 3; NA = not applicable, L = low dose (8E8vg / eye), H = high dose (8E9vg / eye); error bars = standard deviation, threshold = 4 standard deviations above the vehicle mean, *P<0.05, **P<0.01, ***P<0.001. Figure 43B shows the results of rod responses 5 to 7 weeks after injection for the test substances described in Example 16. WT = wild type, Un = uninjected Cnga3 null mice, Ex = vehicle-injected Cnga3 null mice; OPT = codon optimized, NAT = native, V3 = mutant 3; NA = not applicable, L = low dose (8E8vg / eye), H = high dose (8E9vg / eye); error bars = standard deviation, *P<0.05, **P<0.01, ***P<0.001. [Figure 44] Figure 44A shows the results of cone responses 12 to 15 weeks after injection for the test substances described in Example 16. WT = wild type, Un = uninjected Cnga3 null mice, Ex = vehicle-injected Cnga3 null mice; OPT = codon optimized, NAT = native, V3 = mutant 3; NA = not applicable, L = low dose (8E8vg / eye), H = high dose (8E9vg / eye); error bars = standard deviation, threshold = 4 standard deviations above the vehicle mean, *P<0.05, **P<0.01, ***P<0.001. Figure 44B shows the results of rod responses 12 to 15 weeks after injection for the test substances described in Example 16. WT = wild type, Un = uninjected Cnga3 null mice, Ex = vehicle-injected Cnga3 null mice; OPT = codon optimized, NAT = native, V3 = mutant 3; NA = not applicable, L = low dose (8E8vg / eye), H = high dose (8E9vg / eye); error bars = standard deviation, *P<0.05, **P<0.01, ***P<0.001. [Figure 45]Figures 45A and 45B provide the plasmid map of pAAV-hCAR-native-CNGA3-WPRE(p1122) described herein. The sequence of pAAV-hCAR-native-CNGA3-WPRE(p1122) is shown in SEQ ID NO: 46. DETAILED DESCRIPTION OF THE INVENTION

[0036] Detailed Description of the Invention The methods and compositions described herein include compositions and methods for delivering an optimized CHM encoding REP-1 to a mammalian subject for the treatment of ocular diseases, primarily blinding diseases such as choroideremia. Additionally, the methods and compositions described herein include compositions and methods for delivering an optimized CNGA3 or CNGB3 to a mammalian subject for the treatment of ocular diseases, primarily blinding diseases such as color vision deficiency. In one embodiment, such compositions comprise R These include codon optimization of the REP-1, CNGA3, or CNGB3 coding sequences. These features are believed to improve product efficacy and safety as lower doses of reagents are used. This optimization of the transgene cassette may theoretically maximize the level of experimental protein production compared to that which can be produced using the endogenous sequence. However, compositions containing the native REP1, CNGA3, and CNGB3 coding sequences as set forth in SEQ ID NO:3, SEQ ID NO:13, and SEQ ID NO:19, respectively, are also included herein. It should be understood that when embodiments are described with respect to either REP-1, CNGA3, or CNGB3, similar embodiments are intended to be referenced elsewhere.

[0037] The technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published books which provide those skilled in the art with general guidance to many of the terms used in this application. The definitions contained herein are provided for clarity in describing the ingredients and compositions herein and are not intended to limit the invention as claimed.

[0038] The choroideremia gene, CHM, encodes Rab escort protein-1 (REP-1), a 653-amino acid protein thought to be involved in membrane trafficking. As used herein, the terms "REP-1" and "CHM" are used interchangeably when referring to the coding sequence. Because the CHM locus is on the X-chromosome, choroideremia is typically diagnosed only in males. Female carriers of the disease are usually asymptomatic, but retinal examination often reveals patchy degeneration of the retina and RPE, and female subjects may be affected depending on the degree of X-inactivation (lyonization) of the normal X chromosome. See Coussa, cited above. The native amino acid sequence encoding human REP-1 is reported in GenBank accession number P24386 and is reproduced herein as SEQ ID NO:2. The native human nucleic acid sequence of CHM is reproduced herein as SEQ ID NO:3 (Accession number NM_000390.2).

[0039] Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in the retina and olfactory receptors. Genetic defects in CNGA3 and CNGB3, which encode two structurally related subunits of the cone CNG channel, are known to result in color blindness. CNGA3 is a 694-amino acid protein. CNGB is an 809-amino acid protein.

[0040] Color vision deficiency is a heterozygous group of congenital autosomal recessive retinal disorders that manifests with early-onset cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color vision deficiency, and photophobia. The native nucleic acid sequence encoding human CNGA3 is reported in GenBank accession number XM_011210554.1 and reproduced in SEQ ID NO: 13. The native nucleic acid sequence encoding human CNGA3 is reported in GenBank accession number XM_011210554.1 and reproduced in SEQ ID NO: 13. The native nucleic acid sequence for the human CNGA3 X1 variant containing an additional exon is reported in GenBank accession number NM_001298.2 and reproduced in SEQ ID NO: 15. The native nucleic acid sequence encoding human CNGB3 is reproduced in SEQ ID NO: 19.

[0041] In some embodiments of the present invention, the subject has an "ocular disease" for which the components, compositions, and methods of the present invention are designed to treat. As used herein, the term "subject" refers to mammals, including humans, veterinary or farm animals, livestock or pets, and animals commonly used for clinical research. In one embodiment, the subject of these methods and compositions is a human. Further suitable subjects include mice, rats, dogs, cats, pigs, cows, and the like. , sheep, non-human primates, and others. As used herein, the term "subject" is used interchangeably with "patient."

[0042] As used herein, "ocular disease" includes cone-rod dystrophies and retinal diseases, including, but not limited to, Stargardt disease (autosomal dominant or autosomal recessive), retinitis pigmentosa, and pattern dystrophies. In one embodiment, the subject has color vision deficiency. In another embodiment, the subject has choroideremia or an X-linked inherited retinal degeneration. Clinical signs of such ocular diseases include, but are not limited to, decreased peripheral vision, decreased central (reading) vision, decreased night vision, loss of color vision, decreased visual acuity, decreased photoreceptor function, pigmentary changes, and ultimately blindness.

[0043] As used herein, the terms "treatment" or "treating" are defined to include administering to a subject one or more compounds or compositions described herein for the purpose of ameliorating one or more symptoms of an ocular disease. Thus, "treatment" can include one or more of the following in a given subject: reducing the onset or progression of an ocular disease; preventing the disease; reducing the severity of symptoms of the disease or slowing their progression, including the progression of blindness; eliminating symptoms of the disease; delaying the onset of the disease; or monitoring the progression of the disease or the effectiveness of a treatment.

[0044] The term "exogenous," when used to describe a nucleic acid sequence or protein, means that the nucleic acid or protein is not naturally present in the chromosome or host cell at the location where it is present. An exogenous nucleic acid sequence also refers to a sequence that originates from and is inserted into the same host cell or subject, but exists in a non-native state, for example, in a different copy number or under the control of different regulatory elements.

[0045] The term "heterologous," when used to describe a nucleic acid sequence or protein, means that the nucleic acid or protein is derived from a different organism than the host cell or subject in which it is expressed, or from a different species of the same organism. When used in reference to a protein or nucleic acid in a plasmid, expression cassette, or vector, the term "heterologous" indicates that the protein or nucleic acid is present together with another sequence or subsequence that is not found together in the same relationship to each other in nature.

[0046] The terms "percent (%) identity," "sequence identity," "percent sequence identity," or "percent identical" in the context of nucleic acid sequences refer to the bases in two sequences that are the same when aligned for correspondence. Percent identity is determined by comparing two sequences aligned under optimal conditions over the sequences to be compared. The length of sequence identity comparison can be over the entire length of the REP-1, CNGA3, or CNGB3 coding sequence or a fragment of at least about 100-150 nucleotides, or as desired. However, identity between smaller fragments, e.g., of at least about 9 nucleotides, usually at least about 20-24 nucleotides, at least about 28-32 nucleotides, at least about 36 or more nucleotides, can also be desired. Several sequence alignment programs are also available for nucleic acid sequences. Examples of such programs include "Clustal W," "CAP Sequence Assembly," "BLAST," "MAP," and "MEME," which are available via web servers on the Internet. Other sources for such programs will be known to those of skill in the art. Alternatively, the Vector NTI utilities can be used, including those included in the programs listed above. Thus, there are also several algorithms known in the art that can be used to measure nucleotide sequence identity. Another example is Fasta, a program in GCG Version 6.1. TMPolynucleotide sequences can be compared using commonly available sequence analysis software, more particularly BLAST, or analysis tools provided by public databases.

[0047] The term "isolated" means that material is removed from its original environment (e.g., the natural environment if it occurs in nature). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the materials with which it coexists in nature is isolated, even if it is subsequently reintroduced into nature. Such a polynucleotide can be part of a vector, and / or such a polynucleotide or polypeptide can be part of a composition, but such a vector or composition is still isolated in that it is not part of its natural environment.

[0048] By "engineered," it is meant that a nucleic acid sequence encoding a REP-1 or CNGA3 or CNGB3 protein described herein is assembled and placed into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., that carries and transfers the REP-1 or CNGA3 or CNGB3 sequence into a host cell, e.g., for the generation of a non-viral delivery system (e.g., an RNA-based system, naked DNA, etc.), or for the generation of a viral vector in a packaging host cell and / or for delivery to a host cell in a subject. In one embodiment, the genetic element is a plasmid. Methods used to generate such engineered constructs are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic methods. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 1999. Press, Cold Spring Harbor, NY (2012).

[0049] As used herein, the term "transgene" refers to an exogenous or engineered protein-encoding nucleic acid sequence that is under the control of a promoter or expression control sequence in an expression cassette, rAAV genome, recombinant or production plasmid, vector, or host cell described herein. In some embodiments, the transgene is a human CHM(REP-1) sequence that encodes a functional REP-1 protein. In some embodiments, the transgene is a codon-optimized nucleic acid CHM(REP-1) that encodes the REP-1 amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the transgene is encoded by the sequence set forth in SEQ ID NO:1. In some embodiments, the REP-1 transgene is encoded by the sequence set forth in SEQ ID NO:5. SEQ ID NO:5 contains modified ends that include restriction sites for cloning into a plasmid, such as the production plasmids described herein.

[0050] In some embodiments, the transgene is a human CNGA3 sequence encoding a functional CNGA3 protein. In some embodiments, the transgene is a codon-optimized CNGA3 encoding the sequence of SEQ ID NO: 10. In some embodiments, the transgene is encoded by the sequence set forth in SEQ ID NO: 9. In one embodiment, the transgene contains modified ends, such as those set forth in SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, which contain restriction sites for cloning into a plasmid, such as the plasmids described herein. In some embodiments, the transgene is a codon-optimized CNGA3 encoding the sequence of SEQ ID NO: 12. In some embodiments, the transgene is encoded by the sequence set forth in SEQ ID NO: 11. In some embodiments, the transgene is a native CNGA3 protein encoding the sequence of SEQ ID NO: 13. It is encoded by the coding sequence of

[0051] In some embodiments, the transgene is a human CNGB3 sequence encoding a functional CNGB3 protein. In some embodiments, the transgene is a codon-optimized CNGB3 encoding sequence that shares at least 70% identity with SEQ ID NO: 19 or 21. In some embodiments, the transgene is encoded by the sequence set forth in SEQ ID NO: 23. SEQ ID NO: 23 contains modified ends, which contain restriction sites for cloning into a plasmid, such as the production plasmids described herein. Nucleotides 13 to 2448 of SEQ ID NO: 23 provide the ORF for CNGB3. In some embodiments, the transgene is a codon-optimized CNGB3 encoding sequence set forth in SEQ ID NO: 20. In some embodiments, the transgene is encoded by the sequence set forth in SEQ ID NO: 19. In some embodiments, the transgene is encoded by the sequence set forth in SEQ ID NO: 21. In some embodiments, the transgene contains modified ends for cloning into a plasmid, such as the plasmids described herein. SEQ ID NO: 21 is a novel cDNA sequence in which a silent mutation has been introduced into the native coding sequence. In one particular embodiment, the CNGB3 sequence is the codon-optimized sequence set forth in SEQ ID NO: 45. Further modifications to the native sequences described herein are contemplated by the present invention.

[0052] In one embodiment, the nucleic acid sequence encoding REP-1, CNGA3, or CNGB3 further comprises a nucleic acid encoding a tag polypeptide covalently linked thereto. The tag polypeptide may be selected from known "epitope tags," including, but not limited to, myc-tagged polypeptide, glutathione-S-transferase-tagged polypeptide, green fluorescent protein-tagged polypeptide, myc-pyruvate kinase-tagged polypeptide, His6-tagged polypeptide, influenza virus hemagglutinin-tagged polypeptide, Flag-tagged polypeptide, and maltose binding protein-tagged polypeptide.

[0053] As used herein, a "vector" is a nucleic acid molecule into which a foreign or heterologous or engineered nucleic acid transgene may be inserted, which may then be introduced into a suitable host cell. Vectors preferably have one or more origins of replication and one or more sites into which recombinant DNA may be inserted. Vectors often have convenient means by which vector-containing cells can be selected from vector-free cells; for example, they encode drug resistance genes. Common vectors include plasmids, viral genomes, and "artificial chromosomes" (primarily in yeast and bacteria). Some plasmids are described herein.

[0054] A "viral vector" is defined as a replication-deficient virus containing a foreign or heterologous CHM(REP-1) or CNGA3 or CNGB3 transgene. In one embodiment, the expression cassettes described herein may be engineered on a plasmid used for drug delivery or for generating viral vectors. Suitable viral vectors are preferably replication-deficient and selected from those that target ocular cells. Viral vectors may include any virus suitable for gene therapy, including, but not limited to, adenovirus, herpesvirus, lentivirus, retrovirus, parvovirus, etc. However, for ease of understanding, adeno-associated virus will be referred to herein as a representative viral vector.

[0055] "Replication-defective virus" or "viral vector" refers to a synthetic or recombinant viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, and in which any viral genomic sequences also packaged in the viral capsid or envelope are replication-defective; i.e., they are unable to produce progeny virions but retain the ability to infect target cells. In one embodiment, Although the genome of a viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless," containing only the transgene of interest flanked by signals necessary for amplification and packaging of the artificial genome), it may supply these genes during production. It is therefore considered safe for use in gene therapy, as replication and infection by progeny virions cannot occur except in the presence of viral enzymes required for replication.

[0056] In yet another embodiment, an expression cassette comprising any of those described herein is used to generate a recombinant AAV genome.

[0057] As used herein, the term "host cell" can refer to a packaging cell line in which a recombinant AAV is generated from a production plasmid. Alternatively, the term "host cell" can refer to any target cell in which transgene expression is desired. Thus, a "host cell" refers to a prokaryotic or eukaryotic cell that contains exogenous or heterologous DNA that has been introduced into the cell by any means, such as electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion, high velocity DNA-coated pellets, viral infection, and protoplast fusion.

[0058] In some embodiments herein, the term "host cells" refers to cultures of ocular cells of various mammalian species for in vitro evaluation of the compositions described herein. In other embodiments herein, the term "host cells" refers to cells used to generate and package viral vectors or recombinant viruses. In yet another embodiment, the term "host cells" is intended to refer to ocular cells of a subject being treated in vivo for an ocular disease.

[0059] As used herein, the term "ocular cell" refers to any cell in the eye or associated with ocular function. The term may refer to any one of photoreceptor cells, including rod photoreceptors, cone photoreceptors, and light-sensitive ganglion cells, retinal pigment epithelial (RPE) cells, Müller cells, choroidal cells, bipolar cells, horizontal cells, and amacrine cells. In one embodiment, the ocular cell is a photoreceptor cell. In another embodiment, the ocular cell is an RPE cell.

[0060] "Plasmids" are generally referred to herein by a lower case p preceded and / or followed by capital letters and / or numbers, in accordance with standard naming conventions familiar to those of skill in the art. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known to those of skill in the art and are readily available. Moreover, one of skill can readily construct any number of other plasmids suitable for use in the present invention. The properties, construction, and use of such plasmids, as well as other vectors, in the present invention will be readily apparent to those of skill from the present disclosure.

[0061] As used herein, the terms "transcriptional regulatory sequence" or "expression regulatory sequence" refer to DNA sequences such as initiator sequences, enhancer sequences, and promoter sequences that induce, repress, or otherwise regulate the transcription of a protein-coding nucleic acid sequence to which they are operably linked.

[0062] As used herein, the terms "operably linked" or "operably associated" refer to both expression control sequences that are contiguous with nucleic acid sequences encoding REP-1 or CNGA3 or CNGB3 and / or that act in trans or at a distance to regulate their transcription and expression.

[0063] As used herein, the terms "AAV" or "AAV serotype" refer to the numerous naturally occurring and available adeno-associated viruses as well as man-made AAVs. Among the well-characterized AAVs isolated or engineered from humans or non-human primates (NHPs), human AAV2 was the first AAV developed as a gene transfer vector; it has been widely used for effective gene transfer experiments in various target tissues and animal models. Unless otherwise specified, the AAV capsid, ITRs, and other selected AAV components described herein may be readily selected from any AAV, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, and AAVAnc80, or variants of any of the known or referenced AAVs, or undiscovered AAVs, or variants or mixtures thereof. See, e.g., International Publication No. 2005 / 033321, which is incorporated herein by reference. In another embodiment, the AAV capsid is an AAV8bp capsid, which preferentially targets bipolar cells. See, e.g., International Publication No. 2014 / 024282, which is incorporated herein by reference. In another embodiment, the AAV capsid is an AAV7m8 capsid, which has shown preferential delivery to the outer retina. See, e.g., Dalkara et al., "In Vivo-Directed Evolution of a New Adeno-Associated Virus for Therapeutic Outer Retinal Gene Delivery from the Vitreous," Sci Transl Med 5, 189ra76 (2013), which is incorporated herein by reference. In one embodiment, the AAV capsid is an AAV8 capsid. In another embodiment, the AAV capsid is an AAV9 capsid. In another embodiment, the AAV capsid is an AAV5 capsid. In another embodiment, the AAV capsid is an AAV2 capsid.

[0064] As used herein with respect to AAV, the term "variant" refers to an AAV sequence derived from a known AAV sequence, including those that share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or more sequence identity across the entire amino acid or nucleic acid sequence. In other embodiments, the AAV capsid includes variants that may contain up to about 10% variation from any described or known AAV capsid sequence. That is, the AAV capsid shares about 90% identity to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity to an AAV capsid presented herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with an AAV capsid. When determining percent identity of an AAV capsid, comparison may be made with respect to any of a variety of proteins (e.g., vp1, vp2, or vp3). In one embodiment, the AAV capsid shares at least 95% identity with AAV8 vp3. In another embodiment, a self-complementary AAV is used.

[0065] ITRs or other AAV components may be readily isolated or engineered from AAV using methods available to those skilled in the art. Such AAV may be isolated, engineered, or obtained from academic, commercial, or public sources (e.g., American Type Culture Collection, Manassas, VA). Alternatively, AAV sequences may be engineered synthetically or through other suitable means by reference to published sequences available in the literature or in databases such as GenBank, PubMed, etc. AAV viruses can be engineered by conventional molecular biology methods for cell-specific delivery of nucleic acid sequences, to minimize immunogenicity, to tailor stability and particle longevity, for efficient degradation, or for precise targeting to the nucleus. These techniques may allow these particles to be optimized for optimal delivery, etc.

