Recombinant adeno-associated viral vectors for gene delivery

Abstract

To provide recombinant adeno-associated viral vector for gene delivery.SOLUTION: The disclosure provides a recombinant AAV vector for improving gene therapy, AAV viral vector, and capsid protein and methods for production and use thereof. Adeno-associated viral vectors are promising as a delivery vector for gene therapy. Their therapeutic efficacy, however, is lessened by their delivery efficiency or limited tissue tropism. Thus, there is an urgent need for new AAV vectors with a better therapeutic potential.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 775,871, filed December 5, 2018, No. 62 / 801,195, filed February 5, 2019, No. 62 / 863,126, filed June 18, 2019, and No. 62 / 914,856, filed October 14, 2019, each of which is incorporated by reference in its entirety for all purposes.

[0002] Incorporation by reference of sequence listing The contents of the text file submitted electronically herewith are incorporated by reference in their entirety into this specification: Computer-readable copy of the sequence listing (Filename: ABEO_002_04WO_SeqList_ST25, Creation Date: December 3, 2019, File Size: 556kb). [Background technology]

[0003] Adeno-associated virus vectors are promising as delivery vectors for gene therapy.However, their therapeutic effectiveness is hindered by the delivery efficiency or limited tissue tropism of vectors.Therefore, there is an urgent need for new AAV vectors with better therapeutic potential. Summary of the Invention

[0004] The present disclosure relates generally to the field of gene therapy, and specifically to recombinant adeno-associated virus (AAV) vector particles (also known as AAV viral vectors) having novel capsid proteins, their production, and their use to deliver transgenes to treat or prevent a disease or disorder. In certain embodiments, for example, the following are provided: (Item 1) A nucleic acid encoding an AAV capsid protein comprising a VP1 portion, a VP2 portion, and a VP3 portion, wherein the VP3 portion comprises variable regions (VR) I-IX; (a) VR-II comprises the amino acid sequence DNNGVK (SEQ ID NO:54); (b) VR-III comprises the amino acid sequence NDGS (SEQ ID NO:55); (c) VR-IV comprises the amino acid sequence INGSGQNQQT (SEQ ID NO:56); (d) VR-V comprises the amino acid sequence RVSTTTGQNNSNFAWTA (SEQ ID NO:57); (e) VR-VI comprises the amino acid sequence HKEGEDRFFPLSG (SEQ ID NO:58); (f) VR-VII comprises the amino acid sequence KQNAARDNADYSDV (SEQ ID NO:59); (g) VR-VIII comprises the amino acid sequence ADNLQQQNTAPQI (SEQ ID NO: 60); and (h) VR-IX is a nucleic acid comprising the amino acid sequence NYYKSTSVDF (sequence number 61). (Item 2) 2. The nucleic acid of item 1, wherein the VR-I region comprises NSTSGGSS (SEQ ID NO: 53) or SSTSGGSS (SEQ ID NO: 87). (Item 3) 3. The nucleic acid of item 1 or 2, wherein the VR-I region comprises SASTGAS (SEQ ID NO: 52). (Item 4) 4. The nucleic acid according to any one of items 1 to 3, wherein the VP3 portion has the sequence of SEQ ID NO: 41. (Item 5) 3. The nucleic acid of claim 2, wherein the encoded AAV capsid amino acid sequence is at least 95% identical to the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 84. (Item 6) 4. The nucleic acid of claim 3, wherein the encoded AAV capsid amino acid sequence is at least 95% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34. (Item 7) 5. The nucleic acid according to item 4, wherein the nucleic acid sequence is at least 95% identical to a nucleotide sequence selected from SEQ ID NOs: 18 to 23. (Item 8) 8. The nucleic acid according to item 7, wherein the nucleic acid sequence is 100% identical to a nucleotide sequence selected from SEQ ID NOs: 18 to 23. (Item 9) A vector comprising the nucleic acid according to any one of items 1 to 8. (Item 10) An AAV capsid protein encoded by the nucleic acid according to items 1 to 8. (Item 11) 11. The AAV capsid protein of claim 10, wherein the protein comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34. (Item 12) An AAV viral vector comprising the AAV capsid protein encoded by the nucleic acid according to any one of items 1 to 8 and an AAV vector, wherein the AAV vector is, in a 5' to 3' direction, (a) the first AAV inverted terminal repeat; (b) a promoter; (c) a heterologous nucleic acid; (d) a polyA tail, and (e) An AAV viral vector comprising a second AAV inverted terminal repeat. (Item 13) The AAV viral vector of item 12, wherein the heterologous nucleic acid is operably linked to a constitutive promoter. (Item 14) 13. The AAV viral vector of item 12, wherein the heterologous nucleic acid encodes a polypeptide. (Item 15) 13. The AAV viral vector of item 12, wherein the heterologous nucleic acid encodes an antisense RNA, a microRNA, or an RNAi. (Item 16) 13. The AAV viral vector of item 12, wherein the AAV capsid protein comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34. (Item 17) (i) an AAV capsid protein having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34; (ii) an AAV vector, the AAV vector comprising, in a 5' to 3' direction: (a) the first AAV inverted terminal repeat; (b) a promoter; (c) a heterologous nucleic acid; (d) a polyA tail, and (e) a second AAV inverted terminal repeat. (Item 18) 18. The AAV viral vector of item 12 or 17, wherein the heterologous nucleic acid encodes an mRNA, an siRNA, a gRNA, or a microRNA. (Item 19) 18. The AAV viral vector of item 12 or 17, wherein the heterologous nucleic acid encodes a polypeptide. (Item 20) 20. The AAV viral vector of item 19, wherein the heterologous gene sequence encodes cystic fibrosis transmembrane conductance regulator (CFTR), CLN3 protein, alpha galactosidase A (GLA), or acid alpha glucosidase (GAA). (Item 21) 21. The AAV viral vector of item 20, wherein the heterologous sequence encodes CFTR. (Item 22) 22. The AAV viral vector of item 21, wherein the CFTR comprises an amino acid sequence encoded by SEQ ID NO:4. (Item 23) 20. The AAV viral vector of item 19, wherein the heterologous gene sequence encodes a protein comprising an amino acid sequence having at least 70%, 80%, 90%, or 99% identity to any one of SEQ ID NOs: 5, 8, 11, and 14. (Item 24) 20. The AAV viral vector of item 19, wherein the heterologous gene sequence comprises a sequence having at least 70%, 80%, 90%, or 99% identity to any one of SEQ ID NOs: 4, 5, 6, 7, 9, 10, 12, and 13. (Item 25) The promoter is selected from the group consisting of Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, beta actin promoter, phosphoglycerol kinase (PGK) promoter, U6 promoter, H1 promoter, CAG promoter, hybrid chicken beta actin promoter, MeCP2 promoter, EF1 promoter, ubiquitous chicken beta actin hybrid (CBh) promoter, U1a promoter, U1b promoter, 18. The AAV viral vector according to item 12 or 17, wherein the promoter of the AAV viral vector is a MeCP2 promoter, a MeP418 promoter, a MeP426 promoter, a minimal MeCP2 promoter, a VMD2 promoter, a mRho promoter, an EFla promoter, a Ubc promoter, a human β-actin promoter, a TRE promoter, an Ac5 promoter, a polyhedrin promoter, a CaMKIIa promoter, a Gal1 promoter, a TEF1 promoter, a GDS promoter, an ADH1 promoter, a Ubi promoter, or an alpha-1-antitrypsin (hAAT) promoter. (Item 26) A method for treating a disease or disorder, comprising administering to a subject the AAV viral vector according to any one of items 12 to 25. (Item 27) 27. The method of claim 26, wherein the AAV viral vector is administered to the subject orally, rectally, transmucosally, by inhalation, transdermally, parenterally, intravenously, subcutaneously, intradermally, intramuscularly, intrapleurally, intracerebrally, intrathecally, intracerebrally, intraventricularly, intranasally, intraaurally, intraocularly or periocularly, topically, intralymphatically, intracapsularly, intrathecally, or intravitreally. (Item 28) The disease or disorder is amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Fabry disease, Pompe disease, CLN3 disease (or juvenile neuronal ceroid lipofuscinosis), recessive dystrophic epidermolysis bullosa (RDEB), juvenile Batten disease, autosomal dominant disorders, muscular dystrophy, hemophilia A, hemophilia B, multiple sclerosis, diabetes, Gaucher disease, cancer, arthritis, muscle wasting, heart disease, intimal hyperplasia, epilepsy, Huntington's disease, Parkinson's disease, Alzheimer's disease, cystic fibrosis, thalassemia, Hurler syndrome, Sly syndrome, Scheie syndrome, Hurler-Shahi syndrome, 27. The method of item 26, wherein the disease is Yee's syndrome, Hunter syndrome, Sanfilippo syndrome A (mucopolysaccharidosis IIIA or MPSIIIA), Sanfilippo syndrome B (mucopolysaccharidosis IIIB or MPSIIIB), Sanfilippo syndrome C, Sanfilippo syndrome D, Morquio syndrome, Maroteaux-Lamy syndrome, Krabbe disease, phenylketonuria, Batten disease, spinal cerebral ataxia, LDL receptor deficiency, hyperammonemia, arthritis, macular degeneration, retinitis pigmentosa, neuronal ceroid lipofuscinosis 1 (CLN1), or adenosine deaminase deficiency. [Brief description of the drawings]