[0066] As used herein, "artificial AAV" refers to, but is not limited to, an AAV having a capsid protein that does not occur in nature. Such an artificial capsid may be generated by a suitable method using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with a heterologous sequence, which may be obtained from a different selected AAV, a non-contiguous portion of the same AAV, a non-AAV viral source, or a non-viral source. The artificial AAV may be, but is not limited to, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. Pseudotype vectors in which the capsid of one AAV species is replaced with a capsid protein of another species are useful in the present invention. In one embodiment, AAV2 / 5 and AAV2 / 8 are representative pseudotype vectors.

[0067] "Self-complementary AAV" refers to a plasmid or vector containing an expression cassette designed such that the coding region carried by the recombinant AAV nucleic acid sequence forms an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of the scAAV will assemble to form a single double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. See, for example, D. M. McCarty et al., "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis," Gene Therapy, (August 2001), Vol. 8, Number 16, pages 1248-1254. Self-complementary AAVs are described, for example, in U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, the entire disclosures of each of which are incorporated herein by reference.

[0068] By "administration" as used in the methods is meant delivering the composition to a target selected cell characterized by an ocular disease. In one embodiment, the method includes delivering the composition to RPE, photoreceptor cells, or other ocular cells via subretinal injection. In another embodiment, intravitreal injection into ocular cells is used. In yet another embodiment, injection into ocular cells via the palpebral vein may be used. Still other administration methods may be selected by one skilled in the art given this disclosure. "Administration" or "route of administration" refers to delivery of a composition described herein, with or without a pharmaceutical carrier or excipient, to a subject. Routes of administration may be combined if desired. In some embodiments, administration is repeated periodically. The pharmaceutical compositions described herein are designed for delivery to a subject in need by any suitable route or combination of routes. Delivery can be directly to the eye (optionally via intraocular delivery, subretinal injection, intraretinal injection, intravitreal, topical) or via systemic routes, such as intra-arterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration. The nucleic acid molecules and / or vectors described herein can be delivered in a single composition or in multiple compositions. Optionally, more than one type of AAV or multiple viruses can be delivered (see, e.g., WO 2011 / 126808 and WO 2013 / 049493). In another embodiment, the multiple viruses can include different replication-deficient viruses (e.g., AAV and adenovirus), alone or in combination with proteins.

[0069] Some compositions described herein are isolated or synthetically or recombinantly engineered nucleic acid sequences that provide novel codon-optimized sequences encoding REP-1 or CNGA3 or CNGB3. In one embodiment, an isolated nucleic acid sequence encoding human REP-1 is Codon-optimized nucleic acid sequences that have been modified or engineered are provided. In one embodiment, the codon-optimized sequence is SEQ ID NO: 1. In another embodiment, the codon-optimized sequence includes N- and C-terminal restriction sites for cloning. In one embodiment, the REP-1 coding sequence, such as that disclosed in SEQ ID NO: 5, includes an N-terminal NotI restriction site and a C-terminal BamHI restriction site in addition to the Kozak consensus sequence. In further embodiments, the codon-optimized sequence includes one or more additional restriction sites to allow for the addition of markers such as epitope tags. When aligned with a native nucleic acid sequence, the codon-optimized REP-1 may have a percent identity of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, including any integer in between. In one embodiment, the codon-optimized REP-1 has a percent identity of at least 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% with the native sequence. In one embodiment, when aligned with the native nucleic acid SEQ ID NO:3, the codon-optimized REP-1 (SEQ ID NO:1) was found to have a percent sequence identity of only 74% (see Figure 2).

[0070] In another embodiment, an isolated or engineered codon-optimized nucleic acid sequence encoding human CNGA3 is provided. In one embodiment, the codon-optimized sequence is SEQ ID NO:9. In one embodiment, the codon-optimized sequence is the CNGA3 variant set forth in SEQ ID NO:11. In another embodiment, the codon-optimized sequence includes N- and C-terminal restriction sites for cloning. In one embodiment, the CNGA3 coding sequence includes an N-terminal NotI restriction site and a C-terminal BgIII restriction site in addition to the Kozak consensus sequence. Examples of CNGA3 sequences containing such modifications can be found in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18. Furthermore, in some embodiments, the codon-optimized sequence includes one or more additional restriction sites to allow for the addition of markers such as epitope tags. When aligned with a native nucleic acid sequence, the codon-optimized CNGA3 may have a percent identity of at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, including any integer in between. In one embodiment, the codon-optimized CNGA3 has at least 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% percent identity with the native sequence. In one embodiment, when aligned with the native nucleic acid SEQ ID NO: 13, the codon-optimized CNGA3 (SEQ ID NO: 9) was found to have only 80% percent sequence identity (see Figure 3).

[0071] In another embodiment, an isolated or engineered codon-optimized nucleic acid sequence encoding human CNGB3 is provided. In one embodiment, the codon-optimized sequence is a sequence that shares at least 70% identity with SEQ ID NO:19 or SEQ ID NO:21. In one embodiment, the codon-optimized sequence is the sequence set forth in SEQ ID NO:45, which shares approximately 76% identity with the modified CNGB3 sequence of SEQ ID NO:21. In another embodiment, the codon-optimized sequence includes N- and C-terminal restriction sites for cloning, for example, as set forth in SEQ ID NO:23. In further embodiments, the codon-optimized sequence includes one or more additional restriction sites to allow for the addition of markers such as epitope tags. When aligned with a native nucleic acid sequence (such as set forth in SEQ ID NO:19 or the modified sequence of SEQ ID NO:21), the codon-optimized CNGB3 may have a percent identity of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, including any integer in between. In one embodiment, the codon-optimized CNGB3 has at least 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 , 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% percent identity.

[0072] In one embodiment, the optimized nucleic acid sequence encoding the REP-1 or CNGA3 or CNGB3 construct described herein is engineered into a suitable genetic element, such as naked DNA, a phage, a transposon, a cosmid, an RNA molecule (e.g., mRNA), an episome, etc., which transfers the REP-1 or CNGA3 or CNGB3 sequence carried thereon into a host cell, e.g., to generate a DNA or RNA-carrying nanoparticle, a viral vector in a packaging host cell, and / or for delivery to a host cell in a subject. In one embodiment, the genetic element is a plasmid.

[0073] The selected genetic elements may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Methods used to generate such constructs are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic methods. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0074] Various expression cassettes using SEQ ID NO: 1 or 5 for expression of the REP-1 protein are provided. In one embodiment, an example of a plasmid containing such an expression cassette is set forth in SEQ ID NO: 25. In one embodiment, an example of a plasmid containing such an expression cassette is set forth in SEQ ID NO: 26. In one embodiment, an example of a plasmid containing such an expression cassette is set forth in SEQ ID NO: 27. In one embodiment, an example of a plasmid containing such an expression cassette is set forth in SEQ ID NO: 28. As used herein, a "vector genome" is a nucleic acid sequence packaged between the 5' and 3' ITRs, including the ITRs themselves. In some embodiments, the term "vector genome" is used interchangeably with "expression cassette." Thus, in one embodiment, the vector genome comprises a 5' ITR, a CMV enhancer, a chicken beta-actin promoter, a CBA exon 1 and intron, a Kozak sequence, a codon-optimized CHM, a bGH polyA, and a 3' ITR. In one embodiment, the vector genome comprises nucleotides (nt) 1 to 4233 of SEQ ID NO: 25. In another embodiment, the vector genome comprises nucleotides 1 to 4233 of SEQ ID NO: 26. In another embodiment, the vector genome comprises nucleotides 1 to 4233 of SEQ ID NO: 27. In another embodiment, the vector genome comprises nucleotides 1 to 4233 of SEQ ID NO: 28.

[0075] In another embodiment, various expression cassettes using SEQ ID NO: 9, 11, or 13 for expression of CNGA3 protein are provided. In one embodiment, examples of plasmids containing such expression cassettes are set forth in SEQ ID NOs: 30-38. In one embodiment, the vector genome comprises a 5'ITR, an RK1 promoter, a codon-optimized CNGA3, a bGH polyA, and a 3'ITR. In another embodiment, the vector genome comprises a 5'ITR, an RK1 promoter, a native CNGA3, a bGH polyA, and a 3'ITR. In another embodiment, the vector genome comprises a 5'ITR, an RK1 promoter, a codon-optimized CNGA3 variant 3, a bGH polyA, and a 3'ITR. Thus, in one embodiment, the vector genome comprises a 5'ITR, an hCAR promoter, a codon-optimized CNGA3, a bGH polyA, and a 3'ITR. In another embodiment, the vector genome comprises a 5'ITR, an hCAR promoter, a codon-optimized CNGA3, a bGH polyA, and a 3'ITR. In another embodiment, the vector genome comprises a 5'ITR, an hCAR promoter, a codon-optimized CNGA3 variant 3, a bGH polyA, and a 3'ITR. Thus, in one embodiment, the vector genome comprises a 5'ITR, a CMV enhancer, a chicken beta actin promoter, a CBA exon 1 and intron, a codon-optimized CNGA3, a bGH polyA, and a 3'ITR. In another embodiment, the vector genome comprises a 5'ITR, a CMV enhancer, a chicken beta actin promoter, a CBA exon 1 and intron, a native CNGA3, a bGH polyA, and a 3'ITR. In another embodiment, the vector genome comprises a 5'ITR, a CMV enhancer, a chicken beta actin promoter, a CBA exon 1 and intron, a codon-optimized CNGA3 variant 3, a bGH polyA, and a 3'ITR.

[0076] In one embodiment, the vector genome comprises nucleotides 1 to 3189 of SEQ ID NO: 30. In another embodiment, the vector genome comprises nucleotides 1 to 3189 of SEQ ID NO: 31. In another embodiment, the vector genome comprises nucleotides 1 to 3354 of SEQ ID NO: 32. In another embodiment, the vector genome comprises nucleotides 1 to 3583 of SEQ ID NO: 33. In another embodiment, the vector genome comprises nucleotides 1 to 3580 of SEQ ID NO: 34. In another embodiment, the vector genome comprises nucleotides 1 to 3748 of SEQ ID NO: 35. In another embodiment, the vector genome comprises nucleotides 1 to 4357 of SEQ ID NO: 36. In another embodiment, the vector genome comprises nucleotides 1 to 4357 of SEQ ID NO: 37. In another embodiment, the vector genome comprises nucleotides 1 to 4522 of SEQ ID NO: 38.

[0077] In another embodiment, various expression cassettes are provided that use SEQ ID NO: 19, 21, 23, or 45 for expression of CNGB3 protein.

[0078] In one embodiment, examples of plasmids containing such expression cassettes are shown in SEQ ID NOs: 39-44. In one embodiment, the vector genome comprises a 5'ITR, an RK1 promoter, a codon-optimized CNGB3, a bGH polyA, and a 3'ITR. In another embodiment, the vector genome comprises a 5'ITR, an RK1 promoter, a native CNGB3, a bGH polyA, and a 3'ITR. In one embodiment, the vector genome comprises a 5'ITR, an hCAR promoter, a codon-optimized CNGB3, a bGH polyA, and a 3'ITR. In another embodiment, the vector genome comprises a 5'ITR, an hCAR promoter, a native CNGB3, a bGH polyA, and a 3'ITR. In one embodiment, the vector genome comprises a 5'ITR, a CMV enhancer, a chicken beta actin promoter, a CBA exon 1 and intron, a codon-optimized CNGB3, a bGH polyA, and a 3'ITR. In another embodiment, the vector genome comprises the 5'ITR, CMV enhancer, chicken beta actin promoter, CBA exon 1 and intron, native CNGB3, bGH polyA and 3'ITR.

[0079] In one embodiment, the vector genome comprises nucleotides 1 to 3537 of SEQ ID NO: 39. In another embodiment, the vector genome comprises nucleotides 1 to 3536 of SEQ ID NO: 40. In another embodiment, the vector genome comprises nucleotides 1 to 3930 of SEQ ID NO: 41. In another embodiment, the vector genome comprises nucleotides 1 to 3930 of SEQ ID NO: 42. In another embodiment, the vector genome comprises nucleotides 1 to 4704 of SEQ ID NO: 43. In another embodiment, the vector genome comprises nucleotides 1 to 4704 of SEQ ID NO: 44. In another embodiment, the vector genome comprises nucleotides 1 to 4154 of SEQ ID NO: 46.

[0080] As used herein, "expression cassette" refers to a nucleic acid molecule that includes the coding sequence, promoter, and possibly other regulatory sequences for an optimized REP-1 or CNGA3 or CNGB3 protein, which cassette may be engineered into a genetic element or plasmid and / or packaged into the capsid of a viral vector (e.g., a viral particle).

[0081] In one embodiment, the expression cassette comprises a codon-optimized nucleic acid sequence encoding REP-1. In one embodiment, the cassette provides codon-optimized REP-1 in operative association with expression control sequences that direct expression in a host cell of the codon-optimized nucleic acid sequence encoding REP-1.

[0082] In another embodiment, the expression cassette comprises a codon-optimized nucleic acid sequence encoding CNGA3. In one embodiment, the cassette provides the codon-optimized CNGA3 in operative association with an expression control sequence that directs expression of the codon-optimized nucleic acid sequence encoding CNGA3 in a host cell.

[0083] In another embodiment, the expression cassette comprises a codon-optimized nucleic acid sequence encoding CNGB3. In one embodiment, the cassette provides the codon-optimized CNGB3 in operative association with expression control sequences that direct expression of the codon-optimized nucleic acid sequence encoding CNGB3 in a host cell.

[0084] In another embodiment, an expression cassette for use in an AAV vector is provided. In that embodiment, the AAV expression cassette comprises at least one AAV inverted terminal repeat (ITR) sequence. In another embodiment, the expression cassette comprises a 5' ITR sequence and a 3' ITR sequence. In one embodiment, the 5' and 3' ITRs flank a codon-optimized nucleic acid sequence encoding REP-1 or CNGA3 or CNGB3, optionally with additional sequences that direct expression of the codon-optimized nucleic acid sequence encoding REP-1 or CNGA3 or CNGB3 in a host cell. Thus, as described herein, an AAV expression cassette describes the above expression cassette flanked at its 5' end by a 5' AAV inverted terminal repeat (ITR) and at its 3' end by a 3' AAV ITR. The rAAV genome thus comprises the minimal sequences required for packaging the expression cassette into an AAV viral particle, i.e., the AAV 5' and 3' ITRs. AAV ITRs may be derived from any AAV ITR sequence as described herein. These ITRs may be of the same AAV origin as the capsid used in the resulting recombinant AAV or of a different AAV origin (for AAV pseudotyping). In one embodiment, ITR sequences from AAV2 or a deleted version thereof (ΔITR) are used for convenience and to expedite regulatory approval. However, ITRs from other AAV sources may be selected. Each rAAV genome may then be introduced into a production plasmid. In one embodiment, the production plasmid is one described herein or in WO 2012 / 158757, which is incorporated herein by reference. Various plasmids are known in the art for use in generating rAAV vectors and are useful herein. The production plasmid is cultured in a host cell that expresses AAV cap and / or rep proteins. In the host cell, each rAAV genome is rescued and packaged into capsid or envelope proteins to form infectious viral particles.

[0085] One type of production plasmid is shown in SEQ ID NO:7 and is called p584. This plasmid is used in the Examples for the generation of the rAAV-REP-1 vector. Such a plasmid contains a 5'AAVITR sequence; a selected promoter; a polyA sequence; and a 3'ITR; and it also contains a stuffer sequence such as lambda. In one embodiment, a non-coding lambda stuffer region is included in the vector backbone. The nucleic acid sequence encoding REP-1, CNGA3, or CNGB3 is inserted between the selected promoter and polyA sequence or into a similar plasmid. An example of p584 containing the REP-1 coding sequence can be found in SEQ ID NO:8. In another embodiment, the production plasmid is modified for optimized vector plasmid production efficiency. Such modifications include the addition of other neutral sequences (e.g., nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 69, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, Modifications include the addition of non-neutral sequences or the deletion of part or all of the lambda stuffer sequence. Such modifications are encompassed herein. In other embodiments, terminators and other sequences are included in the plasmid.

[0086] In a further aspect, recombinant adeno-associated virus (AAV) vectors for delivery of the REP-1, CNGA3, and CNGB3 constructs and the optimized sequences described herein are provided. Adeno-associated virus (AAV) viral vectors are AAV Dnase-resistant particles having an AAV protein capsid, in which a nucleic acid sequence is packaged for delivery to a target cell. The AAV capsid is composed of 60 capsid protein subunits, VP1, VP2, and VP3, which are arranged in an icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending on the AAV selected. AAVs may be selected as the source of the capsid for the AAV viral vector defined above. See, for example, U.S. Published Patent Application No. 2007-0036760-A1; U.S. Published Patent Application No. 2009-0197338-A1; and European Patent No. 1310571. See also WO 2003 / 042397 (AAV7 and other simian AAVs), U.S. Patent Nos. 7,790,449 and 7,282,199 (AAV8), WO 2005 / 033321 and 7,906,111 (AAV9), and WO 2006 / 110689 and WO 2003 / 042397 (rh.10). Each of these documents is incorporated by reference in its entirety. In some embodiments, AAV capsids are produced using nucleic acid sequences described in the listed documents. These documents also describe other AAVs that may be selected for the production of AAVs, and their disclosures are incorporated herein by reference. In some embodiments, AAV caps for use in viral vectors may be produced by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the above-listed AAV capsids or their encoding nucleic acids. In some embodiments, the AAV capsid is a chimera comprising domains from two, three, four or more of the above-listed AAV capsid proteins.In some embodiments, the AAV capsid is a mosaic of Vp1, Vp2, and Vp3 monomers from two or three AAVs or recombinant AAVs. In some embodiments, the rAAV composition comprises more than one of the Caps listed above.

[0087] In another embodiment, the AAV capsid includes variants that may contain up to about 10% variation from any described or known AAV capsid sequence. That is, the AAV capsid shares about 90% identity to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity to an AAV capsid presented herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with an AAV capsid. When determining the percent identity of an AAV capsid, comparison may be made with respect to any of various proteins (e.g., vp1, vp2, or vp3). In one embodiment, the AAV capsid shares at least 95% identity with AAV8 vp3. In another embodiment, a self-complementary AAV is used. In one embodiment, it is desirable to use an AAV capsid that exhibits tropism for a desired target cell, such as a photoreceptor, RPE, or other ocular cell. In one embodiment, the AAV capsid is a tyrosine capsid-mutant in which some surface-exposed tyrosine residues are replaced with phenylalanine (F). Such AAV mutants are described, for example, in Mowat et al., "Tyrosine capsid-mutant AAV vectors for gene delivery to the canine retina from a subretinal or intravitreal vector." approach,Gene Therapy 21,96-105(January 2014), which is incorporated herein by reference.

[0088] The ITRs are the only AAV components required in cis with the transgene in the same construct for packaging of the expression cassette, rAAV genome, or production plasmid into virions. In one embodiment, coding sequences for replication (rep) and / or encapsidation (cap) are removed from the AAV genome and supplied in trans or by a packaging cell line for production of the AAV vector. For example, as described above, pseudotyped AAV may contain ITRs from a source different from that of the AAV capsid. Additionally or alternatively, chimeric AAV capsids may be used. Additional AAV components may be selected. Sources of such AAV sequences are described herein and may be isolated, engineered, or obtained from academic, commercial, or public sources (e.g., American Type Culture Collection, Manassas, VA). Alternatively, AAV sequences may be obtained synthetically or via other suitable means by reference to published sequences, such as those available in the literature or in databases such as GenBank®, PubMed®, etc.

[0089] Methods for generating and isolating AAV viral vectors suitable for delivery to a subject are known in the art. See, e.g., U.S. Pat. No. 7,790,449; U.S. Pat. No. 7,282,199; WO 2003 / 042397; WO 2005 / 033321; WO 2006 / 110689; and U.S. Pat. No. 7,588,772 B2. In one system, a producer cell line is transiently transfected with a construct encoding a transgene flanked by ITRs and a construct encoding rep and cap. In a second system, a packaging cell line stably supplying rep and cap is transiently transfected with a construct encoding a transgene flanked by ITRs. In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or herpesvirus, which requires separation of the rAAVs from contaminating virus. More recently, systems have been developed in which helper virus infection is not required for AAV recovery—the necessary helper functions (i.e., adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29 and herpesvirus polymerase) are also supplied in trans by the system. In these newer systems, helper functions may be supplied by transient transfection of cells with constructs encoding the necessary helper functions, or cells can be engineered to stably contain genes encoding helper functions, the expression of which can be regulated at the transcriptional or post-transcriptional level.

[0090] In yet another system, the transgene and rep / cap genes flanked by ITRs are introduced into insect cells by infection with a baculovirus-based vector. For a review of these production systems, see generally, e.g., Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the entire disclosure of which is incorporated herein by reference. Methods for making and using these and other AAV production systems are also described in the following U.S. patents, the entire disclosures of each of which are incorporated herein by reference: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065. See generally, e.g., Grieger and Samulski, 2005, "Adeno-associated virus as a gene therapy" See, "Adv. Biochem. Engin / Biotechnol. 99:119-145; Buning et al., 2008, "Recent developments in adeno-associated viral vector technology," J. Gene Med. 10:717-733, and the references cited below, the entire disclosures of each of which are incorporated herein by reference.

[0091] The methods used to construct embodiments of the present invention are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic methods. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Similarly, methods for producing rAAV virions are well known, and the selection of an appropriate method is not a limitation on the present invention. See, e.g., K. Fisher et al. (1993) J. Virol., 70:520-532 and U.S. Pat. No. 5,478,745.

[0092] The rAAV vector comprises an AAV capsid and an AAV expression cassette comprising a sequence encoding REP-1, CNGA3, or CNGB3, as described above. In some embodiments, the rAAV expression cassette comprises an AAV inverted terminal repeat, a codon-optimized nucleic acid sequence encoding REP-1, CNGA3, or CNGB3, and an expression control sequence that directs expression of the encoded protein in a host cell. In other embodiments, the rAAV expression cassette further comprises one or more of an intron, a Kozak sequence, a polyA, and a post-transcriptional regulatory element. Such rAAV vectors for use in pharmaceutical compositions for ocular delivery may use capsids derived from any of the many known AAVs defined above.

[0093] Other typical components of expression cassettes and vectors include other components that can be optimized for a particular species using methods known in the art, including, for example, codon optimization as described herein. Components of cassettes, vectors, plasmids, and viruses or other compositions described herein include a promoter sequence as part of the expression control sequence. In another embodiment, the promoter is cell-specific. The term "cell-specific" means that a particular promoter selected for a recombinant vector may direct expression of an optimized REP-1 or CNGA3 or CNGB3 transgene in a specific ocular cell type. In one embodiment, the promoter is specific for expression of the transgene in photoreceptor cells. In another embodiment, the promoter is specific for expression in rods and cones. In another embodiment, the promoter is specific for expression in rods. In another embodiment, the promoter is specific for expression in cones. In one embodiment, the photoreceptor-specific promoter is the human rhodopsin kinase promoter. The rhodopsin kinase (RK1) promoter has been shown to be active in both rods and cones. See, e.g., Sun et al., Gene Therapy with a Promoter, incorporated herein by reference in its entirety. See, Targeting Both Rods and Cones Rescues Retinal Degeneration Caused by AIPL1 Mutations, Gene Ther. 2010 January;17(1):117-131. In one embodiment, the promoter is modified to add one or more restriction sites to facilitate cloning. In one embodiment, the RK1 promoter is set forth at nucleotides 175-684 of SEQ ID NO:30.

[0094] The human cone arrestin (hCAR) promoter was identified and analyzed in AAV transduction experiments and gene expression studies. It has been used in gene replacement studies. See, for example, Li A, Zhu X, Craft CM. Retinoic acid upregulates cone arrestin expression in retinoblastoma cells through a Cis element in the distal promoter region. Invest Ophthalmol Vis Sci. 2002;43(5):1375-1383; and Carvalho, Livia S. et al., "Long-term and age-dependent restoration of See, "Visual function in a mouse model of CNGB3-associated achromatopsia following gene therapy." Human molecular genetics 20.16(2011):3161-3175. In experiments performed in animals to characterize gene expression, the human cone arrestin promoter drove strong expression in the retina. Dyka, Frank M. et al., "Cone specific promoter for use in gene therapy of retinal degenerative diseases." Retinal Degenerative Diseases. Springer New See, York, 2014, pp. 695-701. Dyka et al. also reported that the specificity of the hCAR promoter is poor, with rods and RPE apparently transduced. The sequence of the hCAR promoter is available through publicly available literature, databases, and commercially available products. In one embodiment, the nucleic acid sequence of the hCAR promoter is reproduced in SEQ ID NO: 33, nucleotides 175 to 1078. In another embodiment, the nucleic acid sequence of the hCAR promoter is reproduced in SEQ ID NO: 33, nucleotides 181 to 1078.

[0095] In another embodiment, the promoter is the human rhodopsin promoter. In one embodiment, the promoter is modified to include terminal restrictions for cloning purposes. See, e.g., Nathans and Hogness, Isolation and nucleotide sequence of the See, "gene encoding human rhodopsin," PNAS, 81:4851-5 (August 1984). In another embodiment, the promoter is a portion or fragment of the human rhodopsin promoter. In another embodiment, the promoter is a variant of the human rhodopsin promoter.

[0096] Other representative promoters include the human G-protein coupled receptor protein kinase 1 (GRK1) promoter (Genbank accession number AY327580). In another embodiment, the promoter is a 292-nucleotide fragment (positions 1793-2087) of the GRK1 promoter (see Beltran et al., Gene Therapy 2010 17:1162-74, incorporated herein by reference in its entirety). In another preferred embodiment, the promoter is the human interphotoreceptor retinoid-binding protein proximal (IRBP) promoter. In one embodiment, the promoter is a 235-nucleotide fragment of the hIRBP promoter. In one embodiment, the promoter is the RPGR proximal promoter (Shu et al., IOVS, May 2102, incorporated herein by reference in its entirety). Other promoters useful in the present invention include the rod opsin promoter, the red-green opsin promoter, the blue opsin promoter, the cGMP-β-phosphodiesterase promoter (Qgueta et al., IOVS, Invest Ophthalmol Vis Sci. 2000 Dec;41(13):4059-63), the mouse opsin promoter (Beltran et al. 2010, cited above), the rhodopsin promoter (Mussolino et al., Gene Ther, July 2011,18(7):637-45); the alpha-subunit of cone transducin (Morrissey et al., BMC Dev, Biol, Jan 2011, 11:3); beta phosphodiesterase (PDE) promoter; retinitis pigmentosa (RP1) promoter (Nicord et al., J. Gene Med, Dec 2007, 9(12):1015-23); NXNL2 / NXNL1 promoter (Lambard et al., PloS One, Oct. 2010, 5(10):el3025), RPE65 promoter; retinal degeneration slow / peripherin 2 (Rds / perph2) promoter (Cai et al., Exp Eye Res. 2010 Ayg;91(2):186-94); and VMD2 promoter (Kachi et al., Human Gene Therapy, 2009(20:31-9)). Each of these documents is incorporated herein by reference in its entirety. In another embodiment, the promoter is selected from the human EF1α promoter, rhodopsin promoter, rhodopsin kinase, inter-photoreceptor binding protein (IRBP), cone opsin promoters (red-green, blue), cone opsin upstream sequences including the red-green cone locus regulatory region, cone transduction and transcription factor promoters (neural retina leucine zipper (Nrl) and photoreceptor-specific nuclear receptor Nr2e3, bZIP).

[0097] In another embodiment, the promoter is a ubiquitous or constitutive promoter. An example of a suitable promoter is the hybrid chicken beta-actin (CBA) promoter with a cytomegalovirus (CMV) enhancer element. In one embodiment, the nucleic acid sequence of the CBA promoter, along with the CMV enhancer element, is set forth in nucleotides 1 to 544 of SEQ ID NO: 36. In one embodiment, the promoter comprises CBA exon 1 and intron sequences, such as the sequence set forth in nucleotides 546 to 823 of SEQ ID NO: 36.

[0098] In another embodiment, the promoter is a CB7 promoter. Other suitable promoters include the human β-actin promoter, human elongation factor-1α promoter, cytomegalovirus (CMV) promoter, simian virus 40 promoter, and herpes simplex virus thymidine kinase promoter. See, e.g., Damdindorj et al. (August 2014) A Comparative Analysis of Constitutive Promoters Located in Adeno-Associated Viral Vecors. PloS ONE 9(8):e106472. Further suitable promoters include viral promoters, constitutive promoters, and regulatable promoters (see, e.g., WO 2011 / 126808 and WO 2013 / 04943). Alternatively, promoters responsive to physiological cues may be used in the expression cassettes, rAAV genomes, vectors, plasmids, and viruses described herein. In one embodiment, due to the size limitation of the AAV vector, the promoter is small, less than 1000 base pairs (bp). In another embodiment, the promoter is smaller than 400 bp. Other promoters may be selected by those skilled in the art. In one embodiment, a ubiquitous promoter is introduced into the REP-1 construct. In another embodiment, a photoreceptor-specific promoter is introduced into the CNGA3 construct. In one embodiment, the REP-1 construct contains a CBA promoter together with a CMV enhancer element.

[0099] In another embodiment, the promoter is an inducible promoter. Inducible promoters may be selected from known promoters, including rapamycin / rapalog promoters, ecdysone promoters, estrogen-responsive promoters, and tetracycline-responsive promoters or heterodimeric repressor switches. See Sochor et al., An Autogenously Regulated Expression System for Gene Therapeutic Ocular Applications.Scientific Reports.2015 Nov 24;5:17105 and Daber R, Lewis M., A novel molecular switch.J Mol Biol 2009 Aug 28;391(4):661-70,Epub See 2009 Jun 21.

[0100] In another embodiment, the cassettes, vectors, plasmids, and viral constructs described herein include other appropriate transcription initiation, termination, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals, TATA sequences, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak consensus sequences), introns, sequences that enhance protein stability, and, if desired, sequences that enhance secretion of the encoded product. In another embodiment, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is included. An expression cassette or vector may include none, one, or more of the elements described herein. Examples of suitable polyA sequences include, for example, SV40, bovine growth hormone (bGH), and TK polyA. Examples of suitable enhancers include, among others, the CMV enhancer, RSV enhancer, alpha-fetoprotein enhancer, TTR minimal promoter / enhancer, and LSP (TH-binding globulin promoter / alpha-microglobulin / bikunin enhancer). In one embodiment, a Kozak sequence is included upstream of the transgene coding sequence to enhance translation from the correct start codon. In another embodiment, CBA exon 1 and an intron are included in the expression cassette. In one embodiment, the transgene is placed under the control of a hybrid chicken beta-actin (CBA) promoter. This promoter consists of the cytomegalovirus (CMV) immediate early enhancer, the proximal chicken beta-actin promoter, and CBA exon 1 flanked by intron 1 sequence. See SEQ ID NO: 36, nucleotides 1-823.

[0101] The adeno-associated virus (AAV) vector comprises a nucleic acid sequence comprising the AAV capsid and AAV inverted terminal repeats, a nucleic acid sequence of SEQ ID NO: 1 encoding human Rab escort protein-1 (REP-1), and an expression control sequence comprising a CBA promoter together with a CMV enhancer that directs expression of REP-1 in a host cell.

[0102] In one embodiment, an adeno-associated virus (AAV) vector is provided, comprising an AAV capsid packaging therein a nucleic acid sequence comprising an AAV inverted terminal repeat and a nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:11 encoding human cyclic nucleotide-gated channel alpha 3 (CNGA3), and an expression control sequence directing expression of CNGA3 in a host cell. In one embodiment, the CNGA sequence comprises SEQ ID NO:9 and a rhodopsin kinase 1 (RK1) promoter. In another embodiment, the RK1 promoter sequence is nucleotides 175 to 684 of SEQ ID NO:30. In another embodiment, the CNGA sequence comprises SEQ ID NO:11 and a human cone arrestin (hCAR) promoter. In another embodiment, the hCAR promoter sequence is the sequence set forth in nucleotides 175 to 1078 of SEQ ID NO:33 or nucleotides 181 to 1078 of SEQ ID NO:33.

[0103] In another embodiment, an adeno-associated virus (AAV) vector is provided, comprising an AAV capsid packaging therein a nucleic acid sequence comprising an AAV inverted terminal repeat and the nucleic acid sequence of SEQ ID NO:45 encoding human cyclic nucleotide-gated channel beta 3 (CNGB3), and expression control sequences that direct expression of CNGB3 in a host cell. In one embodiment, the expression control sequences comprise a CMV / CBA promoter, an RK1 promoter, or an hCAR promoter.

[0104] In another embodiment, the expression cassette comprises a 5' ITR, a CBA promoter, a CMV enhancer, a CBA exon 1 and intron, a Kozak sequence, a human codon-optimized CHM sequence (SEQ ID NO: 1), a bGH polyA, and a 3' ITR.

[0105] In yet another aspect, these nucleic acid sequences, vectors, expression cassettes, and rAAV viral vectors are useful in pharmaceutical compositions, which also include pharmaceutically acceptable carriers, buffers, diluents, and / or additives, etc. Such pharmaceutical compositions are used to express optimized REP-1 or CNGA3 or CNGB3 in ocular cells via delivery by such recombinantly engineered or artificial AAVs.

[0106] For the preparation of these pharmaceutical compositions containing nucleic acid sequences, vectors, expression cassettes and rAAV viral vectors, the sequences or vectors or viral vectors are preferably evaluated for contamination by conventional methods and then formulated into pharmaceutical compositions suitable for administration to the eye. Such formulations may include the addition of a pharmaceutically and / or physiologically acceptable vehicle or carrier, particularly buffered saline or other buffers suitable for administration to the eye, such as HEPES, to maintain pH at an appropriate physiological level, and optionally other medicinal agents. These include the use of pharmaceutical agents, stabilizers, buffers, carriers, excipients, diluents, etc. For injection, the carrier will typically be a liquid. Representative physiologically acceptable carriers include sterile pyrogen-free water and sterile pyrogen-free phosphate-buffered saline. A variety of such known carriers are provided in U.S. Patent Publication No. 7,629,322, incorporated herein by reference. In one embodiment, the carrier is an isotonic sodium chloride solution. In another embodiment, the carrier is a balanced salt solution. In one embodiment, the carrier comprises tween. If the virus is to be stored for long periods of time, it may be frozen in the presence of glycerol or Tween 20.

[0107] In one exemplary specific embodiment, the carrier or excipient composition comprises 180 mM NaCl, 10 mM NaPi, pH 7.3, with 0.0001%-0.01% Pluronic F68 (PF68). The exact composition of the saline component of the buffer ranges from 160 mM to 180 mM NaCl. Optionally, a different pH buffer (perhaps HEPES, sodium bicarbonate, TRIS) is substituted for the specifically described buffer. Additionally, a buffer containing 0.9% NaCl is useful.

[0108] Optionally, in addition to the rAAV and / or variant and carrier, the compositions of the invention may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Suitable representative preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0109] Pharmaceutical compositions comprising at least one replication-deficient rAAV virus as described herein may be prepared using physiologically acceptable carriers, diluents, excipients, and / or additives for use in gene transfer and gene therapy applications. In the case of AAV viral vectors, quantification of genome copies ("GC"), vector genomes ("VG"), or viral particles may be used as a measure of the dose contained in the preparation or suspension. Any method known in the art can be used to determine the genome copy (GC) number of a replication-deficient viral composition of the present invention. One method for performing AAV GC number titration is as follows: a purified AAV vector sample is first treated with Dnase to remove AAV genomic DNA or contaminating plasmid DNA that is not packaged in capsids from the production process. The Dnase-resistant particles are then subjected to heat treatment to release the genome from the capsid. The released genome is then analyzed for specific regions ( The effective dose of recombinant adeno-associated virus carrying a nucleic acid sequence encoding an optimized REP-1 or CNGA3 or CNGB3 transgene is determined as described in S.K. McLaughlin et al., 1988 J. Virol., 62:1963, incorporated herein by reference in its entirety.

[0110] As used herein, the term "dose" can refer to the total dose delivered to a subject during the course of treatment or the amount delivered in a single unit (or multiple unit or split dosage) administration. The pharmaceutical virus composition can be administered in an amount of about 1.0 x 10, including all integer or fractional amounts within the range. 6 GC ~ approx. 1.0 x 10 15 GC's The compositions may be prepared in dosage units to contain an amount of a replication-deficient virus carrying a codon-optimized nucleic acid sequence encoding REP-1 or CNGA3 or CNGB3 described herein within a range. In one embodiment, the compositions contain at least 1 x 10 per dose, including all integers or fractions within the range. 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , or 9 x 10 7 In one embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, with all integer or fractional amounts within the range. 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , or 9 x 10 9In one embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integer or fractional amounts within the range. 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 or 9 x 10 9 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, with all integer or fractional amounts within the range. 10 , 2 × 10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 or 9 x 10 10 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, with all integer or fractional amounts within the range. 11 , 2 × 10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 or 9 x 10 11 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, with all integer or fractional amounts within the range. 12 , 2 × 10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 or 9 x 10 12 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integer or fractional amounts within the range. 13 , 2 × 10 13 , 3×10 13, 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 or 9 x 10 13 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integer or fractional amounts within the range. 14 , 2 × 10 14 , 3×10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 or 9 x 10 14 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integer or fractional amounts within the range. 15 , 2 × 10 15 , 3×10 15 , 4×10 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 or 9 x 10 15 In one embodiment, for human applications, the dose is formulated to contain 1 x 10 GC per dose, including all integer or fractional amounts within the range. 10 ~Approx. 1×10 12 All dosages may be measured by any known method, including, for example, by oqPCR or digital droplet PCR (ddPCR), as described in M. Lock et al., Hum Gene Methods. 2014 Apr. 25(2):115-24. Doi:10.1089 / hgtb.2013.131, incorporated herein by reference.