[0005] [Figure 1] Strategy for constructing the AIM capsid library. [Figure 2A] Comparison of transduction efficiency in tissue culture. HEK293 cells were plated in 96-well plates at 50,000 cells per well. Cells were transduced with capsid-containing AAV9-GFP or AAV214-GFP viruses at an MOI of 5E+5. Images were taken 45 hours after transduction. [Figure 2B] Comparison of transduction efficiency in different tissues of mice administered 2E+11 viral genomes (vg) of AAV9 or AAV214 viruses. [Figure 2C] Comparison of transduction efficiency and expression levels in the brains of mice administered 2E+11 vg of AAV9 or AAV214 virus. [Figure 3A]Scanning laser ophthalmoscopy imaging of mouse retinas following AAV administration. Wild-type C57BL / 6J mice were administered labeled AAV serotypes by both subretinal (right eye) and intravitreal (left eye) injections. 1 μL of 5E+12vg / mL (5E+9vg / eye) AAV vector was injected with both administration methods, and animals were imaged 10 days later with an HRA2 Spectralis Scanning Laser Ophthalmoscope (Heidelberg Engineering, Carlsbad, CA, USA). Images in which cataracts prevented adequate observation were omitted from the figure. [Figure 3B] IHC analysis of mouse eyes injected intravitreally with AAV204-GFP. [Figure 4] Figure 4 compares GFP expression mediated by AAV204 or AAV9 transduction in primates by RT-qPCR. The dotted line corresponds to the background calculated as the -RT mean plus 2 or 4 standard deviations. [Figure 5A] Figures 5A-5F show AAV204-mediated ocular expression. Figure 5A shows the spread of transduction mediated by intravitreal injection of AAV204, mainly peripheral and some foveal. Figure 5B shows transduction (by GFP expression) of various retinal cells, including photoreceptors and RPE cells, in primates after intravitreal injection of AAV204 virus. Figure 5C shows massive photoreceptor and RPE transduction in the macula, where most cones (photoreceptors responsible for color vision) are concentrated. Figures 5D-5F show expression of GFP from AAV204 driven by VMD2 (vitelloid macular degeneration-2 promoter), a cell-specific promoter for the RPE. SLO imaging was performed on days 14 (Figure 5D) and 28 (Figure 5E) after intravitreal injection of 2.5 x 1012 viral genome (vg) vectors. Figure 5F shows GFP expression in the periphery and nuclei (DAPI) on day 28. [Figure 5B]Figures 5A-5F show AAV204-mediated ocular expression. Figure 5A shows the spread of transduction mediated by intravitreal injection of AAV204, mainly peripheral and some foveal. Figure 5B shows transduction (by GFP expression) of various retinal cells, including photoreceptors and RPE cells, in primates after intravitreal injection of AAV204 virus. Figure 5C shows massive photoreceptor and RPE transduction in the macula, where most cones (photoreceptors responsible for color vision) are concentrated. Figures 5D-5F show expression of GFP from AAV204 driven by VMD2 (vitelloid macular degeneration-2 promoter), a cell-specific promoter for the RPE. SLO imaging was performed on days 14 (Figure 5D) and 28 (Figure 5E) after intravitreal injection of 2.5 x 1012 viral genome (vg) vectors. Figure 5F shows GFP expression in the periphery and nuclei (DAPI) on day 28. [Figure 5C] Figures 5A-5F show AAV204-mediated ocular expression. Figure 5A shows the spread of transduction mediated by intravitreal injection of AAV204, mainly peripheral and some foveal. Figure 5B shows transduction (by GFP expression) of various retinal cells, including photoreceptors and RPE cells, in primates after intravitreal injection of AAV204 virus. Figure 5C shows massive photoreceptor and RPE transduction in the macula, where most cones (photoreceptors responsible for color vision) are concentrated. Figures 5D-5F show expression of GFP from AAV204 driven by VMD2 (vitelloid macular degeneration-2 promoter), a cell-specific promoter for the RPE. SLO imaging was performed on days 14 (Figure 5D) and 28 (Figure 5E) after intravitreal injection of 2.5 x 1012 viral genome (vg) vectors. Figure 5F shows GFP expression in the periphery and nuclei (DAPI) on day 28. [Figure 5D]Figures 5A-5F show AAV204-mediated ocular expression. Figure 5A shows the spread of transduction mediated by intravitreal injection of AAV204, mainly peripheral and some foveal. Figure 5B shows transduction (by GFP expression) of various retinal cells, including photoreceptors and RPE cells, in primates after intravitreal injection of AAV204 virus. Figure 5C shows massive photoreceptor and RPE transduction in the macula, where most cones (photoreceptors responsible for color vision) are concentrated. Figures 5D-5F show expression of GFP from AAV204 driven by VMD2 (vitelloid macular degeneration-2 promoter), a cell-specific promoter for the RPE. SLO imaging was performed on days 14 (Figure 5D) and 28 (Figure 5E) after intravitreal injection of 2.5 x 1012 viral genome (vg) vectors. Figure 5F shows GFP expression in the periphery and nuclei (DAPI) on day 28. [Figure 5E] Figures 5A-5F show AAV204-mediated ocular expression. Figure 5A shows the spread of transduction mediated by intravitreal injection of AAV204, mainly peripheral and some foveal. Figure 5B shows transduction (by GFP expression) of various retinal cells, including photoreceptors and RPE cells, in primates after intravitreal injection of AAV204 virus. Figure 5C shows massive photoreceptor and RPE transduction in the macula, where most cones (photoreceptors responsible for color vision) are concentrated. Figures 5D-5F show expression of GFP from AAV204 driven by VMD2 (vitelloid macular degeneration-2 promoter), a cell-specific promoter for the RPE. SLO imaging was performed on days 14 (Figure 5D) and 28 (Figure 5E) after intravitreal injection of 2.5 x 1012 viral genome (vg) vectors. Figure 5F shows GFP expression in the periphery and nuclei (DAPI) on day 28. [Figure 5F]Figures 5A-5F show AAV204-mediated ocular expression. Figure 5A shows the spread of transduction mediated by intravitreal injection of AAV204, mainly peripheral and some foveal. Figure 5B shows transduction (by GFP expression) of various retinal cells, including photoreceptors and RPE cells, in primates after intravitreal injection of AAV204 virus. Figure 5C shows massive photoreceptor and RPE transduction in the macula, where most cones (photoreceptors responsible for color vision) are concentrated. Figures 5D-5F show expression of GFP from AAV204 driven by VMD2 (vitelloid macular degeneration-2 promoter), a cell-specific promoter for the RPE. SLO imaging was performed on days 14 (Figure 5D) and 28 (Figure 5E) after intravitreal injection of 2.5 x 1012 viral genome (vg) vectors. Figure 5F shows GFP expression in the periphery and nuclei (DAPI) on day 28. [Figure 6] FIG. 6 shows IHC analysis of NHP eye explant transduction performed ex vivo. [Figure 7] Neutralizing antibody quantification strategy. The lack of luminescence indicated that the target AAV was bound by the neutralizing antibody. [Figure 8] Figure 8 demonstrates the differential immunogenicity of AAV204 and AAV9. Neutralizing antibody titers were obtained using an in-house developed process. Either AAV9-Luc or pA-AAV204-Luc was incubated with various dilutions of serum at an MOI of 25,000. After incubation, the virus / serum mixture was transferred to wells containing 20,000 Lec2 cells and incubated with the cells for 24 hours, after which luminescence was measured and compared to control values ​​from cells transduced with virus alone at the same MOI. [Figure 9] Figure 9 shows a comparison of pulmonary transduction using AAV204 or AAV6 (benchmark for AAV pulmonary transduction) via intratracheal delivery. [Figure 10A]Figure 10A-C show the functionality of CFTRΔR and full-length expression cassettes upon AAV204 loaded CFTRΔR expression cassette treatment by FLIPR assay (Figure 10A) and dose response (Figure 10B). Figure 10C shows a comparison of CFTRΔR versus full-length codon optimized CFTR expression with respect to membrane potential assayed by FLIPR. 293 cells were transduced with AAV204 expressing the protein and changes in fluorescence were monitored. A baseline was read for 1 min and then 50 μM forskolin was added to the cells. [Figure 10B] Figure 10A-C show the functionality of CFTRΔR and full-length expression cassettes upon AAV204 loaded CFTRΔR expression cassette treatment by FLIPR assay (Figure 10A) and dose response (Figure 10B). Figure 10C shows a comparison of CFTRΔR versus full-length codon optimized CFTR expression with respect to membrane potential assayed by FLIPR. 293 cells were transduced with AAV204 expressing the protein and changes in fluorescence were monitored. A baseline was read for 1 min and then 50 μM forskolin was added to the cells. [Figure 10C] Figure 10A-C show the functionality of CFTRΔR and full-length expression cassettes upon AAV204 loaded CFTRΔR expression cassette treatment by FLIPR assay (Figure 10A) and dose response (Figure 10B). Figure 10C shows a comparison of CFTRΔR versus full-length codon optimized CFTR expression with respect to membrane potential assayed by FLIPR. 293 cells were transduced with AAV204 expressing the protein and changes in fluorescence were monitored. A baseline was read for 1 min and then 50 μM forskolin was added to the cells. [Figure 11A] Figures 11A-C show the ability of AAV204 to transduce cultured CF patient cells (Figure 11A), CFTRΔR expression and proper localization at the cell membrane (Figure 11B), and expression of CFTRΔR to restore CFTR currents in human CF patient cells (Figure 11C). [Figure 11B]Figures 11A-C show the ability of AAV204 to transduce cultured CF patient cells (Figure 11A), CFTRΔR expression and proper localization at the cell membrane (Figure 11B), and expression of CFTRΔR to restore CFTR currents in human CF patient cells (Figure 11C). [Figure 11C] Figures 11A-C show the ability of AAV204 to transduce cultured CF patient cells (Figure 11A), CFTRΔR expression and proper localization at the cell membrane (Figure 11B), and expression of CFTRΔR to restore CFTR currents in human CF patient cells (Figure 11C). [Figure 12A] FIG. 12A shows the in vivo effects of nasal administration of AAV204 particles containing a CFTR transgene on a mouse model of cystic fibrosis. [Figure 12B] FIG. 12B shows the effect of AAV204 / CFTRΔR treatment (by increasing nasal membrane potential) treatment in cells from different CF patients. [Figure 13] FIG. 13 shows the biodistribution of AAV9-CLN3 and AAV214-CLN3 vectors in the CLN3Δex7 / 8 mouse model 30 days after intravenous administration of viral particles. [Figure 14] FIG. 14 shows expression of AAV9-CLN3 and AAV214-CLN3 vectors in CLN3Δex7 / 8 mouse brain tissue as measured by RT-qPCR. [Figure 15] FIG. 15 shows an immunoblot of GLA expression in transduced HEK293 cells. [Figure 16] FIG. 16 shows the enzymatic activity (supraphysiological) of GLA in plasma, brain, liver, spinal cord, heart, kidney, and eye in C57BL / 6 mice following AAV administration. [Figure 17] FIG. 17 shows the enzyme activity of GAA in the brain, biceps, diaphragm, and liver in C57BL / 6 mice after AAV administration by intravenous injection. [Figure 18A]18A-B show immunoblot analysis (FIG. 18A) and enzymatic analysis (FIG. 18B) of recombinant hGAA expressed in HEK293 cells by transfection. [Figure 18B] 18A-B show immunoblot analysis (FIG. 18A) and enzymatic analysis (FIG. 18B) of recombinant hGAA expressed in HEK293 cells by transfection. [Figure 19A] Figures 19A-E show the enzyme activity of GAA in plasma (Figure 19A), brain (Figure 19B), biceps (Figure 19C), diaphragm (Figure 19D), or liver (Figure 19E) in C57BL / 6 mice following AAV administration. Figure 19F shows glycogen levels in GAA- / - mice treated IV with AAV capsid. Data are presented as % of glycogen found in GAA- / - mice. The reduction in glycogen indicates restoration of GAA functionality by AAV9- and AAV214-mediated expression