[0111] These doses may be administered in various volumes of carrier, excipient, or buffer preparations ranging from about 25 to about 1000 microliters, all numbers within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired efficacy of the method. In one embodiment, the volume of the carrier, excipient, or buffer is at least about 25 μL. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 75 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 225 μL. In yet another embodiment, the volume is about 250 μL. In yet another embodiment, the volume is about 275 μL. In yet another embodiment, the volume is about 300 μL. In yet another embodiment, the volume is about 325 μL. In another embodiment, the volume is about 350 μL. In another embodiment, the volume is about 375 μL. In another embodiment, the volume is about 400 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 550 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 650 μL. In another embodiment, the volume is about 700 μL, In another embodiment, the volume is about 700-1000 μL.

[0112] In one embodiment, for small animal subjects such as mice, at least 1 x 10 in a volume of about 1 µL to about 3 µL. 7 ~Approx. 1×10 11 GCs at a dose of 1000 serotypes. For larger veterinary subjects, whose eyes are roughly the same size as the human eye, the larger human doses and volumes discussed above are useful. For a discussion of best practices for administering substances to various veterinary animals, see, e.g., Diehl et al., J. Applied Toxicology, 21:15-23 (2001), which is incorporated herein by reference.

[0113] It is desirable to use the lowest effective concentration of virus or other delivery vehicle to reduce the risk of undesirable effects such as toxicity, retinal dysplasia, and detachment. Further doses within these ranges may be selected by the attending physician, taking into account the physical condition of the subject, preferably a human, being treated, the age of the subject, the particular ocular disease, and the extent to which the disease has manifested, if progressive.

[0114] Yet another aspect described herein is a method for treating, slowing, or halting the progression of vision loss in a mammalian subject having or at risk of developing choroideremia. In one embodiment, a rAAV carrying a REP-1 codon-optimized sequence, preferably suspended in a physiologically compatible carrier, diluent, excipient, and / or additive, may be administered to a desired subject, including a human subject. The method includes administering to a subject in need thereof any of the nucleic acid sequences, expression cassettes, rAAV genomes, plasmids, vectors, or rAAV vectors, or compositions comprising the same. In one embodiment, the composition is delivered subretinal. In another embodiment, the composition is delivered intravitreally. In yet another embodiment, the composition is administered using a combination of administration routes suitable for treating ocular diseases, which may include administration via the palpebral vein or other intravenous or conventional administration routes.

[0115] Yet another aspect described herein is a method for treating, slowing, or halting the progression of blindness in a mammalian subject with or at risk of developing color vision deficiency. In one embodiment, an rAAV carrying a CNGA3 or CNGB3 native, modified, or codon-optimized sequence, preferably suspended in a physiologically compatible carrier, diluent, excipient, and / or additive, may be administered to a desired subject, including a human subject. The method includes administering to a subject in need thereof any of the nucleic acid sequences, expression cassettes, rAAV genomes, plasmids, vectors, or rAAV vectors, or compositions comprising the same. In one embodiment, the composition is delivered subretinally. In another embodiment, the composition is delivered intravitreally. In yet another embodiment, the composition is administered using a combination of administration routes suitable for treating ocular diseases, which may include administration via the palpebral vein or other intravenous or conventional routes of administration.

[0116] For use in these methods, the volume and viral titer of each dose are determined individually as further described herein and may be the same or different from other treatments administered in the same or contralateral eye. The dosage, administration, or regimen may be determined by the attending physician given the description herein. In one embodiment, the composition is administered to one affected eye in a single dose selected from those listed above. In another embodiment, the composition is administered to both affected eyes simultaneously or sequentially in a single dose selected from those listed above. Sequential administration may refer to a time lag of minutes, hours, days, weeks, or months between administrations from one eye to the other. In another embodiment, the method comprises administering the composition to the eye in two or more doses (e.g., split doses). In another embodiment, multiple injections are administered in different parts of the same eye. In another embodiment, a second administration of rAAV containing a selected expression cassette (e.g., a CHM-containing cassette) is administered at a later time point. Such a time point may be weeks, months, or years after the first administration. In one embodiment, such second administration is performed with an rAAV having a different capsid than the rAAV from the first administration, hi another embodiment, the rAAV from the first and second administrations have the same capsid.

[0117] In yet another embodiment, the compositions described herein may be delivered in one composition or multiple compositions. Optionally, two or more AAV or multiple viruses may be delivered (see, e.g., WO 2011 / 126808 and WO 2013 / 049493). In another embodiment, the multiple viruses may include different replication-deficient viruses (e.g., AAV and adenovirus).

[0118] In some embodiments of the present invention, it is desirable to perform non-invasive retinal imaging and functional studies to identify the regions of rod and cone photoreceptors that should be targeted for treatment. In these embodiments, clinical diagnostic tests are used to determine the precise location for one or more subretinal injections. These tests may include electroretinography (ERG), perimetry, topographical mapping of retinal layers and measurement of their thickness by confocal scanning laser ophthalmoscopy (cSLO) and optical coherence tomography (OCT), topographical mapping of cone density via adaptive optics (AO), functional eye exams, and the like, depending on the species, physical condition, or health, and dosage of the subject being treated. In terms of imaging and functional studies, in some embodiments of the present invention, one or more injections are performed in the same eye to target different regions of the affected eye. The volume and viral titer of each injection is determined individually as further described herein and may be the same or different from other injections in the same or contralateral eye. In another embodiment, a single relatively large volume injection is performed to treat the entire eye. In one embodiment, the volume and concentration of the rAAV composition are selected to affect only the region of damaged ocular cells. In another embodiment, the volume and / or concentration of the rAAV composition is greater to reach a larger portion of the eye, including intact photoreceptors.

[0119] In one embodiment of the methods described herein, a single intraocular delivery of a composition described herein, e.g., AAV delivery of an optimized REP-1 cassette, is useful in preventing vision loss and blindness in subjects at risk of developing choroideremia. In another embodiment of the methods described herein, a single intraocular delivery of a composition described herein, e.g., AAV delivery of an optimized CNGA3 or CNGB3 cassette, is useful in preventing vision loss and blindness in subjects at risk of developing color vision deficiency.

[0120] Thus, in one embodiment, the composition is administered prior to the onset of disease. In another embodiment, the composition is administered prior to the onset of vision damage or loss. In another embodiment, the composition is administered after the onset of vision damage or loss. In yet another embodiment, the composition is administered when fewer than 90% of the rods and / or cones or photoreceptors are functioning or remaining compared to an unaffected eye.

[0121] In another embodiment, the method includes conducting additional studies, such as functional and imaging studies, to determine the effectiveness of the treatment. For testing in animals, such tests include retinal and visual function assessment via electroretinograms (ERGs) to look at rod and cone photoreceptor function, optokinetic nystagmus, pupillometry, water maze testing, light-dark preference, optical coherence tomography (to measure the thickness of various layers of the retina), histology (retinal thickness, nuclei arrays in the outer nuclear layer, immunofluorescence to demonstrate transgene expression, cone photoreceptor counting, staining of retinal sections with peanut agglutinin to identify cone photoreceptor sheaths).

[0122] Specifically for human subjects, following administration of the compositions in the dosages described herein, subjects are examined for treatment efficacy using electroretinograms (ERGs) to test rod and cone photoreceptor function, pupillometry, contrast sensitivity color vision testing, visual field testing (Humphrey / Goldmann visual fields), perimetry mobility tests (obstacle course), and reading speed testing. Other useful post-treatment efficacy tests to which subjects may be subjected following treatment with the pharmaceutical compositions described herein are functional magnetic resonance imaging (fMRI), full-field light sensitivity testing, retinal structure studies including optical coherence tomography, fundus photography, fundus autofluorescence, adaptive optics laser scanning ophthalmoscopy, mobility testing, reading speed and accuracy testing, microperimetry, and / or fundusoscopy. These and other efficacy tests are described in U.S. Pat. No. 8,147,823; co-pending International Patent Application Publication No. WO 2014 / 011210 or WO 2014 / 124282, which are incorporated herein by reference.

[0123] In yet another embodiment, any of the above methods are performed in combination with another or secondary treatment. In yet another embodiment, these methods of treating ocular diseases include treating a subject with the compositions described in detail herein in combination with another treatment, such as antibiotic treatment, pain relief treatment, etc. The additional treatment may be any currently known or yet unknown treatment that helps prevent, stop, or ameliorate these mutations or defects or any effects associated therewith. The secondary treatment may be administered before, simultaneously with, or after administration of the above compositions. In one embodiment, the secondary treatment includes non-specific methods for maintaining the health of retinal cells, such as the administration of neurotrophic factors, antioxidants, or anti-apoptotic agents. Non-specific methods are achieved via the injection of proteins, recombinant DNA, recombinant viral vectors, stem cells, fetal tissue, or genetically modified cells. The latter may include encapsulated genetically modified cells.

[0124] In one embodiment, a method for producing a recombinant rAAV comprises obtaining a plasmid containing the AAV expression cassette described above and culturing packaging cells harboring the plasmid in the presence of sufficient viral sequences to allow packaging of the AAV viral genome into an infectious AAV envelope or capsid. As described above, it may be used to generate rAAV vectors capable of delivering codon-optimized REP-1 or CNGA3 or CNGB3 in the expression cassettes and genomes described above and in the Examples below.

[0125] In yet another aspect, a vector is provided that includes any of the expression cassettes described herein. As noted above, such vectors can be plasmids of various origins and are useful in some embodiments for the generation of recombinant replication-defective viruses, as further described herein.

[0126] In another embodiment, the vector is a plasmid comprising an expression cassette, wherein the expression cassette comprises a codon-optimized nucleic acid sequence encoding the AAV inverted terminal repeat and REP-1 and expression control sequences that direct expression of the encoded protein in a host cell.

[0127] In another embodiment, the vector is a plasmid comprising an expression cassette, wherein the expression cassette comprises an AAV inverted terminal repeat and a codon-optimized nucleic acid sequence encoding CNGA3 and expression control sequences that direct expression of the encoded protein in a host cell.

[0128] In another embodiment, the vector is a plasmid comprising an AAV expression cassette, wherein the expression cassette comprises an AAV inverted terminal repeat and a codon-optimized nucleic acid sequence encoding CNGB3 and expression control sequences that direct expression of the encoded protein in a host cell.

[0129] It should be noted that the terms "a" or "an" refer to one or more. Thus, "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0130] The terms "comprise," "comprises," and "comprising" are to be interpreted inclusively and not exclusively. The terms "consist," "consisting," and variations thereof are to be interpreted exclusively and not inclusively. Although various aspects in the specification are presented using the language of "comprising," it is also intended that, under other circumstances, the relevant aspect should be construed using the language of "consisting of" or "consisting essentially of," and will be so described.

[0131] As used herein, the term "about" means a 10% variation from the given reference unless otherwise specified.

[0132] The term "modulate" or variations thereof, as used herein, refers to the ability of a composition to inhibit one or more components of a biological pathway.

[0133] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to published books which provide those skilled in the art with general guidance to many of the terms used in this application. [Example]

[0134] The following examples are illustrative only and are not intended to limit the invention.

[0135] Example 1 - Differentiation of pluripotent stem cells into RPE Choroideremia lacks a relevant mouse model, and there is no canine model, so transduction and expression will be examined in a human retinal cell model of the disease. Because it is not possible to obtain retinal cells from living subjects, RPE will be generated from induced pluripotent stem cells. Pluripotent stem cells will be directed into RPE using the protocol described by Buchholz et al., "Rapid and Efficient Directed Differentiation of Human Pluripotent Stem Cell Into Retinal Pigmented Epithelium," Stem Cells Translational Medicine, 2013;2:384-393, the entire contents of which are incorporated herein by reference. Cereso et al., "Proof of concept for AAV2 / 5-mediated gene therapy in iPSC-derived retinal See also pigment epithelium of a choroideremia patient, Molecular Therapy - Methods & Clinical Development (2014) 1, 14011. Other methods for the production of RPE are known in the art.

[0136] Briefly, human induced pluripotent stem cell lines are maintained on mitomycin C-treated or irradiated mouse embryonic fibroblast feeder layers in Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM / F12) supplemented with 2 mM GlutaMAX-I, 20% knockout serum replacement, 0.1 mM Modified Eagle's Medium Non-Essential Amino Acids (MEM NEAA), 0.1 mM β-mercaptoethanol, and 4 ng / ml bFGF.

[0137] Pluripotent stem cells were subcultured directly onto Matrigel (BD Biosciences) in DMEM / F12 with IX B27, IX N2, and IX NEAA (Invitrogen). From day 0 to day 2, 50 ng / ml Noggin, 10 ng / ml Dkk1, 10 ng / ml IGF1, and 10 mM nicotinamide were added to the basal medium. From days 2 to 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 days 4 to 6, 10 ng / ml Dkk1, 10 ng / ml IGF1, and 100 ng / ml Activin A (R&D Systems) were added to the basal medium. From days 6 to 14, 100 ng / ml Activin A, 10 μM SU5402 (EMD Millipore, Darmstadt, Germany), and 1 mM VIP were added to the basal medium. Control experiments were performed in basal medium only (DMEM / F12, B27, N2, and NEAA).

[0138] Cells with non-RPE morphology are mechanically enriched by scraping. The remaining RPE is then digested using TrypLE Express (Invitrogen) for 5 minutes at 37°C. Cells are passed through a 30-μm single-cell strainer and seeded onto Matrigel-coated tissue culture plastic Transwell membranes or CC2-treated chamber slides. The enriched cells are cultured for 30 days in DMEM-high glucose with 1% fetal bovine serum (FBS), GlutaMAX, and sodium pyruvate.

[0139] Example 2 - Cells transduced with AAV-REP-1 Briefly, we generated the AAV2 / 8CMV.CBA-REP-1 viral vector carrying the REP-1 codon-optimized sequence by transient transfection of HEK293 cells. The vector is purified by double CsCl centrifugation, dialyzed, and titrated by dot blot assay.

[0140] For prenylation experiments, RPE were seeded in 24-well plates at 1.2 × 10 6 At confluence, cells are estimated to contain 100,000 vector genomes (vg) per cell. Prenylation assays are performed 4 weeks after transduction. Experiments are repeated three times.

[0141] Example 3 - Prenylation As described in the above-cited Vasireddy et al., PloS One. 2013 May 7;8(5):e61396, in vitro prenylation assays were performed in the presence of recombinant Rab geranylgeranyltransferase and RAB27 using [3H]-geranylgeranyl pyrophosphate (GGPP) (Perkin Elmer, Boston, MA, USA) as the prenyl group donor. The incorporation of radiolabeled prenyl groups into RAB27 protein was measured by scintillation counting. For consistency, the control value was normalized to 100 and used as the baseline. All experiments were performed in triplicate, and statistical comparisons of prenylation between experimental and control groups were performed using a two-tailed unpaired Student's test. The test is used to evaluate the schizophrenia.

[0142] Briefly, transduced REP cells are washed with cold PBS 48 hours after transduction. The cell pellet is collected and washed extensively with cold PBS. Cells are lysed with RIPA + protease inhibitors for 30 minutes on ice. In an alternative protocol, cells are sonicated. The cytosolic fraction is collected by centrifugation of the cell lysate at 75,000-100,000 g for 1-2 hours at 4°C.

[0143] The stock for the prenylation reaction is prepared as follows.

[0144] [Table 1]

[0145] The final reaction volume used for prenylation is 25 μL.

[0146] [Table 2]

[0147] The reaction mixture is incubated at 37°C for 30 minutes. The reaction is stopped by adding 9:1 ethanol / HCl and incubated for 30 minutes. Protein is collected (0.1 ml) onto glass fiber filters (Whatman papers) by vacuum filtration. The filters are carefully washed three times with cold phosphate buffer to remove unbound material. The membrane is carefully dried. The filters are placed in 5 ml of scintillation cocktail and subjected to scintillation counting. See also Tolmachova et al., CHM / REP1 cDNA delivery by lentiviral vectors provides functional expression of the transgene in the retinal pigment epithelium of choroideremia mice, The Journal of Gene Medicine, 2012;14-158-68, incorporated herein by reference in its entirety.

[0148] Proof-of-concept assays for CNGA3 or CNGB3 may involve the use of naturally occurring mutant animal models (e.g., Cnga3- / - mice or Awassi sheep). These mouse models can be bred with Nrl- / - mice, an "all-cone" photoreceptor mouse, to obtain a double knockout. The latter (Cnga3- / -Nrl- / -) mice may expedite the identification of efficacy. Efficacy can be measured by pupillometry (e.g., using optokinetics), electroretinograms, and visual behavior, which are measures of visual acuity and contrast. Ultimately, histology will demonstrate transgene expression with improved outcome over other measures. Histological methods may also be used to demonstrate the effect of intervention on cone photoreceptors (e.g., total number, density, and location of cone photoreceptors).

[0149] Similar to the choroideremia discussed above, a proof-of-concept assay for gene augmentation therapy for CNGA3- or CNGB3-related color blindness may involve the use of induced pluripotent stem cell (iPSC) models. iPSC models generated from subjects with color blindness due to CNGA3 or CNGB3 mutations are differentiated in vitro into retinal precursor and / or photoreceptor cells. Wild-type CNGA3 (or CNGB3) cDNA is delivered to these cells using recombinant AAV, and the cells are analyzed for preservation of the biogenesis and function of the associated (cyclic nucleotide-gated, CNG) channel composed of these subunits. Channel function is assessed by electrophysiology on membrane patches. Channel restoration should rescue cGMP-activated currents. Additional studies should examine the sensitivity of channel function to physiological ligands before and after delivery of wild-type CNG cDNA. can be done.

[0150] Example 4: In vitro expression of AAV codon-optimized human CHM The objective of this study was to evaluate the potential for AAV-mediated CHM expression in 84-31 and COS-7 cell lines following gene delivery using a series of next-generation AAV2 and AAV8 vectors encoding the codon-optimized CHM gene (SEQ ID NO: 1).

[0151] To maximize CHM expression, a codon-optimized CHM sequence was created (SEQ ID NO: 1). A codon-optimized plasmid was synthesized and used in the construction of all next-generation CHM transgene expression cassettes. To overcome the potential problem of non-functional AAV genome contamination, a non-coding lambda stuffer region was included in the vector backbone. The introduction of the stuffer not only increases the length of the plasmid but also reduces the possibility of plasmid DNA backbone contamination during AAV packaging. The effect of introducing a stuffer region into the vector backbone to remove plasmid DNA impurities was investigated in an independent study. Various constructs were generated using two recombinant AAV proviral plasmid backbones (high-copy and low-copy). The high-copy plasmid was designed based on the pUC vector origin. The low-copy plasmid was designed based on the p15A origin. A Kozak sequence was introduced upstream of the start codon to further enhance translation from the correct start codon.

[0152] A total of four plasmids were constructed for this study and the studies described in the Examples below (Table 1). In addition, a plasmid carrying the CHM native sequence currently being used in clinical trials was also constructed (version 1). Plasmid maps for versions 2a, 2b, 3a, and 3b, as well as version 1, are shown in Figures 6-10, respectively.

[0153] [Table 3]

[0154] In vitro expression of these constructs was examined in COS-7 and 84-31 cell lines. The engineered characteristics of the next-generation CHM constructs are described in Table 1.

[0155] Recombinant AAV proviral high-copy and low-copy plasmids were generated by cloning the codon-optimized human CHM cDNA (hCHM) (SEQ ID NO: 1) into the transgene cassette. The transgene was placed under the control of the hybrid chicken β-actin (CBA) promoter, which consists of the CBA exon 1 flanked by the cytomegalovirus (CMV) immediate-early enhancer, the proximal chicken β-actin promoter, and intron 1 sequences. The proviral high-copy and low-copy plasmids also contain AAV inverted terminal repeats and poly(A) sequences. The next-generation plasmid backbone used in the current study contains a lambda phage fragment stuffer, plus kanamycin A. Bacterial selection gene (kanamycin bacterial selection The high-copy-number vectors are similar to those of pUC plasmids (approximately 300 copies per bacterial cell). The low-copy-number plasmids (approximately 10 copies per bacterial cell) originate from p15A. To enhance translation from the correct start codon, all next-generation constructs contain a Kozak consensus sequence upstream of the ATG start codon. The generated plasmids are sequence-verified using primers that can specifically target either the promoter + enhancer extension sequence or the codon-optimized CHM sequence. Plasmid maps and sequences for all five constructs are shown in Figures 6-10. Standard triple transfection using calcium phosphate was used to generate the AAV vectors listed below (see Table 2 for vector qualification). Both AAV2 and AAV8 serotype vectors were generated, confirming that the results were serotype-independent.

[0156] [Table 4]

[0157] The 84-31 cell line is a subclone of the 293HEK cell line (human embryonic kidney cells) and constitutively expresses the adenovirus E4 protein to enhance AAV viral transduction. COS-7 cells are a fibroblast-like cell line derived from monkey kidney tissue. Both 84-31 and COS-7 cells were cultured separately in 6-well cell culture plates and transduced with one of 10 test articles (either AAV2 or AAV8) at five different multiplicities of infection (MOIs). After 36–48 h, cells were harvested, lysed, and protein samples were prepared for SDS-PAGE and subsequent Western blot analysis to detect the expression of exogenous CHM.

[0158] Both 84-31 and COS-7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM)-High Glucose with 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C in an environment supplied with 5% CO2. The day before transduction (18-24 hours), 3 × 10 5 Cells at a density of (3E5) were seeded in 2 ml of cell culture medium in each well of a 6-well cell culture dish. The seeded cells were incubated at 37°C in an environment supplied with 5% CO2. Wells of both COS-7 and 84-31 cells were infected with the AAV vectors listed below at various multiplicities of infection (MOI) (Tables 3 and 4). No virus was added to negative control cells (untransduced cells). Briefly, the tissue culture medium was removed, and a fresh 2 ml aliquot of medium was added to each well of a 6-well culture dish. A predetermined amount of AAV vector was then measured (directly from the stock solution) and added to each well (Tables 3 and 4). 1 x 10 4 For an MOI of 1, 1 μL of each virus stock was diluted to 10 μL with cell culture medium. A predetermined volume of virus from the solution was added to each well (Tables 3 and 4). Cells were incubated with AAV virus at 37°C with 5% CO for 36-48 hours before harvesting. Cells were examined under a microscope prior to harvesting to check for abnormalities.

[0159] [Table 5]

[0160] [Table 6]

[0161] We first investigated whether in vitro expression of CHM was cell line-independent using both COS7 and 84-31 cell lines. Once this was confirmed, all subsequent experiments were performed exclusively in 84-31 cells, which showed excellent transduction efficiency using AAV. Wells of 84-31 cells were infected with the AAV vectors listed below at various MOIs (see Tables 3 and 4).

[0162] Western blot analysis: 1. Cell lysates were prepared. 36-48 hours after infection, AAV-transduced cells, along with untreated control cells, were harvested after extensive PBS washing. Cells were then lysed on ice using RIPA buffer with protease inhibitors. The cell lysate was clarified by centrifugation at 13,000 rpm for 10 minutes. 2. Protein Quantification and Preparation. Protein quantification of the cell lysate was performed using a ThermoFisher Micro BCA™ Protein Assay Kit according to the manufacturer's instructions. Protein concentration was determined by obtaining an OD reading at 562 nm. To evaluate in vitro expression of CHM, 40-60 μg of measured protein was loaded onto a 4-12% Bis-Tris gel. 3. SDS-PAGE and Blotting. SDS-PAGE and Western blot analysis were performed according to published protocols. Briefly, the protein gel was transferred to a nitrocellulose membrane, blocked in milk, and incubated with primary antibodies. Anti-human REP-1 2F1 antibody (2F1, 1:1000 dilution) and one of the following antibodies were used as the primary antibody for each blot: anti-GAPDH antibody (1:1000 dilution), anti-actin antibody (1:1000 dilution), or anti-tubulin antibody (1:5000 dilution). After washing the blot, HRP-conjugated anti-mouse IgG antibody and / or anti-rabbit IgG antibody at a concentration of 1:5000 was used as the secondary antibody. The blot was developed by chemiluminescence using ECL reagent according to the manufacturer's instructions. Controls: 1. Loading Control: One of the following antibodies was used as a loading control to demonstrate equal loading of proteins in each well of the gel: anti-actin antibody, anti-tubulin antibody, or anti-GAPDH antibody. The anti-tubulin antibody detects a protein of ~51 kDa, the anti-actin antibody detects a protein of ~42 kDa, and the anti-GAPDH antibody detects a protein of ~39 kDa. Initial blots were probed with either anti-tubulin, anti-actin, or anti-GAPDH antibodies depending on their availability. After the initial experiments, anti-GAPDH antibody was used as a loading control for consistency.2. Positive control: After confirming the production of hREP-1 protein in AAV2.V2a-transduced COS-7 cells, AAV2.V2a-Cos-7 cell lysate was used as a positive control in subsequent Western blot experiments. 3. Negative control: Untreated cells were used as a negative control. Analysis of the Western blot results of REP-1 protein production in various cell lines is summarized in Table 5.

[0163] [Table 7]

[0164] A monoclonal human REP-1-specific antibody detected a single ~75-80 kDa hREP-1 protein in cells transduced with next-generation AAV2.copt.CHM / AAV8.copt.CHM. No 75-80 kDa band was observed in cell lysates from untreated control cells. Probing the blot with either anti-actin, anti-tubulin, or anti-GAPDH antibodies revealed bands of equal intensity in all lanes of the Western blot, including the untreated control. The anti-actin antibody detected a protein molecular weight band at ~42 kDa, the anti-tubulin antibody detected a protein at ~51 kDa, and the anti-GAPDH antibody detected a protein at ~39 kDa. All antibodies detected only specific bands of the predicted size and molecular weight. No nonspecific bands were observed in any of the blots. Prestained molecular weight markers were used to compare the molecular weights of the proteins of interest.

[0165] In summary, REP-1 protein was observed at the predicted size in COS-7 and 84-31 cells transduced with AAV2.V2a, AAV2.V2b, AAV2.hCHM.V3a, and AAV2.hCHM.V3b. Untreated controls did not reveal the presence of human REP-1 protein of the predicted size. Labeling of the blot with anti-actin antibody detected a protein band of equal intensity at ∼42 kDa in all lanes of the gel. A prestained protein ladder was used to compare the molecular weights of REP-1 and actin. Data not shown.

[0166] The results demonstrate that AAV2 and AAV8 serotype vectors containing next-generation plasmids can effectively transduce 84-31 and COS-7 cells. Expression of CHM in the next-generation plasmids was detectable and showed a dose-dependent trend. Transduction of cells with next-generation hCHM viruses resulted in the expression of the predicted size of the REP-1 protein. brought about production.

[0167] Example 5: Comparison of in vitro protein expression of AAV codon-optimized human CHM and AAV native human CHM The aim of this study was to delineate the transduction efficiency of AAV vectors (serotypes 2 and 8) containing different versions of CHM-containing transgene cassettes by measuring the levels of REP-1 protein in a research model based on the 84-31 cell line.

[0168] Plasmids and Vectors: A total of five transgene plasmids were compared in either AAV2 or AAV8: Version 1 (previously used in ongoing clinical trials) and four next-generation versions (V2a, V2b, V3a, and V3b). Plasmids were generated as described in Example 4, and their characteristics are shown in Table 1. Table 2 above provides a summary of the AAV2 and AAV8 vectors generated and the concentrations of the virus stocks.

[0169] Study design (e.g., treatment groups) 1. In a pilot experiment, AAV2.hCHM.version 1, version 2a, and version 2b were used to transduce COS-7 and 84-31 cells. Western blot analysis was performed to compare the transduction efficiency levels in the two cell lines.

[0170] 2. 3 × 10 of one of the 10 test articles (versions 1, 2a, 2b, 3a, and 3b in either an AAV2 or AAV8 background) 5 The vectors were used at an MOI of 1000 and transduced 84-31 cells cultured in 6-well plates. After 36-48 hours, cells were harvested and lysed. Cell lysates were loaded onto SDS-PAGE and subjected to further Western blot analysis. REP-1 protein levels were compared among all construct versions. Two separate plates were prepared for each AAV2.CHM or AAV8.CHM experiment and analyzed separately.

[0171] Test material administration 3.4.1 Cell culture Both 84-31 and COS-7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM)-High Glucose with 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C in an environment supplied with 5% CO2.

[0172] 3.4.2 Preparation of cells for transduction The day before transduction (18-24 hours), 84-31 and COS-7 cells were plated at 3 x 10 in 2 ml of cell culture medium per well of a 6-well cell culture dish. 5 The seeds were seeded at a density of (3E5). The seeded cells were incubated at 37°C in an environment supplied with 5% CO2.

[0173] 3.4.3 Transduction 84-31 cells and COS-7 wells were transfected with 3 × 10 AAV vectors as described below. 5Cells were infected at an MOI of 1000 ng / ml (see Table 6 for the pilot experiment and Table 7 for the second set of experiments). No virus was added to negative (untransduced) controls. Briefly, in each well of a 6-well cell culture dish, the tissue culture medium was first removed and replaced with 2 ml of fresh medium per well. A predetermined amount of AAV vector (see Table 2 for the vector volume used for transduction) was then measured (from the stock solution) and added directly to each well. Cells were incubated with AAV virus at 37°C with 5% CO2 for 36-48 hours until harvest. Prior to harvest, cells were examined under a microscope for abnormalities. Western blot analysis was performed as described in Example 4. Analysis was performed.

[0174] [Table 8]

[0175] [Table 9]

[0176] Results: Comparison of expression of native hCHM (AAV2.hCHM.V1) versus codon-optimized CHM AAV2a and 2b versions in 84-31 and COS-7 cells In this experiment, 84-31 and COS-7 cells were transduced with AAV2.hCHM.version 1, AAV2.hCHM.version 2a, or Aav2.hCHM.version 2b without vector (untreated control). Western blot analysis using an anti-human REP-1 antibody showed that REP-1 protein levels were detectable at ∼75–80 kDa in all AAV2 (V1, V2a, V2b)-transduced samples and in both cell lines (data not shown). Slightly superior protein expression was observed in the 84-31 cell line (Table 8). The anti-REP1 antibody detected negligible amounts of REP-1 protein in untreated cells. Labeling of the blot with a GAPDH antibody detected a band at ∼39 kDa in all cell lysates, including untreated cells.

[0177] Densitometric evaluation of the blots (quantification of protein levels) using ImageJ software showed that, after normalizing the values ​​to the expression of endogenous GAPDH protein, the transduction efficiency was similar in 84-31 and COS-7 cells (see Table 8 for results). Based on this, the 84-31 cell line, which is of human origin, was used for further experiments.

[0178] Finally, AAV2.V1, AAV2.V2a, and Aav2.V2b induced REP-1 protein production in both 84-31 and COS-7 cells with similar transduction efficiencies.

[0179] [Table 10]

[0180] Comparison of expression of native CHM versus codon-optimized CHM AAV2 vectors in 84-31 cells: Western blot analysis of 84-31 cells transduced with AAV2.hCHM.V2a, V3a, V2b, V3b, and V1 using an anti-human REP-1 antibody detected a band at ∼75–80 kDa in all conditions (data not shown). The anti-REP1 antibody detected negligible amounts of REP-1 protein in untreated cells. Labeling of blots with a GAPDH antibody detected a band at ∼39 kDa in all cell lysates, including untreated cells. Densitometric evaluation of blots (quantification of expression levels) using ImageJ software showed increased expression of AAV2.hCHM.V2a, 3a, 2b, and 3b compared to AAV2.hCHM.V1 after normalizing values ​​to endogenous GAPDH protein production. See Tables 9 and 10 for results.

[0181] [Table 11]

[0182] [Table 12]

[0183] Comparison of expression of native CHM versus codon-optimized CHM in AAV8.V1, V2a, V3a, V2b, and V3b vectors in 84-31 cells: Western blot analysis of cells transduced with AAV8.V1, AAV8.V2a, AAV8.V3a, AAV8.2b, and AAV8.3b using an anti-human REP-1 antibody detected a band at ~75–80 kDa in all transduced cells (data not shown). The anti-REP-1 antibody detected negligible amounts of REP-1 protein in untreated cells. Labeling of the blot with a GAPDH antibody detected a band at ~39 kDa in all cell lysates, including untreated cells. Densitometric evaluation of the blot using ImageJ software demonstrated higher expression of AAV8.hCHM.V2a;3a;2b;3b compared to AAV8.V1. Values ​​were obtained after first normalizing the CHM values ​​to the expression of each endogenous GAPDH protein and then to the average expression level of version 1. See Tables 11 and 12 for the results.

[0184] [Table 13]

[0185] [Table 14]

[0186] Conclusions: Comparative expression studies showed that application of AAV vectors carrying next-generation AAV.hCHM. versions 2a, 2b, 3a, and 3b in 84-31 cells induced increased REP-1 protein production compared to version 1 (currently used in clinical trials) for both AAV2 and AAV8 serotype vectors.

[0187] Example 6: Evaluation of the effect of lambda stuffer on AAV vector production by qPCR titer analysis To evaluate the effect of the lambda stuffer sequence on the amount of DNA impurities, a single qPCR (quantitative polymerase chain reaction) experiment was performed for all eight AAV vectors listed in Table 2 above. A linearized AAV plasmid standard was used to generate assay standards. Primer-probe sets were designed on either the CMV / CBA promoter region for quantification of correctly packaged AAV genomes or the kanamycin resistance (KanR) coding region for reverse packaging. Standard and vector samples were run in two sets: one using the CMV / CBA primer-probe set and the other using the KanR set. Vector sample values ​​(viral genome copies per mL) were determined from each standard curve. The effect of the stuffer sequence was evaluated by comparing the relative amounts of KanR-containing impurities in each vector lot to the CMV / CBA-containing sequence. Reagents: Transgene-containing viral vector titer: Reference standard: CMV-CBA promoter Primer: CMV-F: CCC ACT TGG CAG TAC ATC AA CMV-R:GCC AAG TAG GAA AGT CCC ATA A FAM-Probe: / 56-FAM / CA TAA TGC C / ZEN / A GGC GGG CCA TTT AC / 3IABkFQ / Impurity-containing viral vector titer: Reference standard: Kanamycin resistance gene Primer: KAN-F:GAT GGT CGG AAG TGG CAT AA KAN-R:TGC GCC AGA GTT GTT TCT FAM-Probe: / 56-FAM / CC GTC AGC C / ZEN / A GTT TAG TCT GAC CA / 3IABkFQ / Diluent Reagents: Diluent Q (0.001% PF-68 in nuclease-free water): Dilute 1% PF-68 solution 1000-fold with sterile water. Diluent S: Diluent Q + 2ng / µL salmon sperm DNA (Agilent technologies Cat# 201190). ABI TaqMan™ Universal Master Mix (App lied Biosystems 4304437 / 4326708) Quiagen PCR Product Purification Kit(Quiagen 28104) ·ABI QuantStudio 6 Flex System

[0188] Sample preparation Dnase digest solution was prepared by mixing the following: 5 μL Dnase buffer (10X), 30 μL nuclease-free H2O, 5 μL Dnase I (Invitrogen, 18068-015).

[0189] Ten microliters of each AAV vector sample was mixed and incubated at ambient temperature for 10 minutes. The digestion mixture was inactivated by adding 50 μL of SDS / EDTA / NaCl solution (0.2% SDS / 5 mM EDTA / 0.2 M NaCl) and incubated at 95°C for 10 minutes. Each AAV vector sample was diluted 10-100,000 times in Diluent S for qPCR analysis.

[0190] qPCR standard preparation Reference standard DNA (linearized) was prepared by digesting plasmid p1008 (a stuffer-free low-copy transgene plasmid) with XhoI and purifying it using the Quiagen PCR purification kit. The purified material was analyzed on an agarose gel to confirm identity and quantified by Nanodrop. The equivalence is as follows: 1 bp = 1.096 x 10 -21 The DNA copy number was determined from the stock solution concentration using g. CR standards were prepared according to the following table:

[0191] [Table 15]

[0192] PCR reaction setup Extracted DNA samples were analyzed in triplicate (three wells) in a single qPCR experiment. Reference DNA standards were included at concentrations ranging from 103 to 108 copies per well, and the same conditions were repeated three times. A no-template control (NTC) was included as a negative control. Each AAV vector preparation was analyzed using both the CMV / CBA and KanR primer / probe sets. Similarly, for quantification of each set, a standard was also analyzed using both the CMV / CBA and KanR primer / probe sets.

[0193] [Table 16]

[0194] The PCR reaction conditions were set as follows: 1 cycle of 50°C for 2 minutes; 95°C for 10 minutes. 1 cycle; 95℃ 15 seconds, 40 cycles; 60℃ 1 minute, 40 cycles Experimental Performance. Standards were prepared and run at 103–108 DNA copies per well. Because assay sensitivity was not a critical factor for this experiment, the lower limit of the assay was set at 1,000 copies. A standard curve for the experiment was constructed using the standard copy numbers and the standard CT (threshold cycle) values. Linear regression of the standards was performed using ABI software (data not shown). The standard curve fit had a correlation coefficient (R2 value) of 0.998 or greater, indicating a reliable fit model. The slope of the standard curve was -3.5. The slope was used to calculate the efficiency of the amplification reaction; values ​​of -3.2 to -3.6 indicated an amplification efficiency of 90%–110%. Both standard reactions were run at a high efficiency of 92.6–93.8%. The precision of triplicate wells ranged from 2–10%, indicating excellent agreement between replicate products. The no-template control (NTC) produced unquantifiable amplification below the lower limit of the assay.

[0195] [Table 17]

[0196] result: Sample value determination: Sample values ​​(AAV genome and reverse-packaging copy numbers) were interpolated from each fitted standard curve (CMV / CBA or KanR) using the C values. The interpolated DNA copy numbers were corrected for the initial dilution and / or digestion dilution. An additional correction factor of 2 was applied to account for differences between the double-stranded DNA standard and single-stranded DNA in the samples.

[0197] The analytical results for the eight AAV vectors are summarized in the table below, along with a quantitative comparison between transgene-containing AAV concentrations (CMV / CBA) and KanR-containing impurity concentrations. Analysis of the results indicates that insertion of the lambda stuffer into the transgene effectively reduces the occurrence of plasmid-backbone DNA (i.e., KanR) packaging during AAV production by 7-20 fold (Figure 11).