of the codon-optimized GAA enzyme. [Figure 19B] Figures 19A-E show the enzyme activity of GAA in plasma (Figure 19A), brain (Figure 19B), biceps (Figure 19C), diaphragm (Figure 19D), or liver (Figure 19E) in C57BL / 6 mice following AAV administration. Figure 19F shows glycogen levels in GAA- / - mice treated IV with AAV capsid. Data are presented as % of glycogen found in GAA- / - mice. The reduction in glycogen indicates restoration of GAA functionality by AAV9- and AAV214-mediated expression of the codon-optimized GAA enzyme. [Figure 19C] Figures 19A-E show the enzyme activity of GAA in plasma (Figure 19A), brain (Figure 19B), biceps (Figure 19C), diaphragm (Figure 19D), or liver (Figure 19E) in C57BL / 6 mice following AAV administration. Figure 19F shows glycogen levels in GAA- / - mice treated IV with AAV capsid. Data are presented as % of glycogen found in GAA- / - mice. The reduction in glycogen indicates restoration of GAA functionality by AAV9- and AAV214-mediated expression of the codon-optimized GAA enzyme. [Figure 19D]Figures 19A-E show the enzyme activity of GAA in plasma (Figure 19A), brain (Figure 19B), biceps (Figure 19C), diaphragm (Figure 19D), or liver (Figure 19E) in C57BL / 6 mice following AAV administration. Figure 19F shows glycogen levels in GAA- / - mice treated IV with AAV capsid. Data are presented as % of glycogen found in GAA- / - mice. The reduction in glycogen indicates restoration of GAA functionality by AAV9- and AAV214-mediated expression of the codon-optimized GAA enzyme. [Figure 19E] Figures 19A-E show the enzyme activity of GAA in plasma (Figure 19A), brain (Figure 19B), biceps (Figure 19C), diaphragm (Figure 19D), or liver (Figure 19E) in C57BL / 6 mice following AAV administration. Figure 19F shows glycogen levels in GAA- / - mice treated IV with AAV capsid. Data are presented as % of glycogen found in GAA- / - mice. The reduction in glycogen indicates restoration of GAA functionality by AAV9- and AAV214-mediated expression of the codon-optimized GAA enzyme. [Figure 19F] Figures 19A-E show the enzyme activity of GAA in plasma (Figure 19A), brain (Figure 19B), biceps (Figure 19C), diaphragm (Figure 19D), or liver (Figure 19E) in C57BL / 6 mice following AAV administration. Figure 19F shows glycogen levels in GAA- / - mice treated IV with AAV capsid. Data are presented as % of glycogen found in GAA- / - mice. The reduction in glycogen indicates restoration of GAA functionality by AAV9- and AAV214-mediated expression of the codon-optimized GAA enzyme. [Figure 20-1] FIG. 20 shows an alignment of VP1 amino acid sequences from AAV204 (SEQ ID NO:2) and AAV6 (SEQ ID NO:63). [Figure 20-2] FIG. 20 shows an alignment of VP1 amino acid sequences from AAV204 (SEQ ID NO:2) and AAV6 (SEQ ID NO:63). [Figure 21-1]Figure 21 shows an alignment of VP1 protein amino acid sequences from AAV214 (sequence number 3), AAV214A (sequence number 30), AAV214AB (sequence number 84), AAV214e (sequence number 31), AAV214e8 (sequence number 32), AAV214e9 (sequence number 33), AAV214e10 (sequence number 34), ITB204_45 (sequence number 49), AAV9 (sequence number 71), and AAV8 (sequence number 67). [Figure 21-2] Figure 21 shows an alignment of VP1 protein amino acid sequences from AAV214 (sequence number 3), AAV214A (sequence number 30), AAV214AB (sequence number 84), AAV214e (sequence number 31), AAV214e8 (sequence number 32), AAV214e9 (sequence number 33), AAV214e10 (sequence number 34), ITB204_45 (sequence number 49), AAV9 (sequence number 71), and AAV8 (sequence number 67). [Figure 21-3] Figure 21 shows an alignment of VP1 protein amino acid sequences from AAV214 (sequence number 3), AAV214A (sequence number 30), AAV214AB (sequence number 84), AAV214e (sequence number 31), AAV214e8 (sequence number 32), AAV214e9 (sequence number 33), AAV214e10 (sequence number 34), ITB204_45 (sequence number 49), AAV9 (sequence number 71), and AAV8 (sequence number 67). [Figure 22-1] Figure 22 shows an alignment of VP2 protein amino acid sequences from AAV214 (sequence number 35), AAV214A (sequence number 36), AAV214AB (sequence number 85), AAV214e (sequence number 37), AAV214e8 (sequence number 38), AAV214e9 (sequence number 39), AAV214e10 (sequence number 40), ITB204_45 (sequence number 50), AAV9 (sequence number 72), and AAV8 (sequence number 68). [Figure 22-2]Figure 22 shows an alignment of VP2 protein amino acid sequences from AAV214 (sequence number 35), AAV214A (sequence number 36), AAV214AB (sequence number 85), AAV214e (sequence number 37), AAV214e8 (sequence number 38), AAV214e9 (sequence number 39), AAV214e10 (sequence number 40), ITB204_45 (sequence number 50), AAV9 (sequence number 72), and AAV8 (sequence number 68). [Figure 22-3] Figure 22 shows an alignment of VP2 protein amino acid sequences from AAV214 (sequence number 35), AAV214A (sequence number 36), AAV214AB (sequence number 85), AAV214e (sequence number 37), AAV214e8 (sequence number 38), AAV214e9 (sequence number 39), AAV214e10 (sequence number 40), ITB204_45 (sequence number 50), AAV9 (sequence number 72), and AAV8 (sequence number 68). [Figure 23-1] Figure 23 shows an alignment of VP3 protein amino acid sequences from AAV214 (sequence number 41), AAV214A (sequence number 42), AAV214AB (sequence number 86), AAV214e (sequence number 43), AAV214e8 (sequence number 44), AAV214e9 (sequence number 45), AAV214e10 (sequence number 46), ITB204_45 (sequence number 51), AAV9 (sequence number 73), and AAV8 (sequence number 69). [Figure 23-2] Figure 23 shows an alignment of VP3 protein amino acid sequences from AAV214 (sequence number 41), AAV214A (sequence number 42), AAV214AB (sequence number 86), AAV214e (sequence number 43), AAV214e8 (sequence number 44), AAV214e9 (sequence number 45), AAV214e10 (sequence number 46), ITB204_45 (sequence number 51), AAV9 (sequence number 73), and AAV8 (sequence number 69). [Figure 23-3]Figure 23 shows an alignment of VP3 protein amino acid sequences from AAV214 (sequence number 41), AAV214A (sequence number 42), AAV214AB (sequence number 86), AAV214e (sequence number 43), AAV214e8 (sequence number 44), AAV214e9 (sequence number 45), AAV214e10 (sequence number 46), ITB204_45 (sequence number 51), AAV9 (sequence number 73), and AAV8 (sequence number 69). [Figure 24A] Figure 24A shows expression of the GFP transgene delivered by AAV110 and AAV9 vector particles in muscle after intramuscular administration. The upper panel shows the left and right legs in white light, showing the overall tissue structure. The lower panel shows GFP fluorescence. [Figure 24B] FIG. 24B provides a quantitative analysis of the fluorescence obtained in FIG. 24A for AAV110 particles (ITCord1.10) compared to AAV9. [Figure 25A] Figures 25A-C illustrate the expression of transgenes delivered by AAV110 particles (ITCord1.10) relative to AAV9 following intramuscular administration. The data show that expression of AAV110 in muscle is particularly high. [Figure 25B] Figures 25A-C illustrate the expression of transgenes delivered by AAV110 particles (ITCord1.10) relative to AAV9 following intramuscular administration. The data show that expression of AAV110 in muscle is particularly high. [Figure 25C] Figures 25A-C illustrate the expression of transgenes delivered by AAV110 particles (ITCord1.10) relative to AAV9 following intramuscular administration. The data show that expression of AAV110 in muscle is particularly high. [Figure 26A] Figure 26A shows muscle tissue immunohistochemistry to detect GFP expression after intramuscular administration of AAV110 and AAV9 particles expressing GFP. The figure shows GFP expressed from AAV9 vector particles (lower left panel), GFP expressed from AAV110 particles (lower right panel), and control muscle (upper panel). Tissue was stained with anti-GFP antibody. [Figure 26B] IM delivered AAV214 transduces a larger muscle area than AAV9. A whole muscle (biceps femoris) from a rat was analyzed for GFP or mCherry expression by immunohistochemistry 10 days after IM injection. Fixed frozen sections were probed with GFP and mCherry pAbs. AAV214 shows a significantly larger transduced area compared to AAV9, which was mostly restricted to the upper part of the muscle coinciding with the injection site. [Figure 27] Figure 27 shows a bioluminescence image showing luciferase expressed as a transgene and exposed to luciferin. Data was obtained 28 days after AAV214 administration. [Figure 28] FIG. 28 compares muscle expression of SMN-1 protein delivered intravenously with AAV214 and AAV9. [Figure 29] Figure 29 shows the expression of GFP in heart and biceps as a transgene mediated by AAV214 versus AAV9 variants. The y-axis shows the log10 value of viral copy number per microgram of genomic DNA. [Diagram 30] Figure 30 shows a diagram of the VP1, VP2, and VP3 capsid proteins. The VP1-specific and VP2-specific portions are shown along with the VP3 portion, which is identical to the produced VP3 protein. The amino acid sequence of AAV214 VP3 (SEQ ID NO:41) is shown, as are the variable regions I-IX. The complete VP1 protein amino acid sequence for AAV214 is provided as SEQ ID NO:3. [Figure 31A]Figures 31A-C illustrate animals treated with AAV214 demonstrating reduced production of AAV9 neutralizing antibodies. Figure 31A shows animals administered either AAV9 or AAV214 by IM assayed for neutralizing antibodies to AAV9. Analysis was performed by measuring the ability of animal sera to inhibit transduction of AAV9 luciferase vector into the permissive cell type Lec2. Three days after transduction, cells were assayed for luciferase activity. Each group consisted of two or three rats for either control, AAV9, or AAV214. Figures 31B and 31C show cross-reactivity to AAV9 and AAV204. Figure 31B shows the development of various AAV neutralizing antibodies after AAV9 challenge. Figure 31C shows the development of various AAV neutralizing antibodies after AAV204 challenge. [Figure 31B] Figures 31A-C illustrate animals treated with AAV214 demonstrating reduced production of AAV9 neutralizing antibodies. Figure 31A shows animals administered either AAV9 or AAV214 by IM assayed for neutralizing antibodies to AAV9. Analysis was performed by measuring the ability of animal sera to inhibit transduction of AAV9 luciferase vector into the permissive cell type Lec2. Three days after transduction, cells were assayed for luciferase activity. Each group consisted of two or three rats for either control, AAV9, or AAV214. Figures 31B and 31C show cross-reactivity to AAV9 and AAV204. Figure 31B shows the development of various AAV neutralizing antibodies after AAV9 challenge. Figure 31C shows the development of various AAV neutralizing antibodies after AAV204 challenge. [Figure 31C]Figures 31A-C illustrate animals treated with AAV214 demonstrating reduced production of AAV9 neutralizing antibodies. Figure 31A shows animals administered either AAV9 or AAV214 by IM assayed for neutralizing antibodies to AAV9. Analysis was performed by measuring the ability of animal sera to inhibit transduction of AAV9 luciferase vector into the permissive cell type Lec2. Three days after transduction, cells were assayed for luciferase activity. Each group consisted of two or three rats for either control, AAV9, or AAV214. Figures 31B and 31C show cross-reactivity to AAV9 and AAV204. Figure 31B shows the development of various AAV neutralizing antibodies after AAV9 challenge. Figure 31C shows the development of various AAV neutralizing antibodies after AAV204 challenge. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] Some embodiments according to the present disclosure are described more fully below. However, aspects of the present disclosure may be embodied in different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art. The terminology used in the description of this specification is for the purpose of describing specific embodiments only, and is not intended to be limiting.