[0198] [Table 18]

[0199] Example 6: In vitro expression of next-generation AAV8 vectors in iPS cells by Western blot The objective of this study was to evaluate the ability of AAV-mediated CHM expression in induced pluripotent cell lines (iPSCs) after gene delivery using a series of next-generation AAV2 and AAV8 vectors carrying a codon-optimized REP-1-encoding gene.

[0200] Induced pluripotent stem (iPS) cell technology has been successfully used as a platform for testing gene therapy vectors in several proof-of-concept and gene therapy studies, including ocular diseases. These subject-specific iPS cells provide a useful in vitro model system for studying disease pathogenesis and establishing models for proof-of-concept gene therapy testing when relevant animal models are unavailable. As a preliminary step to testing our AAV-mediated gene augmentation therapy for choroideremia (CHM), we generated iPS cells from a human subject with a mutation in the causative gene CHM, encoding Rab escort protein 1 (REP-1) (see Example 1) (methods are described in NCP.003). The generated iPS cells were used to evaluate the in vitro expression of our next-generation AAV.codon-optimized.CHM construct.

[0201] Plasmids and vectors were as described in Example 4. Induced pluripotent stem (iPS) cells are stem cells generated in the laboratory from somatic cells, peripheral blood mononuclear cells, which are reprogrammed back to a pluripotent state. Reprogramming blood cells allows for the development of individualized in vitro cell models for therapeutic applications. In this report, iPS cells from subjects with CHM were used to examine the in vitro production of REP-1 protein via Western blot analysis. The following table (Table 17) describes the details of the iPS cells studied and their respective CHM disease-causing mutations.

[0202] [Table 19]

[0203] Study design (e.g., treatment groups) 1. Culture iPS cells on 12-well cell culture plates with 1 x 10 AAV2.hCHM version 1, version 2a; version 2b; version 3a; version 3b (AAV2.V1; V2a; V2b; V3a; V3b). 5 or 3 x 10 5 Cells were infected at an MOI of either 0.01 or 0.1. After 24 hours of transduction, 1 ml of iPS cell medium was added to the cells. After 36-48 hours of transduction, cells were harvested, lysed, and processed for SDS-PAGE and subsequent Western blot analysis. REP-1 protein production was assessed in cells transduced with all versions of the construct and compared to untreated controls.

[0204] 2. In a pilot experiment, three iPS cell lines cultured on 12-well cell culture plates were transfected with AAV8.hCHM version 1 and AAV8.hCHM version 2a (AAV8.V1; AAV8.V2a) at 1 × 10 6 Transduce iPS cells at an MOI of 100. The cell lines were derived from three CHM-affected subjects with unrelated mutations in the REP1 gene and cultured in separate plates for this purpose. After 36-48 hours, the cells were harvested, lysed, and subjected to Western blot analysis compared with untreated cell lysates.

[0205] Test material administration 3.4.1 Cell culture Culture of iPS cells from CHM subjects. Briefly, iPS cells were cultured on mouse embryonic fibroblasts (MEFs, feeder cells) in iPS cell medium at 37°C in an environment supplied with 5% CO2 and 5% O2.

[0206] 3.4.2 Preparation of cells for transduction The day before seeding the cells, 12-well dishes were coated with Matrigel as described in reference NCP.003 (NCP.003: Culturing of iPS cells from CHM patients and controls). Cells cultured on MEFs were seeded on Matrigel without MEFs (feeder cell-free culture) before transduction of iPS cells with AAV2 or AAV8 viral vectors, respectively. 4.5 × 10 cells were added to 1 ml of iPS cell medium in each well of a 12-well cell culture dish. 5 (4.5+E5)~6×10 5 The cells were seeded at a density of 1 / 4, and incubated at 37°C in an environment supplied with 5% CO2 and 5% O2.

[0207] 3.4.3 Transduction To infect iPS cells with viral vectors, cells were grown to approximately 50-60% confluence (which can take 2-4 days without feeder cells). Upon reaching 50-60% confluence, one well of the 12-well plate was isolated and a cell count was performed to determine the total number of cells per well. The iPS cell wells were then infected with the AAV vectors listed below at a predetermined MOI (see Tables 18 and 19). Prior to transduction, old iPS cell medium was removed from the plate, and 1 ml of fresh iPS cell medium was added to each well. A predetermined volume of virus was added directly from the stock solution to each well. See Tables 18 and 19 for information on the total number of cells infected, the MOI, and the volume of virus used for infection. The cells were then incubated for 18-48 hours at 37°C in an environment supplied with 5% CO2 and 5% O2. After 18-24 hours of transduction, cells were examined under a microscope for abnormalities or cell death. At this time point, an additional 1 ml of fresh iPS cell medium was added to each well containing infected and uninfected cells, and the cells were incubated for an additional 18-24 hours at 37°C in a 5% CO2, 5% O2 atmosphere. Prior to harvest, cells were examined under a microscope to assess cell death or abnormal appearance.

[0208] [Table 20]

[0209] [Table 21]

[0210] Outcome Measures—Western blot analysis was performed as described herein.

[0211] result 5.1 Expression of AAV2-hCHM V1, V2a, V2b, V3a, and V3b in the JB588 iPS cell line: A monoclonal human REP-1-specific antibody detected a single ~75-80 kDa hREP-1 protein in transduced JB588 iPS cells (data not shown). No bands were observed in untreated controls, confirming the presence of disease (data not shown). 3 x 10 5 The intensity of the REP-1 protein band at an MOI of 1 x 10 for all vectors was 1 x 10. 5 It was observed that the production of REP-1 protein was stronger compared to an MOI of 1000. Recombinant AAV2.hCHM virus-mediated delivery of the hCHM gene into iPS cells resulted in dose-dependent production of REP-1 protein. Probing the blot with GAPDH antibody showed bands of equal intensity in all cell lysates. GAPDH detected a protein at ~39 kDa. Both REP-1 and GAPDH antibodies detected only a specific band of the predicted molecular weight. No nonspecific bands were observed in the blot.

[0212] Expression of AAV8-hCHM.V1, V2a in iPS cells: A monoclonal human REP-1-specific antibody detected a single ∼75-80 kDa REP-1 protein in transduced iPS cells from subjects JB527, JB500, and JB588 (data not shown). No protein band was observed in untreated controls (data not shown). Probing the blot with GAPDH antibody showed bands of equal intensity in all cell lysates, including those from untreated cells. Anti-GAPDH antibody detected a specific ∼39 kDa protein band. REP-1 and GAPDH Both H antibodies detected only specific protein bands at the predicted size and molecular weight. No detectable nonspecific protein bands were observed in the blots.

[0213] conclusion Preliminary results presented in this report revealed the following observations: Western blot analysis confirmed the presence of CHM (lack of REP-1 protein) in each of three subject-derived iPSCs (JB588, JB500, and JB527). In vitro expression studies showed that infection of iPSCs from CHM subjects with AAV2.hCHM.versions 2a, 2b, 3a, and 3b and AAV2.hCHM.version 1 (current clinical trial candidate) induced production of REP-1 protein at all MOIs examined. Infection of iPSCs from CHM subjects with AAV2.hCHM.version 2a and AAV2.hCHM.version 1 (current clinical trial candidate) at 1 × 10 6 Infection of iPS cells at an MOI of 1 resulted in production of REP1 protein in all three CHM iPS cell lines. The level of REP1 production was higher in iPSCs infected with AAV8.hCHM.V2a than in those infected with AAV8.hCHM.V1.

[0214] Example 7: Comparison of in vivo expression of AAV8 codon-optimized human CHM versus AAV native human CHM Gene therapy for multiple retinal diseases relies on effective transduction of appropriate target cells, and in the case of choroideremia, these cells are the retinal pigment epithelium (RPE) and photoreceptor cells. This study focuses on comparing in vivo expression in wild-type mice induced by constructs based on the native CHM sequence (version 1) and four next-generation transgene cassettes packaged in the AAV8 backbone. Here, we evaluated the AAV8 serotype for the purpose of improving gene transfer to photoreceptor cells.

[0215] Our experiments were designed to answer the following questions: a. How do these vectors compare with respect to in vivo transduction of photoreceptors: specifically, how effectively does Next Generation AAV8.CHM transduce photoreceptors compared to version 1 after subretinal injection of each test substance? b. Dose response: Does Next Generation AAV8.CHM differ from AAV8.CHM-version 1 vectors in the dose response of gene expression?

[0216] Experimental Details: Plasmids and vectors were as described in Example 4. Mice (animals): To examine the in vivo expression of CHM, as assessed by the production of REP-1 protein, wild-type CD1 mice were used. The CD1 mouse strain is an outbred Swiss mouse strain, a colony of which we maintain in-house. Details of the study are described under CAROT research protocol PCPR02.01.

[0217] 3.3 Study design (e.g., treatment groups) 3.3.1 Animal Husbandry: Both male and female mice (~3-4 months old) weighing ~20-30 grams were injected with the test substances described. Animals were housed in the University of Pennsylvania's John Morgan University Laboratory Animal Resources (ULAR) facility in accordance with University of Pennsylvania's ULAR regulations. Mice were kept on a 12-hour light / 12-hour dark cycle. Food and water were provided ad libitum. All animals were identified by ear tag number.

[0218] 3.4 Test Material Administration: Test article preparations provided by the CAROT Vector Core were used for dose administration. Test materials were stored at -60 to -80°C. Test materials were thawed on ice prior to dosing. For intraocular injection, the preparations were prepared as described in Preparation Table 20. Test substances are diluted to target concentrations using phosphate-buffered saline. A total of 60 μl of master mix is ​​prepared.

[0219] [Table 22]

[0220] Preparation of Injection Log before Subretinal Injection Prior to subretinal injection of test substances, an injection log was kept with the following information: Gauge number / mouse number Research Identification ·KK ·date of birth ·Injection date Researcher / injector name The eye to be injected (left or right) Injected material (vector / serotype) Dosage and volume Route of Administration (ROA)

[0221] Subretinal injection: Injections were performed by subretinal injection by the surgeon. Briefly, animals were anesthetized prior to injection. Subretinal injection of test articles was performed using a Hamilton 33G syringe. Test articles and injection details are described in Table 21. A volume of 1.5 μl was administered per injection from the prepared injection master mix. 5×10 per eye was administered to one eye per animal. 8 Inject 5 x 10 vector genomes (vg) per eye into the contralateral eye. 9 vector genome was injected.

[0222] [Table 23]

[0223] Outcome measurement method Animal Sacrifice: a. After the animals were injected with the test substance, all animals were observed for 48 hours post-injection for any associated abnormalities. B. 21-35 days after injection, animals were observed for ocular abnormalities using funduscopy. C. 90-12 days after injection, animals were sacrificed and ocular tissues were collected for evaluation of exogenous REP-1 protein production by SDS-PAGE followed by Western blot analysis.

[0224] Ocular tissue collection: Ocular tissues for Western blot analysis were collected after removing the lenses from the eyes using a sharp scalpel blade. Eyes (lens-free) were collected into freezer tubes, which were labeled appropriately.

[0225] Western blot analysis In short: 1. Preparation of tissue cell lysates a. Ocular tissues from animals injected with two doses of next-generation AAV8.CHM and AAV8.V1, along with tissues from uninjected control animals, were collected 21-35 days after injection by sacrificing the animals. B. Tissues were then lysed on ice using RIPA buffer with protease inhibitors. c. Tissue cell lysates were clarified by centrifugation at 13,000 rpm for 10 minutes.

[0226] 2. Protein Quantification and Preparation a. Protein quantification of cell lysates was performed using the ThermoFisher Micro BCA™ Protein Assay Kit according to the manufacturer's instructions. B. Protein concentrations were determined by obtaining OD readings at 562 nm. C. To assess in vivo expression of CHM, 20-40 μg of measured protein was loaded onto a 4-12% Bis-Tris gel.

[0227] 3. SDS-PAGE and Western Blot Protein gels were transferred to nitrocellulose membranes, blocked in milk, and incubated with primary antibodies. Anti-human REP-1 2F1 antibody (2F1, 1:1000 dilution) and / or anti-GAPDH antibody (1:1000 dilution) were used as primary antibodies. After washing the blots, HRP-conjugated anti-mouse IgG antibody and / or anti-rabbit IgG antibody at a concentration of 1:5000 were used as secondary antibodies. The blots were developed by chemiluminescence using ECL reagent according to the manufacturer's instructions.

[0228] 4. Control Standard a) Loading control: Anti-GAMMA antibody was used to demonstrate equal loading of proteins in each well of the gel. A PDH antibody was used as a loading control. Anti-GAPDH antibody detects a protein of ~39 kDa. B) Positive control: AAV2.V2a-transduced COS-7 cell lysate was used as a positive control. C) Negative control: Ocular tissue from uninjected animals was used as a negative control.

[0229] Sample value determination Quantification of Western blot analysis was performed using Image J software. Briefly, the densitometric evaluations presented in this report were first normalized to the endogenous expression level of GAPDH protein in the corresponding sample. The expression level was then normalized again to the mean REP-1 expression level of the uninjected control.

[0230] Details of densitometric assessment and fold change calculations to demonstrate REP-1 protein expression are presented as Tables 22 and 23.

[0231] Short Description: 1. In Tables 22 and 23, column 2 shows the raw values ​​for REP-1 protein and column 3 shows the raw values ​​for GAPDH protein. 2. The GAPDH value for each sample was first normalized to the GAPDH value for AAV8.V1 animal-1, which is shown in column 4 in Table 22. 3. The values ​​for each sample were also normalized to the GAPDH value for AAV8.V1 animal-2, which is shown in column 5 in Table 22. 4. The REP-1 values ​​(column 2) are then normalized to either GAPDH normalized to Animal 1 (column 4) or GAPDH previously normalized to Animal 2 (column 5). These are shown in columns 6 and 7, respectively. 5. The normalized REP-1 values ​​are then converted to relative values ​​(fold change). 6. Each REP-1 value was normalized to the expression of REP-1 in either animal 1 or animal 2 of the AAV8.V1-injected group and is expressed as a relative value (columns 8 and 9). 7. Column 10 shows the mean relative values ​​for REP-1 protein expression.

[0232] result Comparison of CHM expression using native CHM AAV8.V1 versus codon-optimized CHM vectors: AAV8.V2a, V2b, V3a, and V3b. Two doses of each AAV8 vector in wild-type CD1 mice: 5 x 10 per eye 9 High dose of vector genomes and 5 x 10 per eye 8 A low dose of the vector genome was injected. The results below describe the levels of REP1 protein after injection with high and low doses of AAV8.V1, AAV8.V2a, and AAV8.V3a.

[0233] High dose (5 × 10 per eye) 9Comparison of AAV8.V1 expression versus AAV8.V2a and AAV8.V3a (stuffer-containing vectors) in animals injected with viral vectors (each containing 1 vector genome). Western blot analysis using a human anti-REP-1 antibody detected a ∼75-80 kDa hREP-1 protein band in both (low- and high-dose injected) eye tissues from each animal treated with either next-generation AAV8.V2a or V3a or the original AAV8.V3a version 1. A very weak (minimal) band was observed in both uninjected control mice. Increased intensity bands were observed in tissues transduced with next-generation vectors (AAV8V.2a and AAV8.V3a) compared with tissues transduced with version 1. Anti-GAPDH antibody revealed a ∼39 kDa band of equal intensity in all lanes of the Western blot, including the uninjected control. For comparison of the molecular weight of the protein of interest, pre-stained ocular tissues were used. Densitometric evaluation of the blots (quantification of expression levels) using ImageJ software showed increased production of REP-1 in animals injected with one of the next generation AAV8 high and low dose constructs (V2a or V3a). (See Table 22 for values.)

[0234] [Table 24]

[0235] [Table 25]

[0236] Low dose (5 × 10 per eye) 8 Comparison of AAV8.V1 versus AAV8.V2a and AAV8.V3a expression in animals injected with viral vectors (each vector genome) 5×10 8Western blot analysis using a human anti-REP-1 antibody of ocular tissues from animals injected with next-generation AAV8.V2a, V3a, and AAV8.Version 1 at doses of 1000-10000 detected a ~75-80 kDa hREP-1 protein band in the tissues of injected mice. A weak (minimal) REP-1 band was observed in ocular tissue cell lysates from both uninjected control mice. A band of increased intensity was observed in tissue cell lysates transduced with the next-generation vector compared to cell lysates transduced with Version 1. Anti-GAPDH antibody detected a protein band of equal intensity at ~39 kDa in all tissue lysates. This data supports the AAV8-mediated delivery of next-generation V2. a) Delivery of CHM results in robust levels of REP-1 protein compared to levels produced after injection of AAV8.V3a or AAV8.V1.

[0237] Densitometry evaluation of the blots (quantification of expression levels) using ImageJ software further demonstrated increased production of REP-1 in animals injected with next generation AAV8.CHM constructs (particularly V2a) compared to version 1. See Table 23 for values.

[0238] Expression of AAV8.V2b in CD1 mice Concurrent with this ongoing study and evaluation of the effect of lambda stuffer on AAV vector production by qPCR titer analysis, we performed all animal injections and collected all samples for the in vivo expression studies described in study protocol PCPR.02. After completing the qPCR studies for the lambda stuffer element (described above), we decided to perform Western blot experiments only to examine the expression of stuffer-free AAV vectors, such as AAV8.2b and AAV8.3b, and to exclude them from further analysis (such as comparison with version 1).

[0239] Human anti-REP-1 antibody was 5 × 10 9A protein of ∼75–80 kDa was detected in the ocular tissues of CD-1 mice injected with AAV8.2b at 5 × 10 (high dose) vector genome copies (Figure 12A). 8 Animals injected with AAV8.2b at a low dose showed a very weak protein band at ∼75–80 kDa (Figure 12A). Lysates of ocular tissue from uninjected control animals showed no presence of REP-1 protein. Anti-GAPDH antibodies detected a ∼39 kDa protein in all ocular tissue lysates, including the uninjected control. This data allows us to determine a minimum dose for AAV8.2b.

[0240] Expression of AAV8.V3b in CD1 mice We performed Western blot analysis using anti-REP-1 antibody on ocular tissues from AAV8.3b-injected CD1 mice (two mice per group), which revealed the presence of a protein of ∼75–80 kDa in one eye injected with a low dose and in both eyes injected with a high dose of AAV8.3b. No REP-1 expression was detected in ocular tissues from uninjected mice (Fig. 12B). The level of REP-1 produced was dose-dependent in animals injected with AAV8.3b. High doses of AAV8.3b (5 × 10 9 Injection with a low dose (5 x 10 vector genomes) was performed in the injected eye. 8 The vector induced higher amounts of REP-1 compared to the uninjected vector (Figure 1). Anti-GAPDH antibodies detected a protein of ∼39 kDa in ocular tissue lysates from all injected and uninjected animals.

[0241] These results revealed the following observations: 1) Next-generation vectors AAV8.versions 2a, 2b, 3a, and 3b can effectively transduce ocular tissue. 2) Transgene (codon-optimized CHM) expression was detectable for all of the next-generation vectors. 3) Transgene (codon-optimized CHM) expression is dose-dependent. 4) AAV8.version 2a and AAV8.version 2b induced increased REP-1 protein production compared to AAV8.version 1 in ocular tissues of CD-1 mice. 5) There was variability in the exact level of transgene protein production between eyes injected with the same dose, reflecting variability in surgical delivery methods. However, the difference in levels was low (5 x 10 8 pcs) and high (5 × 10 9 6) AAV8.CHM.V2a and AAV8.V3a delivered high doses (5 × 10) subretinally in mice. 9 After in vivo administration of vectors of this type (each vector genome), they produce much higher levels of REP-1 protein than AAV8.V1.