[0007] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of this application and related art, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0008] Unless otherwise indicated by the context, it is specifically intended that the various features of the present invention described herein can be used in any combination.Furthermore, the present disclosure also contemplates that in embodiments, any feature or combination of features described herein can be excluded or omitted.For illustration, when the present specification describes that a complex comprises components A, B, and C, it is specifically intended that any of A, B, or C, or combinations thereof, can be omitted and abandoned, either alone or in any combination.

[0009] Unless expressly indicated otherwise, all specified embodiments, features, and terms are intended to include both the recited embodiment, feature, or term and its biological equivalents.

[0010] Incorporation by Reference All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, mention of any reference, article, publication, patent, patent publication, or patent application cited herein should not be construed as an admission or any form of suggestion that it constitutes valid prior art or forms part of the common general knowledge in any country throughout the world.

[0011] definition The practice of this technology, unless otherwise indicated, employs conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the skill of the art.See, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); Current Protocols In Molecular Biology (FMA Usubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (RI. Freshney, ed. (1987)).

[0012] As used in the description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0013] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude others. As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) should be interpreted as including the recited materials or steps and those that do not substantially affect the basic and novel feature(s) of the recited embodiment. Thus, as used herein, the term "consisting essentially of" should not be interpreted as the equivalent of "comprising." "Consisting of" is intended to mean excluding other components beyond trace elements and substantial method steps for administering the compositions disclosed herein. The aspects defined by each of these transitional terms are within the scope of this disclosure.

[0014] All numerical designations, including ranges (e.g., pH, temperature, time, concentration, and molecular weight, etc.), are approximations that vary (+) or (-) in increments of 1.0 or 0.1, as appropriate, or alternatively, by a variation of + / -15%, 10%, 5%, 2%. Although not always explicitly stated, it should be understood that all numerical designations are preceded by the term "about". It should also be understood, although not always explicitly stated, that the reagents described herein are merely exemplary, and that equivalents of such are known in the art. When referring to measurable values, such as amounts or concentrations and the like, the term "about" as used herein is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0015] The terms "acceptable," "effective," or "sufficient," when used to describe the selection of any component, range, dosage form, etc. disclosed herein, intend that the component, range, dosage form, etc. is appropriate for the purpose disclosed.

[0016] Additionally, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items as well as the lack of combinations when interpreted in the alternative ("or").

[0017] Unless specifically recited, the term "host cell" includes eukaryotic host cells, including, for example, fungal cells, yeast cells, higher plant cells, insect cells, and mammalian cells. Non-limiting examples of eukaryotic host cells include monkey, bovine, porcine, murine, rat, avian, reptilian, and human, e.g., HEK293 and 293T cells.

[0018] As used herein, the term "isolated" refers to molecules or biological or cellular material that is substantially free from other materials.

[0019] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers that contain, consist essentially of, or consist of purine and pyrimidine bases or other naturally occurring bases, chemically or biochemically modified bases, non-natural bases, or derivatized nucleotide bases.

[0020] "Gene" refers to a polynucleotide containing at least one open reading frame (ORF) capable of encoding a particular polypeptide or protein. "Gene product," or alternatively, "gene expression product," refers to the amino acid sequence (e.g., peptide or polypeptide) produced when a gene is transcribed and translated.

[0021] As used herein, "expression" refers to the two-step process in which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is then translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells.

[0022] "Under transcriptional control" is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, is dependent on being operably linked to elements that contribute to or facilitate the initiation of transcription. "Operably linked" refers to being arranged in a manner that allows the polynucleotide to function within a cell. In one aspect, the present invention provides a promoter operably linked to a downstream sequence.

[0023] The term "encodes," as applied to a polynucleotide, refers to a polynucleotide that is said to "encode" a polypeptide if, in its natural state, or when manipulated by methods well known to those of skill in the art, it can be transcribed to produce mRNA for the polypeptide and / or fragments thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0024] As used herein, the term "promoter" refers to a control sequence, which is a region of a polynucleotide sequence where the initiation and transcription rate of a coding sequence, such as a gene or transgene, is controlled. A promoter may be, for example, constitutive, inducible, repressible, or tissue-specific. A promoter may contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and transcription factors, may bind. Non-limiting exemplary promoters include the Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, the U6 promoter, the H1 promoter, the ubiquitous chicken β-actin hybrid (CBh) promoter, the small nuclear RNA (U1a or U1b) promoter, the MeCP2 promoter, the MeP418 promoter, the MeP426 promoter, the minimal MeCP2 promoter, the VMD2 promoter, the mRho promoter, or the EFI promoter.

[0025] Additional non-limiting exemplary promoters provided herein include, but are not limited to, EFla, Ubc, human β-actin, CAG, TRE, Ac5 polyhedrin, Gal1, TEF1, GDS, ADH1, Ubi, and alpha-1-antitrypsin (hAAT). It is known in the art that the nucleotide sequences of such promoters may be modified to increase or decrease the efficiency of mRNA transcription. See, for example, Gao et al. (2018) Mol. Ther.: Nucleic Acids 12:135-145 (modifying the TATA box of the 7SK, U6, and H1 promoters to abolish RNA polymerase III transcription and stimulate RNA polymerase II-dependent mRNA transcription). Synthetic derived promoters may be used for ubiquitous or tissue-specific expression. Additionally, viral derived promoters, some of which are described above, may be useful in the methods disclosed herein, e.g., CMV, HIV, adenovirus, and AAV promoters. In embodiments, promoters are used with enhancers to increase transcription efficiency. Non-limiting examples of enhancers include interstitial retinoid binding protein (IRBP) enhancer, RSV enhancer, or CMV enhancer.

[0026] Enhancers are regulatory elements that increase the expression of a target sequence. A "promoter / enhancer" is a polynucleotide that contains a sequence capable of providing both promoter and enhancer functions. For example, retroviral long terminal repeats contain both promoter and enhancer functions. Enhancers / promoters may be "endogenous" or "exogenous" or "heterologous". An "endogenous" enhancer / promoter is one that is naturally linked to a given gene in a genome. An "exogenous" or "heterologous" enhancer / promoter is juxtaposed to a gene by genetic engineering (i.e., molecular biology techniques) such that transcription of that gene is directed by the linked enhancer / promoter. Non-limiting examples of linked enhancers / promoters for use in the methods, compositions, and constructs provided herein include PDE promoter + IRBP enhancer or CMV enhancer + U1a promoter. It is understood in the art that an enhancer can operate from a distance and regardless of its orientation relative to the location of the endogenous or heterologous promoter. Thus, an enhancer that acts at a distance from a promoter is therefore further understood to be "operably linked" to that promoter, regardless of its location within the vector, or its orientation relative to the location of the promoter.

[0027] The terms "protein", "peptide" and "polypeptide" are used interchangeably and in their broadest sense to refer to a compound of two or more subunits of amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and no limit is placed on the maximum number of amino acids that a protein or peptide can contain, consist essentially of, or consist of. As used herein, the term "amino acid" refers to any of the natural and / or unnatural or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics.

[0028] As used herein, the term "signal peptide" or "signal polypeptide" refers to an amino acid sequence that is usually present at the N-terminus of newly synthesized secretory or membrane polypeptides or proteins. It acts to direct the polypeptide to a specific cellular location, for example across the cell membrane, into the cell membrane, or into the nucleus. In an embodiment, the signal peptide is removed after localization. Examples of signal peptides are well known in the art. Non-limiting examples are those described in U.S. Patent Nos. 8,853,381, 5,958,736, and 8,795,965. In an embodiment, the signal peptide can be an IDUA signal peptide.