[0242] Example 8 - Expression of CNGA3 To maximize expression of CNGA3, a codon-optimized CNGA3 sequence was generated (SEQ ID NO: 9). Additionally, a CNGA3 mutant was codon-optimized (SEQ ID NO: 11). These sequences, as well as the native CNGA3 coding sequence, were incorporated into production plasmids (SEQ ID NOs: 30-38) described herein to generate AAV vectors. Vectors using AAV8 and AAV9 capsids were generated as described below.

[0243] [Table 26]

[0244] Protein expression was assessed as described above for REP-1. AAV8-CMV-CBA expression is observed in 84-31 cells transduced with each of the three CNGA3 vectors at two different MOIs. Figure 28. The positive control used was mouse retinal protein recovered after injection of AAV8-CMV-CBA-native CNGA3. CNGA Codon optimization of the three plasmids demonstrated enhanced expression from three different promoters: CMV / CBA (Figure 28), RK-1, and hCAR (Figure 29). Enhancement from the CMV / CBA promoter was more pronounced at lower doses (presumably more saturated at higher doses).

[0245] Exogenous hCNGA3 expression using AAV8&9 was tested in vivo.

[0246] Subretinal injections were performed on WT mice between 30 and 120 days of age. Mice were sacrificed 3 to 4 weeks after injection, and tissues were collected to measure endpoints. In normal mice, CNGA3 expression was measured by qPCR, as shown in Figure 30. In null mice, CNGA3 protein expression in the retina was measured using Western and IHC, and retinal histopathology was examined.

[0247] CNGA3 mouse model: CNGA3 null mice with a CPFL5 (exon 5) containing missense mutation

[0248] Phenotype: 5 weeks: Severely reduced cone-specific ERG 10 weeks: Cone opsin pigment loss and mislocalization 5 months: Decreased optokinetic reflexes

[0249] CNGA3-Gene replacement therapy for color blindness: CNGA3 null mice were injected subretinally at p16-18 with one of two doses of the described vector: low dose: 8E8vg / eye; high dose: 8E9vg / eye. At least five animals per group were injected. ERG and OKR were performed at weeks 5-7 and 12-15. At sacrifice, IHC, Western blot, and histology were performed.

[0250] To provide objective information on retinal function and serve as an efficacy parameter in preclinical studies, electroretinograms (ERGs), the electrical response of retinal cells to flashes of light, were assessed in mice according to conventional methods and user manuals. See, e.g., Marmor, Michael F. et al., "Standard for clinical electroretinography (2004 update)." Documenta Ophthalmologica 108.2(2004):107-114; and Cronin, Therese, Arkady Lyubarsky, and Jean Bennett. “Dark-rearing the rd10 mouse: implications for therapy.” Retinal Degenerative Diseases. Springer US, 2012. 129-136.

[0251] Briefly, ophthalmoscopic evaluation of the animals was completed prior to ERG measurements. Mice with ocular defects that could potentially compromise ERG results were excluded. These included corneal opacities such as cataracts, corneal injury, or inflammation. Mice were then dark-adapted for at least 4 hours, weighed in the dark, and intraperitoneally injected with an anesthetic (ketamine / xylazine cocktail, 100 mg / kg and 10 mg / kg, respectively, in phosphate-buffered saline (PBS; pH 7.2)). The pupils of pigmented mice were dilated using 1% tropicamide solution; albino mice also required pupil dilation. Animals were housed on absorbent bedding on a heated platform. A reference electrode was placed in contact with the mouse's body, and a recording electrode was placed above the cornea of ​​each eye, gently touching the cornea. A magnifying glass was used, if necessary.

[0252] The stimulator was set up as shown below. Unless otherwise indicated, any color or achromatic stimulus was Test Protocol I: Step 1: 0.01076 scotopic cd s m -2 (candela-seconds per square meter (cd / m 2 ));Step 2: 500 scotopic cd sm -2 , achromatic xenon flash; and Step 3: background intensity 100 scotopic cd m -2 , Stimulus: 500 scotopic cd sm -2 In Set II, the following stimuli were observed at all steps: 100 scotopic cd m -2 (candela per square meter (cd / m 2 )) Emitted with green (520 nm) background illumination. Test Protocol II, Step 1: 500 scotopic cd sm -2 , achromatic xenon flash; Step 2: 0.0015 cd / m -2 , UV (365 nm), isi (stimulus time interval, time interval between successive flashes) 1.5 s; step 3: 0.004 cd sm -2 , UV (365 nm), isi 1.5 s; step 4: 0.01 cd sm -2, UV (365 nm), isi 2 s; step 5: 0.03 cd sm -2 , UV (365 nm), isi 2 s; step 6: 4scot cd sm -2 , Green (520nm), ISI 2s; Step 7: 10scot cd sm -2 , Green (520nm), ISI 2s; Step 8: 25scot cd sm -2 , Green (520nm), ISI 2s; and step 9: 500 scotopic cd sm -2 , including an achromatic xenon flash. Test protocols I and II were performed sequentially on each animal.

[0253] Because the CNG3A / B3 null mice used had normally functioning rods, rod-generated ERGs were used as a signature of retinal status. The parameter was the amplitude of the saturated a-wave. When stimulated with a bright light flash, the fast corneal negative voltage was the first (in terms of temporal order) component of the ERG. The a-wave amplitude was the difference between the most negative point of the ERG during the time interval up to 20 ms after the flash and the baseline value of the ERG signal measured 3 ms after the flash. The selection of the 3 ms data point as "zero" allowed for the elimination of light flash artifacts and the minimization of the effects of drift. With a gradual increase in stimulus intensity, the a-wave amplitude increased until saturation was reached. The a-wave of the mouse ERG is directly proportional to the magnitude of the retinal rod photocurrent (Lyubarsky, Arkady L., and Edward N. Pugh Jr., "Recovery phase of the murine rod photoresponse reconstructed from electroretinographic recordings." Journal of Neuroscience 16.2 (1996): 563-571) and is therefore the most direct measure of rod function.

[0254] The amplitude of the a-wave, defined as the difference in potential between the baseline and the first trough occurring 7–10 ms after the flash, was measured for both eyes using data obtained by Test Protocol I. The amplitude of the cone ERG was measured using data obtained by Test Protocol II. The magnitude of the cone ERG was divided by the amplitude of the saturated a-wave from each eye. If the amplitude of the a-wave from the injected eye was less than 50% of that from the control eye, the animal was excluded from further treatment and considered to have been severely injured by the injection. The results were calculated and normalized. A paired t-test was performed between the sets of normalized cone ERGs from the injected and uninjected eyes. A statistically significant improvement in the treated eye was considered a signature of successful treatment.

[0255] Retinal function was assessed 5-7 weeks after birth following administration of the indicated vectors, compared with that of wild-type, vehicle-injected, and uninjected eyes. For each vector / dose, three light intensities (0.01 cd.s / m 2 , 10 cd.s / m 2 and 25 cd.s / m 2 ) are shown in Table 25 below and Figure 31. These results demonstrate that the RK-1 promoter, together with the optimized CNGA3 expression cassette, can correct ERG function in a mouse model. [Table 27]

[0256] Calcium uptake in transduced versus non-transduced 84-31 cells after addition of cGMP is shown in FIG.

[0257] Example 9 - CNGB3 The CNGB3 sequence was incorporated into the vector as described above for REP-1 and CNGA3. Vectors using AAV8 and AAV9 capsids were generated as described below.

[0258] CNGB3 null mice were injected subretinally at p16-18 with one of two doses of the described vector: low dose: 1E9 vg / eye; high dose: 1E10 vg / eye. At least five animals per group were injected. ERG and OKR were performed at weeks 5-7 and 12-15. At sacrifice, IHC, Western blot, and histology were performed.

[0259] Retinal function was assessed at 12-16 weeks of age following administration of the indicated vectors, compared with that of wild-type, vehicle-injected, and uninjected eyes. Three light intensities (0.01 cd.s / m) were used for each vector / dose. 2 , 10 cd.s / m 2 and 25 cd.s / m 2 The results measured from the IR spectrum are shown below and in Figure 33. Series 5, 8 and 9 are 0.01 cd.s / m 2 , 10 cd.s / m 2 and 25 cd.s / m 2 is the light intensity.

[0260] These results reveal that both the hCAR and RK-1 promoters, together with an optimized CNGB3 expression cassette, are able to correct ERG function in the CNGB3 mouse model.

[0261] ERG studies suitable for use with the constructs described herein are described, for example, in WO 17 / 27529, filed April 14, 2017, which is incorporated herein by reference.

[0262] Example 10. Mouse electroretinogram (ERG) To provide objective information on retinal function and serve as an efficacy parameter in preclinical studies, electroretinograms (ERGs), the electrical response of retinal cells to flashes of light, were assessed in mice according to conventional methods and user manuals. See, e.g., Marmor, Michael F. et al., "Standard for clinical electroretinography (2004 update)." Documenta Ophthalmologica 108.2(2004):107-114; and Cronin, Therese, Arkady Lyubarsky, and Jean Bennett. “Dark-rearing the rd10 mouse: implications for therapy.” Retinal Degenerative Diseases. Springer US, 2012. 129-136.

[0263] Briefly, ophthalmoscopic evaluation of the animals was completed prior to ERG measurements. Mice with ocular defects that could potentially compromise ERG results were excluded. These included corneal opacities such as cataracts, corneal injury, or inflammation. Mice were then dark-adapted for at least 4 hours, weighed in the dark, and intraperitoneally injected with an anesthetic (ketamine / xylazine cocktail, 100 mg / kg and 10 mg / kg, respectively, in phosphate-buffered saline (PBS; pH 7.2)). The pupils of pigmented mice were dilated using 1% tropicamide solution; albino mice also required pupil dilation. Animals were housed on absorbent bedding on a heated platform. A reference electrode was placed in contact with the mouse's body, and a recording electrode was placed above the cornea of ​​each eye, gently touching the cornea. A magnifying glass was used, if necessary.

[0264] The stimulus apparatus was set up as shown below. Any chromatic or achromatic stimulus could be used unless otherwise indicated. Test Protocol I consisted of Step 1: 0.01076 scotopic cd s m -2 (candela-seconds per square meter (cd / m 2));Step 2: 500 scotopic cd sm -2 , achromatic xenon flash; and Step 3: background intensity 100 scotopic cd m -2 , Stimulus: 500 scotopic cd sm -2 In Set II, the following stimuli were observed at all steps: 100 scotopic cd m -2 (candela per square meter (cd / m 2 )) Emitted with green (520 nm) background illumination. Test Protocol II, Step 1: 500 scotopic cd sm -2 , achromatic xenon flash; Step 2: 0.0015 cd / m -2 , UV (365 nm), isi (stimulus time interval, time interval between successive flashes) 1.5 s; step 3: 0.004 cd sm -2 , UV (365 nm), isi 1.5 s; step 4: 0.01 cd sm -2 , UV (365 nm), isi 2 s; step 5: 0.03 cd sm -2 , UV (365 nm), isi 2 s; step 6: 4scot cd sm -2 , Green (520nm), ISI 2s; Step 7: 10scot cd sm -2 , Green (520nm), ISI 2s; Step 8: 25scot cd sm -2 , Green (520nm), ISI 2s; and step 9: 500 scotopic cd sm -2 , including an achromatic xenon flash. Test protocols I and II were performed sequentially on each animal.

[0265] Because the CNG3 mice used have normally functioning rods, the rod-generated ERG was utilized as a signature of retinal status. The parameter was the amplitude of the saturated a-wave. When stimulated with a bright light flash, the fast corneal negative voltage was the first (in terms of time order) component of the ERG. The a-wave amplitude was the difference between the most negative point of the ERG during the time interval up to 20 ms after the flash and the baseline value of the ERG signal measured 3 ms after the flash. The selection of the 3 ms data point as "zero" allowed for the elimination of flash artifacts and the minimization of the effect of drift. With a gradual increase in stimulus intensity, the a-wave amplitude increased until it reached saturation. The a-wave of the mouse ERG is directly proportional to the magnitude of the retinal rod photocurrent (Lyubarsky, Arkady L., and Edward N. Pugh Jr. "Recovery phase of the murine rod photoresponse reconstructed"). extracted from electroretinographic recordings.” Journal of Neuroscience 16.2 (1996): 563-571), and is therefore the most direct measure of rod function.

[0266] The amplitude of the a-wave, defined as the difference in potential between the baseline and the first trough occurring 7–10 ms after the flash, was measured for both eyes using data obtained by Test Protocol I. The amplitude of the cone ERG was measured using data obtained by Test Protocol II. The magnitude of the cone ERG was divided by the amplitude of the saturated a-wave from each eye. If the amplitude of the a-wave from the injected eye was less than 50% of that from the control eye, the animal was excluded from further treatment and considered to have been severely injured by the injection. The results were calculated and normalized. A paired t-test was performed between the sets of normalized cone ERGs from the injected and uninjected eyes. A statistically significant improvement in the treated eye was considered a signature of successful treatment.

[0267] Example 11 - Evaluation of Lambda Stuffer Effect In addition to the experiments described in Example 6, the percentage of residual plasmid DNA was assessed and the resulting data are plotted in Figure 34. The results showed that the oversized stuffer sequence reduced DNA impurities by ∼80% during triple transfection preparations for both AAV2 and AAV8.

[0268] Example 12 - In vivo expression of rAAV In addition to the experiment described in Example 7, various doses of AAV8.V2a, AAV8.V3a, and AAV8.V1 (5E7 vector genome copies per eye (vg / eye), 1E8 vg / eye, 5E8 vg / eye, 5E9 vg / eye, and 1E10 vg / eye) were injected into CD-1 mice approximately 3 to 4 months old. Eyeballs were harvested, and their REP1 protein expression was assessed by Western blot. Representative results are shown in Figures 35 to 39. Quantification was performed and shown in the table below. The results revealed that AAV8.V2a and AAV8.V3a showed higher expression levels of REP1 protein at 5E8 vg / eye and 5E9 vg / eye compared to AAV8.V1. Subretinal delivery of AAV8.V2a and AAV8.V3a resulted in robust and reproducible delivery of the CHM transgene to retinal cells. Recombinant AAV8.CHM-mediated delivery of the CHM gene resulted in a dose-dependent effect on REP1 protein production.

[0269] [Table 28]

[0270] [Table 29]

[0271] Example 13 - Retinal histopathology diagnosis by injection of rAAV Retinal histopathology analysis was performed on retinas harvested from mice treated as described in Examples 7 and 12. H&E staining was performed to reveal changes on photoreceptors and the presence of immune infiltrates. Tunel staining was performed to reveal the presence of apoptosis. Ve-treated cells served as a positive control. Representational images are not shown. A summary of observations is provided below as a table. Results showed that recombinant AAV8.CHM-mediated delivery of the CHM gene resulted in a dose-dependent effect on REP1 protein production and retinal histopathology. Mouse eyes injected with the highest doses of AAV8.V2a, AAV8.V3, and AAV8.V1 exhibited inflammation, retinal degeneration, and apoptosis.

[0272] [Table 30]

[0273] Example 14 - iPSCs

[0274] To determine the multiplicity of infection (MOI) required for AAV8 to achieve comparable transduction / expression of the GFP reporter as an AAV2 vector in human iPSCs, and to examine the cytotoxicity of AAV8.V2a and AAV8.V3a at high MOI, the following experiments were performed. Experiments were performed. iPSC cells were transduced with AAV2.CMV / CβA-GFP and AAV8.CMV / CβA-GFP at multiple MOIs (1E4 to 1E7). Culture wells were imaged and GFP quantified to determine the comparability of AAV2 vectors with AAV8 MOIs. The results are plotted in Figure 40 and show that the transduction efficiency achieved with AAV8.CMV / CβA-GFP at 1E7 vg / cell is comparable to that of AAV2.CMV / CβA-GFP at approximately 2E5 vg / cell.

[0275] Additionally, cells were transduced with 1E7 vg of AAV8.V2a and AAV8.V3a, and 2E5 vg of AAV2.V1. Cells were then stained and counted for caspase 3 (an apoptosis marker). Immunofluorescence staining of iPSCs treated with 1 μM staurosporine, untreated, transduced with 2E5 vector genome copies per cell of AAV2.V2a, transduced with 1E7 vector genome copies per cell of AAV8.V3a, and transduced with 1E7 vector genome copies per cell of AAV8.V1 was performed (images not shown). Staurosporine was used to induce apoptosis. Cells treated with it served as a positive control. Substance 1 is AAV2.V2a; substance 2 is AAV8.V3a; and substance 3 is AAV8.V1.

[0276] The resulting data are presented in Figure 41 and show that AAV8 serotype vectors at an MOI of 1E7vg / cell resulted in robust transgene expression in transduced iPSCs without causing apoptosis.

[0277] Example 15 - Prenylation of RAB CHM encodes Rab escort protein 1 (REP1), which is required for the prenylation of target RAB proteins. Therefore, as described in Example 3, the prenylation of target RAB proteins in CHM patient-derived iPSCs after transduction with the test rAAV ( 3 The effects of iPSCs on the development of CHM (using incorporation of the HGGPP substrate) were evaluated. iPSCs were generated and treated as described in Example 1. Briefly, the CHM patient-derived iPSC cell line JB 588, JB 527, and JB 415 were generated and maintained. Transduction was performed with AAV8.V2a, AAV8.V3a, and AAV8.V1 at an MOI of 1E7. Untreated cells served as negative controls.

[0278] The results are shown in Figure 42. Compared to non-transduced iPSCs, the transduced cells 3 H showed an increase in both absolute and normalized incorporation of the GGPP substrate.

[0279] Example 16 - Therapeutic effect of hCNGA3 gene therapy on Cnga3 null mice A proof-of-concept study was conducted to test the feasibility of gene augmentation as a therapy for hCNGA3-mediated ACHM disease using a Cnga3 knockout mouse model of ACHM. To do so, we generated 14 unique transgene cassettes, each carrying one of three different promoters (RK1, CMV / CBA, and hCAR), and packaged them into AAV vector serotypes 8 or 9. The cDNA sequences used among these 14 test vectors were native, codon-optimized, or a codon-optimized version of naturally occurring variant 3. Subretinal injections of test substances at one of two different doses were performed between P16 and P19, and general clinical observations and fundusscopic examinations were performed in all study animals. Retinal and visual function of study animals was assessed by electroretinogram (ERG) and optokinetic reflex (OKR) testing. Cohorts of injected animals were euthanized at the study endpoint, and retinal histopathology was evaluated. Additional histological analysis of retinal photoreceptor slices was performed using TUNEL staining and fluorescent immunohistochemistry for CNGA3.

[0280] Fundus examinations performed between 5 and 7 weeks revealed dose-dependent histopathological changes. OKR results were inconclusive. ERG analysis revealed a dose-dependent effect as well as a morphological change. Improved cone photoreceptor function was demonstrated compared to drug-treated eyes. Optical responses were observed using a transgene cassette incorporating a photoreceptor-specific promoter.