[0029] The terms "equivalent" or "biologically equivalent" are used interchangeably when referring to a particular molecule, biological material, or cellular material, and are intended to have minimal homology while still maintaining a desired structure or functionality. Non-limiting examples of equivalent polypeptides include polypeptides having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% identity, or at least about 99% identity to a reference polypeptide (e.g., a wild-type polypeptide), or polypeptides encoded by a polynucleotide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% identity, at least about 97% sequence identity, or at least about 99% sequence identity to a reference polynucleotide (e.g., a wild-type polynucleotide).

[0030] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Percent identity can be determined by comparing positions in each sequence that may be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. The degree of identity between sequences is a function of the number of matching positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, less than 25% identity with one of the sequences of the present disclosure. Alignment and percent sequence identity may be determined for nucleic acid or amino acid sequences by importing the nucleic acid or amino acid sequences provided herein into and using ClustalW (available at https: / / genome.jp / tools-bin / clustalw / ). For example, the ClustalW parameters used to perform the protein sequence alignments found herein (e.g., Figures 20-23) were generated using the Gonnet (for proteins) weight matrix. In embodiments, the ClustalW parameters used to perform nucleic acid sequence alignments using the nucleic acid sequences found herein are generated using the ClustalW (for DNA) weight matrix.

[0031] As used herein, an amino acid modification may be an amino acid substitution, an amino acid deletion, or an amino acid insertion. An amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution. A conservative substitution (also called a conservative mutation, conservative substitution, or conservative variation) is a replacement of an amino acid in a protein that changes a given amino acid into a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, or size). As used herein, a "conservative variation" refers to the replacement of an amino acid residue with another biologically similar residue. Examples of conservative variations include the replacement of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another, or the replacement of one charged or polar residue for another, such as the replacement of arginine with lysine, the replacement of aspartic acid with glutamic acid, the replacement of asparagine with glutamine, and the like. Illustrative examples of conservative substitutions include changing an alanine to a serine, changing an asparagine to a glutamine or a histidine, changing an aspartate to a glutamate, changing a cysteine ​​to a serine, changing a glycine to a proline, changing a histidine to an asparagine or a glutamine, changing a lysine to an arginine, glutamine, or glutamate, changing a phenylalanine to a tyrosine, changing a serine to a threonine, changing a threonine to a serine, changing a tryptophan to a tyrosine, changing a tyrosine to a tryptophan or a phenylalanine, and the like.

[0032] As used herein, the term "vector" refers to a nucleic acid that comprises, consists essentially of, or consists of an intact replicon, such that the vector may be replicated when placed in a cell, for example, by a process of transfection, infection, or transformation. Once inside the cell, it is understood in the art that the vector may be replicated as an extrachromosomal (episomal) element or may be integrated into the host cell chromosome. A vector may comprise a nucleic acid derived from a retrovirus, adenovirus, herpes virus, baculovirus, modified baculovirus, papovavirus, or an otherwise modified naturally occurring virus. Exemplary non-viral vectors for delivering nucleic acids include the use of naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles consisting essentially of, or consisting of, DNA, including DNA condensed with cationic polymers (such as heterogeneous polylysine, oligopeptides of defined length, and polyethyleneimine), in some cases contained within liposomes; and ternary complexes comprising, consisting essentially of, or consisting of viruses and polylysine-DNA.

[0033] Regarding general recombinant techniques, vectors that contain both promoters and cloning sites that can operably link polynucleotides into them are well known in the art. Such vectors have the ability to transcribe RNA in vitro or in vivo, and are commercially available from suppliers such as Agilent Technologies (Santa Clara, Calif., USA) and Promega Biotech (Madison, Wis., USA). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated parts of the cloned transgene to remove excess, potentially inappropriate alternative translation initiation codons or other sequences that may prevent or reduce expression, either at the transcription or translation level. Alternatively, consensus ribosome binding sites can be inserted immediately at the 5' initiation codon to enhance expression.

[0034] "Viral vector" is defined as a recombinantly produced virus or virus particle that contains a polynucleotide that is delivered into a host cell either in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, AAV vectors, lentiviral vectors, adenoviral vectors, alphavirus vectors, and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, for example, Schlesinger et al., "Virology of the Human Cells," vol. 14, no. 1, pp. 1171-1175, 1999. See, e.g., Ying, et al. (1999) Nat. Med. 5(7):823-827, and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439.

[0035] As used herein, the term "recombinant expression system" or "recombinant vector" refers to a genetic construct or constructs for the expression of certain genetic material formed by recombinant means.

[0036] "Gene delivery vehicle" is defined as any molecule capable of carrying an inserted polynucleotide into a host cell. Examples of gene delivery vehicles are liposomes, micelles, biocompatible polymers, including natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; bacteria; viruses, such as baculoviruses, adenoviruses, and retroviruses; bacteriophages, cosmids, plasmids, and fungal vectors; and other recombinant vehicles typically used in the art, which have been described for expression in various eukaryotic and prokaryotic hosts and may be used for simple protein expression as well as gene therapy. Liposomes that also contain, consist essentially of, or consist of targeting antibodies or fragments thereof can also be used in the methods disclosed herein. In addition to delivery of a polynucleotide to a cell or cell population, directly introducing the proteins described herein into a cell or cell population can be done by the non-limiting technique of protein transfection, alternatively refining conditions that can enhance expression and / or promote activity of the proteins disclosed herein are other non-limiting techniques.

[0037] The polynucleotides disclosed herein can be delivered to cells or tissues using gene delivery vehicles. "Gene delivery", "gene transfer", "transduction" and the like, as used herein, are terms that refer to the introduction of an exogenous polynucleotide into a host cell, regardless of the method used for the transfer (sometimes also referred to as a "transgene"). Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (e.g., by viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes), as well as techniques that facilitate the delivery of "naked" polynucleotides (electroporation, "gene gun" delivery, and various other techniques used for the transfer of polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide contains an origin of replication compatible with the host cell, or integrates into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. Numerous vectors are known in the art and are known to have the ability to mediate the transfer of genes into mammalian cells, as described herein.

[0038] A "plasmid" is a DNA molecule that typically separates from chromosomal DNA and has the ability to replicate independently of chromosomal DNA. It is often circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a microbial population and typically provide a selective advantage under a given environmental condition. Plasmids may carry genes that provide resistance to antibiotics that occur naturally in competing environmental niches, or alternatively, the proteins produced may act as toxins under similar circumstances. Although plasmid vectors often exist as extrachromosomal circular DNA molecules, it is known in the art that plasmid vectors may be designed to stably integrate into host chromosomes in either a random or targeted manner, and such integration may be achieved using either circular plasmids or plasmids that are linearized prior to introduction into the host cell.

[0039] "Plasmids" used in genetic engineering are called "plasmid vectors". Many plasmids are commercially available for such use. The gene to be replicated is inserted into a copy of the plasmid, which contains a gene that makes the cell resistant to a particular antibiotic, and into a multiple cloning site (MCS, or polylinker), a short region that contains several commonly used restriction sites that allow easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria or eukaryotic cells that contain a plasmid carrying the gene of interest and can induce them to produce large amounts of the protein from the inserted gene.

[0040] In embodiments in which gene transfer is mediated by a DNA viral vector, such as adenovirus (Ad) or adeno-associated virus (AAV), vector construct refers to a polynucleotide that comprises, consists essentially of, or consists of the viral genome or a portion thereof, and a transgene.

[0041] As used herein, the term "adeno-associated virus" or "AAV" refers to a member of the class of viruses associated with this name and belonging to the genus Dependoparvovirus, family Parvoviridae. Adeno-associated viruses are single-stranded DNA viruses that grow only in cells in which certain functions are provided by a coinfecting helper virus. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). It is well known that various serotypes are structurally and functionally very closely related, even at the genetic level, and it is fully expected that the same principles described in these reviews can be applied to additional AAV serotypes that are characterized after the publication date of the reviews. (See, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R. Puttison, ed.; and Rose, Comprehensive Virology 3:1-61 (1974).) For example, all AAV serotypes clearly exhibit very similar replication properties mediated by homologous rep genes, and all have three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the length of the genome, and the presence of similar self-annealing segments at the ends that correspond to the "inverted terminal repeats" (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. Multiple serotypes of this virus are known to be suitable for gene delivery, and all known serotypes can infect cells from a variety of tissue types. At least eleven consecutively numbered AAV serotypes are known in the art.Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the eleven serotypes (e.g., AAV2, AAV8, AAV9), or variant serotypes (e.g., AAV-DJ and AAV PHP.B). AAV particles comprise, consist essentially of, or consist of three major viral proteins, VP1, VP2, and VP3. In embodiments, AAV refers to serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, or AAVrh74.

[0042] As used herein, "AAV vector" refers to a vector that comprises, consists essentially of, or consists of one or more heterologous nucleic acid (HNA) sequences and one or more AAV inverted terminal repeats (ITRs). Such AAV vectors can replicate and be packaged into infectious viral particles when present in a host cell that provides the functionality of the rep and cap gene products, for example, by transfection of the host cell. In embodiments, the AAV vector contains a promoter, at least one nucleic acid that can code for at least one protein or RNA, and / or enhancer and / or terminator in the adjacent ITRs that are packaged into infectious AAV particles. The encapsidated nucleic acid portion may be referred to as the AAV vector genome. A plasmid containing an AAV vector may also contain elements for manufacturing purposes, such as, for example, antibiotic resistance genes, but these are not encapsidated and therefore do not form part of the AAV particle.

[0043] As used herein, the term "viral capsid" or "capsid" refers to the proteinaceous shell or coating of a viral particle. The capsid functions to encapsidate, protect, transport, and release the viral genome into the host cell. Capsids are generally composed of oligomeric structural subunits of proteins ("capsid proteins"). As used herein, the term "encapsidated" means enclosed within a viral capsid. The viral capsid of AAV is composed of a mixture of three viral capsid proteins, VP1, VP2, and VP3. The mixture of VP1, VP2, and VP3 contains 60 monomers arranged in a T=1 icosahedral symmetry in a ratio of 1:1:10 (VP1:VP2:VP3) or 1:1:20 (VP1:VP2:VP3) as described in Sonntag F et al., (June 2010) "A viral assembly factor promotes AAV2 capsid formation in the nucleolus". Proceedings of the National Academy of Sciences of the United States of America. 107(22):10220-5, and Rabinowitz JE, Samulski RJ (December 2000) "Building a better vector: the manipulation of AAV virions". Virology. 278(2):301-8, each of which is incorporated by reference in its entirety.

[0044] "AAV virion" or "AAV virus particle" or "AAV virus vector" or "AAV vector particle" or "AAV particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. Thus, production of an AAV vector particle necessarily includes production of an AAV vector, whereby the vector is contained within the AAV vector particle.

[0045] As used herein, the term "helper" with respect to a virus or plasmid refers to a virus or plasmid used to provide additional components necessary for replication and packaging of any one of the AAV vectors disclosed herein. The components encoded by the helper virus may include any genes required for virion assembly, encapsidation, genome replication, and / or packaging. For example, the helper virus or plasmid may encode an enzyme required for replication of the viral genome. Non-limiting examples of helper viruses and plasmids suitable for use in AAV constructs include pHELP (plasmid), adenovirus (virus), or herpesvirus (virus). In an embodiment, the pHELP plasmid may be a pHELPK plasmid in which the ampicillin expression cassette is replaced with a kanamycin expression cassette, and pHELPK has the sequence shown in SEQ ID NO: 92.

[0046] As used herein, packaging cell (or helper cell) is the cell used to produce viral vector. The production of recombinant AAV viral vector requires not only Rep and Cap proteins provided in trans, but also gene sequences from adenovirus that help AAV replication. In some embodiments, the packaging / helper cell containing the plasmid is stably integrated into the genome of the cell. In other embodiments, the packaging cell may be transiently transfected. Typically, the packaging cell is a eukaryotic cell, such as a mammalian cell or an insect cell.

[0047] As used herein, a reporter protein is a detectable protein that is operably linked to a promoter to assay the expression (e.g., tissue specificity and / or strength) of the promoter. In embodiments, a reporter protein may be operably linked to a polypeptide. In embodiments, a reporter protein may be used for monitoring DNA delivery methods, functional identification and characterization of promoter and enhancer elements, translation and transcription regulation, mRNA processing, and protein-protein interactions. Non-limiting examples of reporter proteins include β-galactosidase; fluorescent proteins such as green fluorescent protein (GFP) or red fluorescent protein (RFP); luciferase; glutathione S-transferase; and maltose binding protein.

[0048] A "composition" is intended to mean a combination of an active polypeptide, polynucleotide or antibody and another compound or composition, inert (e.g., a detectable label) or active (e.g., a gene delivery vehicle).

[0049] A "pharmaceutical composition" is intended to include an active polypeptide, polynucleotide, or antibody combined with an inert or active carrier (such as a solid support), making the composition suitable for in vitro, in vivo, or ex vivo diagnostic or therapeutic use.

[0050] As used herein, the term "pharmaceutical acceptable carrier" includes standard pharmaceutical carriers such as phosphate buffered saline, water, and emulsions such as oil / water or water / oil emulsions, and any of the various types of wetting agents. The compositions can also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington"s Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0051] A "subject" of diagnosis or treatment is a cell or an animal, such as a mammal or human. Subjects are not limited to a particular species and include non-human animals that are the subject of diagnosis or treatment, and non-human animals that are the subject of infection or animal models, including, but not limited to, monkey, murine, rat, dog, or rabbit species, as well as other farm animals, sport animals, or pets. In an embodiment, the subject is a human.

[0052] The term "tissue" is used herein to refer to tissue of a living or dead organism, or any tissue derived from or designed to mimic a living or dead organism. Tissues may be healthy, diseased, and / or genetically mutated. Biological tissues may include any single tissue (e.g., a collection of cells that may be interconnected), or a group of tissues making up an organ or part or region of the body of an organism. Tissues may include homogenous cellular material, may consist essentially of homogenous cellular material, may consist of homogenous cellular material, or may be composite structures such as found in regions of the body including the thorax, which may include, for example, lung tissue, skeletal tissue, and / or muscle tissue. Exemplary tissues include, but are not limited to, those derived from the liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidney, brain, biliary system, duodenum, abdominal aorta, iliac vein, heart, and intestine, including any combination thereof.

[0053] As used herein, "treating" a disease in a subject or "treatment" of a disease refers to (1) preventing a symptom or disease from occurring in a subject who is susceptible to the disease or who does not yet exhibit symptoms of the disease, (2) inhibiting or halting the development of the disease, or (3) causing an improvement or regression of the disease or symptoms of the disease. As understood in the art, "treatment" is an approach to obtain beneficial or desired results, including clinical results. For purposes of the present technology, beneficial or desired results can include, but are not limited to, one or more of the following: alleviation or amelioration of one or more symptoms, reduction in the severity of a condition (including a disease), stabilization (i.e., not worsening) of a condition (including a disease), delay or slowing of a condition (including a disease), progression, improvement, or palliation of a condition (including a disease), and remission (whether partial or total) (whether detectable or undetectable).

[0054] As used herein, the term "effective amount" is intended to mean an amount sufficient to achieve a desired effect. In the context of therapeutic or prophylactic applications, the effective amount depends on the type and severity of the condition in question, as well as the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to pharmaceutical compositions. In the context of gene therapy, in embodiments, the effective amount is an amount sufficient to cause partial or full function restoration of a gene that is missing in a subject. In some other embodiments, an effective amount of AAV viral particles is an amount sufficient to cause expression of a gene of interest. In embodiments, the effective amount is the amount required to increase galactose metabolism in a subject in need thereof. Those skilled in the art can determine the appropriate amount depending on these and other factors.

[0055] In embodiments, effective amount depends on the size and nature of the application in question.It also depends on the nature and sensitivity of the target object and the method of use.Those skilled in the art will be able to determine effective amount based on these and other considerations.Effective amount may comprise, consist essentially of, or consist of one or more administrations of composition depending on the embodiment.

[0056] As used herein, the term "administer" or "administration" is intended to mean the delivery of a substance to a subject, such as an animal or human. Administration can be performed in one dose, continuously, or intermittently throughout the course of treatment. Methods for determining the most effective means and dosage of administration are known to those skilled in the art and will vary with the composition used for treatment, the purpose of the treatment, as well as the age, health, or sex of the subject being treated. Single or multiple administrations can be performed with the dose level and pattern selected by the treating physician, or in the case of pets and other animals, by the treating veterinarian.

[0057] AAV structure and function AAV is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb in length and contains two 145-nucleotide inverted terminal repeats (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided under GenBank Accession No. NC_002077, the complete genome of AAV-2 is provided under GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983), the complete genome of AAV-3 is provided under GenBank Accession No. NC_l829, the complete genome of AAV-4 is provided under GenBank Accession No. NC_001829, the complete genome of AAV-5 is provided under GenBank Accession No. AF085716, the complete genome of AAV-6 is provided under GenBank Accession No. NC_001862, at least portions of the AAV-7 and AAV-8 genomes are provided under GenBank Accession Nos. AX753246 and AX753249, respectively, and the AAV-9 genome is provided by Gao et al. al., J.Virol.,78:6381-6388(2004); AAV-10 genome is provided in Mol.Ther.,13(1):67-76(2006), and AAV-11 genome is provided in Virology,330(2):375-383(2004). The sequence of the AAV rh.74 genome is provided in U.S. Pat. No. 9,434,928, which is incorporated herein by reference in its entirety. U.S. Pat. No. 9,434,928 also provides the sequence of the capsid protein and the self-complementary genome. In one embodiment, the genome is a self-complementary genome. The cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the AAV ITR. Three AAV promoters (designated p5, p19, and p40 for their relative map locations) drive expression of two AAV internal open reading frames encoding the rep and cap genes.Two rep promoters (p5 and p19) combined with differential splicing of a single AAV intron (at nucleotides 2107 and 2227) result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. The rep proteins have multiple enzymatic properties that are ultimately responsible for the replication of the viral genome.

[0058] The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation start sites are responsible for the production of the three related capsid proteins. More specifically, after a single mRNA into which each of the VP1, VP2, and VP3 proteins is translated is transcribed, it can be spliced ​​in two different ways, i.e., either a longer or a shorter intron can be excised, resulting in the formation of two pools of mRNAs (2.3 kb and 2.6 kb long mRNA pools). Often, the longer intron is preferred, and therefore the 2.3 kb long mRNA can be referred to as the major splice variant. This form lacks the first AUG codon, from which the synthesis of the VP1 protein is initiated, resulting in a reduced overall level of VP1 protein synthesis. The first AUG codon remaining in the major splice variant is the start codon for the VP3 protein. However, upstream of that codon in the same open reading frame is an ACG sequence (coding threonine) surrounded by an optimal Kozak (translation initiation) context, which contributes to low-level synthesis of the VP2 protein, which is in fact Becerra SP et al., (December 1985). "Direct mapping of adeno-associated virus capsid proteins B and C: a possible ACG initiation codon". Proceedings of the National Academy of Sciences of the United States of America. 82 (23): 7919-23, Cassinotti P et al., (November 1988). "Organization of the adeno-associated virus (AAV) capsid gene: mapping of a minor spliced As described in Muralidhar S et al., (January 1994) "Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons; effects on regulation of synthesis and biological activity". Journal of Virology. 68(1):170-6, and Trempe JP, Carter BJ (September 1988) "Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein". Journal of Virology. 62 (9):3356-63, each of which is incorporated herein by reference, VP3 protein, like VP1, has additional N-terminal residues. A single consensus polyA site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are described in Muzyczka, Current Topics in Reviewed in Microbiology and Immunology, 158:97-129 (1992).

[0059] Each VP1 protein contains a VP1 portion, a VP2 portion, and a VP3 portion. The VP1 portion is the N-terminal portion of the VP1 protein that is unique to the VP1 protein. The VP2 portion is an amino acid sequence present in the VP1 protein that is also found in the N-terminal portion of the VP2 protein. The VP3 portion and the VP3 protein have the same sequence. The VP3 portion is the C-terminal portion of the VP1 protein that is shared with the VP1 protein and the VP2 protein. See Figure 30.

[0060] The VP3 protein can be further divided into individual variable surface regions I-IX (VR-I-IX). Each of the variable surface regions (VR) can comprise or contain a specific amino acid sequence that can confer a unique infection phenotype (e.g., reduced antigenicity, improved transduction, and / or tissue-specific tropism compared to other AAV serotypes) to a particular serotype, either alone or in combination with the specific amino acid sequences of each of the other VRs, as described in DiMatta et al., "Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9," J. Virol., Vol. 86(12):6947-6958, June 2012, the contents of which are incorporated herein by reference.