[0281] Results demonstrated that hCNGA3 gene delivery using AAV8 and AAV9 viral vectors demonstrated excellent preliminary safety when injected at 8E8vg / eye. Minimal ocular inflammation was observed, and no adverse reactions occurred following subretinal delivery of the test substance. Furthermore, histopathological analysis of tissues from test substance-injected eyes revealed minimal retinal toxicity with 8E8vg / eye, with transgene protein localization restricted to photoreceptors. Furthermore, AAV-mediated delivery of hCNGA3 was sufficient to improve cone responses, while AAV8.RK1-hCNGA3.Opt and AAV8.hCAR-hCNGA3.V3 had minimal impact on rod responses.

[0282] overview The in vivo expression and function of hCNGA3 were investigated in B6.RHJ-Cnga 3cpfl5 / BocJ (Future Cnga3 - / - The test substances were tested in mice (referred to as rhodopsin kinase (RK1), human cone arrestin (hCAR), and CMV / CβA) combinations. The rhodopsin kinase (RK1) promoter drives photoreceptor-specific expression, the human cone arrestin (hCAR) promoter, with or without the woodchuck posttranscriptional regulatory element (WPRE) enhancer, drives cone expression, and the cytomegalovirus enhancer, with the chicken beta-actin promoter (CMV / CβA), drives ubiquitous expression. The three alleles used were the native allele (Nat), the codon-optimized allele (Opt), and the naturally occurring variant 3 (V3), which was also codon-optimized. These promoter / enhancer and allele combinations were encapsulated in AAV8 or AAV9 serotypes. Test substance dilution: All test substances were diluted in a vehicle consisting of Dulbecco's phosphate-buffered saline. Vehicle or a low dose was injected into the left eye, and a high dose was injected into the right eye.

[0283] Subretinal injections were performed to deliver one of the following test substances: Group 1—AAV8 capsid packaging native human CNGA3 transgene vector controlled by an upstream RK1 promoter sequence; Group 2—AAV8 capsid packaging codon-optimized human CNGA3 transgene vector controlled by an upstream RK1 promoter sequence; Group 3—AAV8 capsid packaging codon-optimized human mutant 3CNGA3 transgene vector controlled by an upstream RK1 promoter sequence; Group 4—AAV8 capsid packaging native human CNGA3 transgene vector controlled by an upstream human CAR promoter sequence; Group 5—AAV8 capsid packaging codon-optimized human CNGA3 transgene vector controlled by an upstream human CAR promoter sequence; Group 6—AAV8 capsid packaging codon-optimized human mutant 3CNGA3 transgene controlled by an upstream human CAR promoter sequence. Group 8 - AAV8 capsid packaging codon-optimized human CNGA3 transgene vector controlled by an upstream cytomegalovirus enhancer / chicken beta actin promoter (CMV / CβA) sequence; Group 10 - AAV8 capsid packaging native human CNGA3 transgene vector controlled by an upstream human CAR promoter sequence with a woodchuck post-transcriptional regulatory element (WPRE); Group 11 - AAV9 capsid packaging codon-optimized human CNGA3 transgene vector controlled by an upstream RK1 promoter sequence; Group 12 - AAV9 capsid packaging codon-optimized human CNGA3 transgene vector controlled by an upstream human CAR promoter sequence; or Group 14 - AAV9 capsid packaging native human CNGA3 transgene vector controlled by an upstream human CAR promoter sequence with a woodchuck post-transcriptional regulatory element (WPRE).

[0284] General clinical observations and fundusscopic examinations were performed on all study animals. Visual function in study animals was assessed by electroretinogram (ERG) and optokinetic reflex (OKR) testing. Cohorts of injected animals were euthanized at the study endpoint, and eyes were processed and stained with hematoxylin and eosin (H&E) for histopathological diagnosis. Additional histological analysis of retinal photoreceptor slices was performed using Tunel staining and fluorescent immunohistochemistry for CNGA3.

[0285] Results - Summary Subretinal administration of all test materials carrying the hCNGA3 transgene resulted in mild inflammatory changes in the eye after surgery. No animals had to be euthanized as a result of inflammation or adverse reactions to the delivered test material. Of the 182 pups injected with vectors, 169 remained in the study at weaning time and 162 remained in the study until completion. Fundus examinations performed between 5 and 7 weeks revealed that the majority of eyes showed low to moderate evidence of inflammation associated with degenerative histopathological changes (Appendix 3). Specifically, injection of all vectors at the high dose (8E9vg / eye) resulted in signs of photoreceptor loss to a greater extent than eyes injected with vectors at the low dose (8E8vg / eye). The variability in the extent of retinal changes among eyes injected at the low doses within the same cohort likely reflects the variability of the surgical delivery procedure. Overall, the best results were observed in eyes that received low doses of AAV8.RK1-hCNGA3 native, AAV8.RK1-hCNGA3 codon-optimized, AAV8.RK1-hCNGA3 codon-optimized variant 3, AAV8.hCAR-hCNGA3 native, and AAV8.hCAR-hCNGA3 codon-optimized variant 3.

[0286] Examination of retinal and visual function by ERG revealed superior functionality in eyes injected with low doses of AAV8.RK1-hCNGA3 native, AAV8.RK1-hCNGA3 codon-optimized, AAV9.RK1-hCNGA3 codon-optimized, and AAV8.hCAR-hCNGA3 mutant 3 compared to controls. See Figures 43A-44B.

[0287] Electroretinogram results ERG recordings were collected following 12 hours of dark adaptation for the animals. Each recording consisted of 10 cycles of light stimulation eliciting rod-, S-type, or L / M-type cone-driven responses. Cone responses were used to measure treatment efficacy, and rod responses were used to measure toxicity.

[0288] Group 1 (AAV8.RK1-hCNGA3 native): Eyes injected with 8E8vg / eye and 8E9vg / eye showed robust and significant improvements in cone responses compared with vehicle-injected controls at 5-7 and 12-15 weeks after injection. Eyes injected with 8E8vg / eye vector showed no change in rod function compared with vehicle-injected eyes at 5-7 or 12-15 weeks after injection. However, injection of 8E9vg / eye caused a large and significant decrease in rod function at 5-7 weeks after injection. Rod function improved slightly by 12-15 weeks after injection.

[0289] Group 2 (AAV8.RK1-hCNGA3 codon optimized): Eyes injected with 8E8vg / eye and 8E9vg / eye showed robust and significant improvements in cone responses at 5-7 and 12-15 weeks post-injection compared with vehicle-injected controls. Eyes injected with 8E8vg / eye had a mild but significant decrease in rod responses at 5-7 and 12-15 weeks post-injection. The decrease in rod responses was much greater in eyes injected with 8E9vg / eye.

[0290] Group 3 (AAV8.RK1-hCNGA3 mutant 3): Eyes injected with 8E8vg / eye showed significantly higher mean morbidity at 5-7 and 12-12 post-injection compared with vehicle-injected controls. Although they showed no change in cone responses at week 5, they did show a large and significant improvement in rod responses at weeks 5-7. Eyes injected with 8E9vg / eye showed robust and significant improvements in cone responses at weeks 5-7 and 12-15 after injection compared to vehicle-injected controls. However, eyes injected with 8E9vg showed a large and significant decrease in rod responses at weeks 5-7 and 12-15 after injection.

[0291] Group 4 (AAV8.hCAR-hCNGA3 native): Eyes injected with 8E8vg or 8E9vg showed robust and significant improvements in cone responses at 5-7 and 12-15 weeks post-injection compared with vehicle-injected controls. While no changes in rod responses were observed in 8E8vg-injected eyes at 5-7 weeks post-injection, rod responses in these eyes were indeed significantly reduced by 12-15 weeks post-injection compared with vehicle-injected controls. 8E9vg-injected eyes showed reduced rod responses at both time points.

[0292] Group 5 (AAV8.hCAR-hCNGA3 codon optimized): Eyes injected with 8E8 or 8E9vg showed significant, moderate to robust improvements in cone responses at 5-7 and 12-15 weeks post-injection compared to vehicle-injected controls. In eyes receiving 8E8vg, no changes in rod responses were observed at 5-7 weeks post-injection, but there was a significant decrease in function by 12-15 weeks post-injection. Eyes injected with 8E9vg also showed a decrease in rod responses at both time points.

[0293] Group 6 (AAV8.hCAR-hCNGA3 variant 3): Eyes injected with 8E8vg showed robust and significant improvements in cone responses at 5-7 and 12-15 weeks post-injection compared to vehicle-injected controls. No changes in rod responses were observed in these eyes at either time point. Eyes injected with 8E9vg showed robust and significant increases in cone function at 5-7 weeks post-injection, but not at 12-15 weeks post-injection, compared to vehicle-injected controls. These eyes showed significant decreases in rod function at both time points.

[0294] Group 8 (AAV8.CMV / CBA-hCNGA3 codon optimized): Eyes injected with either dose showed no change in cone responses compared to vehicle-injected eyes at 5-7 or 12-15 weeks after injection. Furthermore, both doses produced significant decreases in rod responses at both time points.

[0295] Group 10 (AAV8.hCAR-hCNGA3 native with WPRE): Eyes injected with either dose showed significant improvement in cone responses compared with vehicle-injected controls at both 5-7 and 12-15 weeks post-injection. Cone responses were unchanged in 8E8vg-injected eyes at 5-7 weeks post-injection, whereas rod responses were significantly reduced in 8E9vg-injected eyes by 5-7 weeks post-injection. Furthermore, both doses resulted in a reduction in rod responses at 12-15 weeks post-injection.

[0296] Group 11 (AAV9.RK1-hCNGA3 codon optimized): Eyes injected with 8E8vg showed robust and significant improvements in cone responses, with no change in rod responses, at both 5-7 and 12-15 weeks after injection. Eyes injected with 8E9vg also showed significant improvements in cone responses at both time points, but these eyes also showed decreases in rod responses.

[0297] Group 12 (AAV9.hCAR-hCNGA3 codon optimized): Eyes injected with 8E8vg did not show significant improvement in cone responses at either time point, while eyes injected with 8E9vg did show significant improvement in cone responses at both time points, but rod responses were significantly reduced by 5–7 weeks post-injection.

[0298] Group 14 (AAV9.hCAR-hCNGA3 native with WPRE): Eyes injected with 8E8 or 8E9vg showed a moderate to robust increase in cone responses at both 5-7 and 12-15 weeks after injection, but injection of either dose resulted in a significant decrease in rod responses by 12-15 weeks after injection.

[0299] Low doses (8E8vg / eye) of AAV in groups 1 (AAV8.RK1-hCNGA3 native), 2 (AAV8.RK1-hCNGA3 codon-optimized), 11 (AAV9.RK1-hCNGA3 codon-optimized), 6 (AAV8.hCAR-hCNGA3 codon-optimized variant 3), and 14 (AAV9.hCAR-hCNGA3 native with WPRE) resulted in the preservation of rod function and improvement of cone function at both time points after injection.

[0300] There is no significant difference in S- or L / M-type cone function between these test groups.

[0301] AAV group 11 (AAV9.RK1-hCNGA3 codon-optimized) has significantly less impact on rod function than the AAVs used in groups 1 (AAV8.RK1-hCNGA3 native), 2 (AAV8.RK1-hCNGA3 codon-optimized), and 14 (AAV9.hCAR-hCNGA3 native with WPRE).

[0302] High doses of AAV (8E9vg / eye) in groups 11 (AAV9.RK1-hCNGA3 codon optimized) and 12 (AAV9.hCAR-hCNGA3 codon optimized) resulted in the preservation of rod function and improvement of cone function at both time points.

[0303] Rod function is significantly better with 8E8vg / eye than with 8E9vg / eye in Group 11 (AAV9.RK1-hCNGA3 codon-optimized) at 12–15 weeks post-injection.

[0304] At 12–15 weeks post-injection, there was no difference in rod function between groups 11 (AAV9.RK1-hCNGA3 codon-optimized) and 12 (AAV9.hCAR-hCNGA3 codon-optimized) at 8E9vg / eye.

[0305] Histological diagnosis, immunohistochemistry, and Tunel assay conclusions: Histological evaluation revealed photoreceptor loss, inflammation, and the presence of abnormal retinal structures in mice injected with all test substances at the higher doses.

[0306] The retinal structure of mice injected with AAV.hCNGA3, in which the expression of hCNGA3 was driven by the promoter CMV.CβA, was found to be significantly damaged.

[0307] Mice injected with the test substance at the lower dose of 8E8vg / eye did not reveal any significant histological changes or the presence of inflammation.

[0308] Mice injected with the test substance at the higher dose of 8E9 vg / eye showed significant histological changes or inflammation.Vector-to-vector variability in effecting retinal structure was noted.

[0309] Transduction of hCNGA3-null mouse retinal tissue with codon-optimized and codon-optimized mutant 3 hCNGA3 driven by the RK1 and hCAR promoters, respectively, resulted in localization of hCNGA3 to photoreceptor cells, which was similar among all test substances at the injected doses.

[0310] The variability of hCNGA3 expression was relatively low between mice injected with AAV8.RK1-hCNGA3 codon-optimized and AAV8.hCAR-hCNGA3 codon-optimized variant 3. In mice injected with AAV9.RK1-hCNGA3 codon-optimized, hCNGA3 expression was found to be variable between animals in the same cohort.

[0311] The presence of apoptotic cells was not evident in the retinas of mice injected with any of the test substances.

[0312] Overall conclusions: Subretinal injection of AAV.RK1-hCNGA3 and AAV.hCAR-hCNGA3 resulted in robust delivery of the hCNGA3 transgene to retinal photoreceptor cells. Subretinal delivery of all test substances at the higher dose (8E9 vg / eye) resulted in inflammatory and degenerative changes in the retina. The severity of retinal histopathological changes was dependent on the promoter used. Vectors in which hCNGA3 expression was driven by CMV-CβA resulted in more severe retinal degenerative changes, followed by RK1 and then hCAR. Inflammatory changes were noted after injection of the lower dose (8E8 vg / eye) of vectors, but were less severe. The results of this report establish that delivery of hCNGA3 codon-optimized cDNA driven by a photoreceptor-specific promoter via AAV8 or AAV9 capsids, or delivery of hCNGA3 codon-optimized mutant 3 cDNA driven by a photoreceptor-specific promoter via AAV8 capsids, at a viral dose of 8E8vg / eye is sufficient for production of hCNGA3 protein in retinal photoreceptors with minimal toxicity.

[0313] Postmortem histopathology revealed that subretinal delivery of all test substances at high doses resulted in inflammatory and degenerative changes in the retina (Appendix 6). Among eyes injected at low doses, eyes receiving vectors with the CMV / CβA promoter showed severe retinal histopathological changes. Histopathological findings revealed the presence of infiltrating cells in 108 samples, including those injected with vehicle. Retinal layers were deteriorated in 98 retinas, including those injected with vehicle. Forty of the 320 eyes scored showed inflammatory cells in the choroid or vitreous, which were distributed throughout the group, and 32 samples contained rare macrophages observed in the subretinal space.

[0314] Three test substances, AAV8.CMV / CβA-hCNGA3 native, AAV8.CMV / CβA-hCNGA3 codon-optimized mutant 3, and AAV9.CMV / CβA-hCNGA3 codon-optimized, all caused tissue damage when driven by a constitutive promoter and were therefore not included in this study. The observed decreased cone function was due to the Cnga - / - This was consistent with previously reported phenotypes in mice. Ocular inflammation was detected in only one animal following subretinal injection with the gene therapy vector or vehicle control. Animals receiving the viral test substance showed no increase in morbidity or mortality. Delivery of 8E8vg / eye of AAV8.RK1-hCNGA3 codon-optimized or AAV8.hCAR-hCNGA3 codon-optimized variant 3 resulted in significant preservation of retinal histology and improved visual function compared with vehicle-treated eyes. Based on these results, we conclude that subretinal delivery of AAV8.RK1-hCNGA3 codon-optimized or AAV8.hCAR-hCNGA3 codon-optimized variant 3 significantly improved retinal histology and improved visual function compared with vehicle-treated eyes. - / - We conclude that this is sufficient to halt the progression of ACHM in mice.

[0315] All published documents cited herein, including Provisional Patent Application No. 62 / 266,789, filed December 14, 2015, Provisional Patent Application No. 62 / 519,821, filed June 14, 2017, and International Publication No. WO 2017 / 106202, are incorporated herein by reference in their entirety. Similarly, any SEQ ID NOs referred to herein and appearing in the accompanying Sequence Listing are incorporated herein by reference. While the invention has been described with reference to specific embodiments, it will be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to be within the scope of the appended claims. [Sequence List Free Text]

[0316] The following information is the identification number <223> The sequence is provided with free text below.

[0317] Table 31-1

[0318] Table 31-2

[0319] Table 31-3

[0320] Table 31-4

[0321] Table 31-5

[0322] Table 31-6

[0323] Table 31-7

[0324] Table 31-8

[0325] Table 31-9

[0326] Table 31-10

[0327] Table 31-11

Claims

1. A cDNA comprising a nucleic acid sequence encoding cyclic nucleotide-gated channel beta 3 (CNGB3) comprising SEQ ID NO:

45.

2. An expression cassette comprising the cDNA sequence of claim 1.

3. An adeno-associated virus (AAV) vector comprising an AAV8 capsid, a nucleic acid sequence comprising an AAV inverted terminal repeat (ITR) sequence, a nucleic acid sequence of SEQ ID NO: 45 encoding human cyclic nucleotide-gated channel beta 3 (CNGB3), and an expression control sequence that directs expression of the CNGB3 in a host cell, wherein the expression control sequence comprises an RK1 promoter.

4. 4. The AAV vector of claim 3, wherein the expression control sequence comprises an ocular cell-specific promoter, and optionally the promoter is selected from the group consisting of human EF1α promoter, metabotropic glutamate receptor 6 (mGluR6) promoter, rhodopsin promoter, cone opsin promoter, and transcription factor promoters (neural retinal leucine zipper (Nrl) and photoreceptor-specific nuclear receptor Nr2e3, bZIP).

5. 4. The AAV vector of claim 3, wherein the expression control sequence comprises a promoter selected from an inducible promoter, a constitutive promoter, and a tissue-specific promoter, and optionally, the promoter is an inducible promoter selected from a rapamycin / rapalog promoter, an ecdysone promoter, an estrogen-responsive promoter, a tetracycline-responsive promoter, and a heterodimeric repressor switch.

6. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and at least one AAV vector according to any one of claims 3 to 5.

7. A plasmid for producing an AAV vector for expressing CNGB3, comprising the nucleic acid sequence of SEQ ID NO: 45, wherein the plasmid comprises a nucleic acid sequence comprising SEQ ID NO: 44 or a sequence sharing at least 80% identity therewith.

8. 10. A method for producing a recombinant AAV (rAAV) virus, comprising culturing packaging cells harboring the plasmid of claim 7 in the presence of sufficient viral sequences to allow packaging of a gene expression cassette viral genome into an infectious AAV envelope or capsid.

9. A viral vector comprising a vector genome comprising nucleotides 1-3930 of SEQ ID NO:42 or nucleotides 1-4704 of SEQ ID NO:

44.

10. A composition for use in a method for treating color vision deficiency, comprising an AAV vector according to any one of claims 3 to 5.

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