[0061] AAV has unique features that make it attractive as a vector for delivering foreign DNA to cells, for example in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. In addition, AAV can infect numerous mammalian cells, allowing the possibility of targeting numerous different tissues in vivo. Furthermore, AAV can transduce slowly dividing and nondividing cells, and persist essentially for the life of the cell as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is inserted as cloned DNA into a plasmid, which makes the construction of recombinant genomes feasible. Furthermore, signals directing AAV replication and genome encapsidation are housed within the ITRs of the AAV genome, so that some or all of the internal approximately 4.3 kb of the genome (encoding the replication and structural capsid protein, rep-cap) may be replaced with foreign DNA to generate AAV vectors. The rep and cap proteins may be provided in trans. Another important feature of AAV is that it is an extremely stable and robust virus. It easily withstands the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making cryopreservation of AAV less hazardous. AAV can even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0062] Several studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle. Clark et al., Hum Gene Ther, 8:659-669 (1997); Kessler et al., Proc Nat. Acad. Sci. Sc. USA, 93:14082-14087 (1996); and Xiao et al., J Virol, 70:8098-8108 (1996). See also Chao et al., Mol Ther, 2:619-623 (2000), and Chao et al., Mol Ther, 4:217-222 (2001). Furthermore, because muscle is highly vascularized, recombinant AAV transduction results in the appearance of the transgene product in the systemic circulation following intramuscular injection, as described in Herzog et al., Proc Natl Acad Sci USA, 94:5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94:13921-13926 (1997). Additionally, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the cellular factors necessary for correct antibody glycosylation, folding, and secretion, indicating that muscle is capable of stable expression of secreted protein therapeutics. The recombinant AAV (rAAV) genome of the present invention comprises, consists essentially of, or consists of a nucleic acid molecule encoding a therapeutic protein (e.g., CFTR) and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in the rAAV genome may be from any AAV serotype capable of inducing recombinant virus, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV PHP.B, and AAV rh74. The production of pseudotyped rAAV is disclosed, for example, in International Patent Publication No. 2001083692. Other types of rAAV variants, such as rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0063] AAV vector particles, capsid proteins, and AAV vectors Provided herein are AAV vector particles, AAV vectors, and capsid proteins that have desirable tissue specificity and find use in delivering a variety of therapeutic payloads, including nucleic acids, and proteins useful in the treatment of diseases.

[0064] AAV Capsid Protein The present disclosure provides AAV particles with the characteristics of high gene transfer efficiency and increased tissue tropism.The delivery of AAV vectors currently relies on the use of serotype selection for tissue targeting based on the natural tropism of the virus or by direct injection into target tissue.When systemic delivery is required to achieve maximum therapeutic benefit, serotype selection is the only option available for tissue targeting in combination with tissue-specific promoters.Therefore, many of the AAV vectors currently available are therefore not optimal for gene therapy.

[0065] The present disclosure provides AAV capsid protein sequences that confer high gene transfer efficiency and increased tissue specificity on the AAV capsids that contain them. In embodiments, the AAV capsid sequences provided herein are produced using the AAV capsid production platform shown in FIG.

[0066] In embodiments, the VP1 capsid protein comprises any one of the amino acid sequences listed in Table 1, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids mutated, deleted, or added compared to any one of the amino acid sequences listed in Table 1. In aspects, up to 20 amino acids, up to 30 amino acids, or up to 40 amino acids may be mutated, deleted, or added compared to these sequences. In embodiments, the VP1 capsid protein is encoded by any one of the nucleic acid sequences listed in Table 1, or by a sequence having up to 5, up to 10, up to 30, or up to 60 nucleotide changes relative to any one of the nucleic acid sequences listed in Table 1. [Table 1]

[0067] In embodiments, the AAV VP1 protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1-3, 30-34, 49, or 84, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that differ from SEQ ID NO: 1-3, 30-34, 49, or 84. Polynucleotides encoding these VP1 proteins are also provided. In embodiments, the polynucleotides encoding the VP1 protein comprise, consist essentially of, or consist of the sequence of SEQ ID NO: 15, 18-23, 47, 82, and 98, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes relative to SEQ ID NO: 15, 18-23, 47, 82, and 98.

[0068] In embodiments, the AAV capsid sequence is AAV-110 capsid protein (SEQ ID NO:1), AAV204 capsid protein (SEQ ID NO:2), AAV214 capsid protein (SEQ ID NO:3), or AAV ITB102_45 capsid protein (SEQ ID NO:49). In embodiments, the AAV capsid protein is a variant of the AAV214 capsid protein. In embodiments, the AAV capsid protein is AAV214A (SEQ ID NO:30), AAV-214-AB (SEQ ID NO:84), AAV214e (SEQ ID NO:31), AAV214e8 (SEQ ID NO:32), AAV214e9 (SEQ ID NO:33), or AAV214e10 (SEQ ID NO:34).

[0069] Sequences for exemplary VP2 and VP3 proteins are provided in Tables 2 and 3. Given the VP2 and VP3 sequences, the VP1 portion may be determined by alignment with the complete VP1 protein sequence. [Table 2]

[0070] An exemplary nucleic acid for ITB102_45 is SEQ ID NO: 47. Exemplary nucleic acids for other capsid VP2 portions can be derived from corresponding portions of the VP1 capsid protein nucleic acid. [Table 3]

[0071] The VP3 proteins of AAV214, AAV214e, AAV214e8, AAV214e9, and AAV214e10 have the same amino acid (SEQ ID NO:41) and nucleic acid (SEQ ID NO:24) sequences.

[0072] In embodiments, the AAV VP2 protein comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 35-40, 50, or 85, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that differ from SEQ ID NOs: 35-40, 50, or 85. Polynucleotides encoding these VP2 proteins are also provided. In embodiments, the polynucleotide encoding the VP2 protein comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 47, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes relative to SEQ ID NO: 47.

[0073] In embodiments, the AAV VP3 protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17, 41-46, 51, or 86, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that differ from SEQ ID NO: 17, 41-46, 51, or 86. Polynucleotides encoding these VP3 proteins are also provided. In embodiments, the polynucleotides encoding the protein comprise, consist essentially of, or consist of the sequence of SEQ ID NO: 16, 24-29, 48, and 83, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes relative to SEQ ID NO: 16, 24-29, 48, and 83.

[0074] In embodiments, the AAV capsid protein is a chimeric protein. In embodiments, the VP1, VP2, or VP3 portion of an AAV capsid protein disclosed herein may be replaced with a VP1, VP2, or VP3 portion from a different AAV capsid protein disclosed herein.

[0075] In embodiments, provided herein is an AAV capsid protein that includes a leucine residue at amino acid 129, an asparagine residue at amino acid 586, and a glutamic acid residue at amino acid 723, where the amino acid positions of the AAV capsid protein are numbered relative to the amino acid positions of the amino acid sequence of SEQ ID NO: 2. In some cases, the protein includes the amino acid sequence of SEQ ID NO: 2. In other cases, these amino acids may be introduced into other capsid proteins.

[0076] In embodiments, provided herein is an AAV VP1 capsid protein comprising a VP1 portion, a VP2 portion, and a VP3 portion, wherein the VP1 portion comprises a leucine (L) residue at amino acid 129, the VP2 portion comprises a threonine (T) or asparagine (N) residue at amino acid 157, and a lysine (K) or serine (S) residue at amino acid 162, and the VP3 portion comprises an asparagine (N) residue at amino acid 223, an alanine (A) residue at amino acid 224, a histidine (H) residue at amino acid 272, a threonine (T) residue at amino acid 410, a histidine (H) residue at amino acid 724, and a proline (P) residue at amino acid 734, wherein amino acid positions within the AAV capsid protein are numbered relative to the amino acid positions in the amino acid sequence of SEQ ID NO:3 (i.e., VP1 capsid subunit numbering).

[0077] In embodiments, the VP1 portion further comprises an aspartic acid (D) or an alanine (A) residue at amino acid 24, where the amino acid positions in the AAV capsid protein are numbered relative to the amino acid positions in the amino acid sequence of SEQ ID NO: 3. In embodiments, the VP2 portion further comprises one or more of: (i) a proline (P) residue at amino acid 148, (ii) an arginine (R) residue inserted at amino acid 152, (iii) an arginine (R) residue at amino acid 168, (iv) an isoleucine (I) residue at amino acid 189, and (v) a serine (S) residue at amino acid 200, where the amino acid positions in the AAV capsid protein are numbered relative to the amino acid positions in the amino acid sequence of SEQ ID NO: 3.

[0078] In embodiments, one or more of variable regions I-IX in the disclosed VP3 partial capsid protein (see FIG. 30) may be removed and replaced with alternative regions. Suitable alternatives are identified in the table below. The locations for these as well as the identities of additional alternatives may be identified by alignment with SEQ ID NO:41, as shown in FIG. 30. In embodiments, one or more of the VRs may have an insertion of one, two, or three amino acids. In embodiments, one or more of the VRs may have a deletion of one, two, or three amino acids. [Table 9]

[0079] The present disclosure provides a nucleic acid encoding any one of the AAV capsid proteins disclosed herein. The present disclosure also provides a vector comprising any one of the nucleic acids disclosed herein.

[0080] In an embodiment, the AAV is an AAV9 serotype. Alternative serotypes or modified capsid viruses can be used to optimize neuronal tropism. Alternative vectors include modified AAV9 serotype vectors for higher neuronal tropism than standard AAV9, such as PHP.B, which uses the Cre-lox recombination system to specify neuronal targeting vectors. Alternatively, AAV9 PHP.B has modified amino acid 498 of VP1 from asparagine to lysine to reduce liver tropism. Further variants of AAVrh74 with several amino acids mutated can be used for very broad tissue tropism, including the brain.

[0081] AAV vectors AAV vectors provide not only the nucleic acid that is encapsidated into AAV vector particles that contain the element(s) responsible for controlling the expression of the nucleic acid in a subject, but also ITRs to facilitate encapsidation.In an embodiment, the AAV vector disclosed herein comprises at least one heterologous nucleic acid (HNA) sequence that is effective for treating a disease or disorder when expressed in a subject's cell.In an embodiment, the HNA sequence comprises a transgene.In an embodiment, the AAV vector comprises at least one ITR sequence and at least one transgene.In an embodiment, the transgene encodes a therapeutic protein or a therapeutic RNA.

[0082] In embodiments, the control of transgene expression in a host cell may be regulated by regulatory elements contained within the AAV vector, including a promoter sequence and a polyA site. In embodiments, the AAV vector may also code for a signal peptide. In embodiments, the AAV vector has 5' and 3' inverted terminal repeats (ITRs). The 5'ITR is located upstream of the promoter, which is also upstream of the transgene. In embodiments, the 5' and 3'ITRs have the same sequence. In embodiments, they have different sequences. In embodiments, the AAV vector of the present disclosure may include, in a 5' to 3' orientation, a first (5')ITR, a promoter, a transgene, a polyA site, and a second (3')ITR.

[0083] In embodiments, the AAV vector has a nucleotide sequence as set forth in SEQ ID NO:88 (pA_CF1), SEQ ID NO:89 (pA_CF3), SEQ ID NO:90 (pA_CF5), or SEQ ID NO:91 (pA_CF7). These vectors contain the following components: [Table 10]

[0084] In embodiments, the HNA (e.g., HNA comprising a transgene) is operably linked to a constitutive promoter. The constitutive promoter can be any constitutive promoter known in the art and / or provided herein. In embodiments, the constitutive promoter comprises, consists essentially of, or consists of Rous sarcoma (RSV) LTR promoter (optionally with RSV enhancer), cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, beta actin promoter, phosphoglycerol kinase (PGK) promoter, U6 promoter, H1 promoter, hybrid chicken beta actin promoter, MeCP2 promoter, H1 promoter, U1a promoter, mMeP418 promoter, mMeP426 promoter, minimal MeCP2 promoter, CAG promoter, or EF1 promoter. It is known in the art that the nucleotide sequence of such promoters may be modified to increase or decrease the efficiency of mRNA transcription. See, for example, Gao et al. (2018) Mol. Ther.: Nucleic Acids 12:135-145 (modifying the TATA box of the 7SK, U6, and H1 promoters to abolish RNA polymerase III transcription and stimulate RNA polymerase II-dependent mRNA transcription). In embodiments, the HNA sequence is operably linked to a tissue-specific control promoter, or an inducible promoter. In embodiments, the tissue-specific control promoter is a central nervous system (CNS) cell-specific promoter, a lung-specific promoter, a skin-specific promoter, a muscle-specific promoter, a liver-specific promoter, an eye-specific promoter (e.g., VMD2, or mRho promoter).

[0085] In embodiments, the promoter may comprise, consist essentially of, or consist of a polynucleotide having the sequence of SEQ ID NO:96 (mouse U1 promoter) or SEQ ID NO:97 (H1 promoter). In embodiments, the promoter is a U1a or U1b promoter, an EF1 promoter, or CBA (chicken beta actin). In embodiments, the promoter may comprise, consist essentially of, or consist of any one of the nucleic acid sequences listed in Table 5, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes relative to any one of the nucleic acid sequences listed in Table 5. [Table 5]

[0086] In an embodiment, the HNA sequence is operably linked to additional regulatory elements.The additional regulatory element can be Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE).In an embodiment, the AAV vector can include regulatory components suitable for the growth and culture of the vector in bacterial hosts for vector production purposes.For example, the vector can include genes for antibiotic resistance and associated regulatory elements for controlling protein expression in bacteria as well as maintaining plasmid in bacteria.

[0087] In an embodiment, the HNA sequence is operably linked to a polyA site. The polyadenylation site comprises, consists essentially of, or consists of MeCP2 poly-A site, retinol dehydrogenase 1 (RDH1) poly-A site, bovine growth hormone (BGH) poly-A site, SV40 poly-A site, SPA49 poly-A site, sNRP-TK65 poly-A site, sNRP poly-A site, or TK65 poly-A site. Exemplary SPA49 poly-A sequences are described in Ostedgaard et al., Proc. Nat'l Acad. Sci. USA (Feb. 22, 2005) 102: 2952-2957 (hereby incorporated by reference).

[0088] Heterologous nucleic acid (HNA) The AAV vectors disclosed herein infect and deliver one or more heterologous nucleic acids (HNA) to a target tissue. In embodiments, the HNA sequences are transcribed, and optionally translated, within the cells of the target tissue.

[0089] In some cases, HNA encodes antisense RNA, microRNA, siRNA, or guide RNA (gRNA). CRISPR technology has been used to target genomes of living cells for modification. Cas9 protein is a large enzyme that needs to be efficiently delivered to target tissues and cells to mediate gene repair through the CRISPR system, and current CRISPR / Cas9 gene correction protocols suffer from many shortcomings. Long-term expression of Cas9 can elicit host immune responses. Additional guide RNAs may be delivered via separate vectors due to packaging constraints. In embodiments, HNA encodes Cas9 protein or its equivalent.

[0090] In embodiments, HNA comprises a transgene encoding a protein that may be expressed in a subject's cells to treat a disease or disorder resulting from the reduction or elimination of activity of a native protein. Thus, in embodiments, the transgene is selected from the group consisting of cystic fibrosis transmembrane conductance regulator (CFTR), N-acetylglucosaminidase (NAGLU), N-sulfoglucosamine sulfohydrolase (SGSH), palmitoyl-protein thioesterase 1 (PPT1), survival of motor neuron 1, telomere (SMN1), alkaline phosphatase, associated biomineralization (ALPL, also known as TNALP), glial cell-derived neurotrophin (GLN), and the like. The protein may encode a protein selected from GDNF, glucosylceramidase beta (GBA1), iduronidase alpha-L- (IDUA), cytochrome P450 family 4 subfamily V member 2 (CYP4V2), retinoschisin 1 (RS1), phosphodiesterase 6B (PDE6B), methyl-CpG binding protein 2 (MeCP2), rhodopsin (Rho), or ceroid lipofuscinosis, neuron, 1 (CLN1).

[0091] In an embodiment, the transgene encodes CFTR. In an embodiment, the CFTR comprises a mutated sequence, a codon-optimized sequence, and / or a truncated sequence of CFTR. An exemplary suitable CFTR sequence is disclosed in US Patent Publication No. 20110035819, which is incorporated herein by reference in its entirety. In an embodiment, the CFTR comprises a deletion of amino acids 708-759 (CFTRΔR). See Ostedgaard et al., Proc. Nat'l Acad. Sci. USA (Feb. 22, 2005) 102:2952-2957, which is incorporated herein by reference in its entirety.

[0092] In embodiments, the transgene comprises, consists essentially of, or consists of a nucleic acid having a sequence of SEQ ID NO:4 (codon optimized CFTRΔR) or SEQ ID NO:93 (full length codon optimized CFTR), or a sequence having up to 5, up to 10, or up to 30 nucleotide changes relative to SEQ ID NO:4 or 93. In embodiments, the transgene encodes a protein comprising, consisting essentially of, or consisting of an amino acid having a sequence of SEQ ID NO:95 (CFTRΔR) or SEQ ID NO:94 (full length CFTR), or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids differing from SEQ ID NO:94 or 95.

[0093] In embodiments, the transgene encodes the CLN3 lysosomal / endosomal transmembrane protein, battenin (CLN3) protein, alpha galactosidase A (GLA), or acid alpha glucosidase (GAA).

[0094] In embodiments, the GAA protein is encoded by the nucleotide sequence of SEQ ID NO:5, 6, or 7, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes relative to SEQ ID NO:5, 6, or 7. In embodiments, the GAA protein comprises the amino acid sequence of SEQ ID NO:8, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that differ from SEQ ID NO:8.

[0095] In embodiments, the GLA protein is encoded by the nucleotide sequence of SEQ ID NO:9 or 10, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes relative to SEQ ID NO:9 or 10. In embodiments, the GLA protein comprises the amino acid sequence of SEQ ID NO:11, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that differ from SEQ ID NO:11.

[0096] In embodiments, the CLN3 protein is encoded by the nucleotide sequence of SEQ ID NO: 12 or 13, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes relative to SEQ ID NO: 12 or 13. In embodiments, the CLN3 protein comprises the amino acid sequence of SEQ ID NO: 14, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that differ from SEQ ID NO: 14.

[0097] In embodiments, the transgene comprises any one of the nucleic acid sequences listed in Table 4, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes relative to any one of the DNA sequences in Table 4. In embodiments, the transgene encodes any one of the amino acid sequences listed in Table 4, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that differ from any one of the amino acid sequences listed in Table 4. [Table 4-1] [Table 4-2]

[0098] In embodiments, the transgene comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 99-133, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes relative to any one of SEQ ID NOs: 99-133. In embodiments, the transgene encodes an amino acid sequence set forth in any one of SEQ ID NOs: 134-151, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that differ from any one of the amino acid sequences of SEQ ID NOs: 134-151.

[0099] In embodiments, the heterologous nucleic acid encodes a reporter protein, for example a fluorescent protein.

[0100] Methods for Producing AAV Viral Vectors Various approaches may be used to produce AAV viral vector.In an embodiment, packaging is achieved by using helper virus or helper plasmid and cell line.Helper virus or helper plasmid contains elements and sequences that facilitate viral vector production.In another aspect, helper plasmid is stably integrated into the genome of packaging cell line, so that packaging cell line does not need additional transfection with helper plasmid.

[0101] In an embodiment, the cell is a packaging or helper cell line. In an embodiment, the helper cell line is a eukaryotic cell, for example, a HEK293 cell or a 293T cell. In an embodiment, the helper cell is a yeast cell or an insect cell.

[0102] In an embodiment, the cell comprises a nucleic acid encoding a tetracycline activator protein and a promoter that regulates the expression of the tetracycline activator protein. In an embodiment, the promoter that regulates the expression of the tetracycline activator protein is a constitutive promoter. In an embodiment, the promoter is a phosphoglycerate kinase promoter (PGK) or a CMV promoter.

[0103] The helper plasmid may, for example, contain at least one viral helper DNA sequence derived from a replication-incompetent viral genome to encode in trans all virion proteins required for packaging replication-incompetent AAV and to produce virion proteins capable of packaging replication-incompetent AAV at high titers without producing replication-competent AAV.

[0104] Helper plasmids for packaging AAV are known in the art, see, for example, US Patent Publication No. 2004 / 0235174 A1 (hereby incorporated by reference).As described therein, AAV helper plasmids may contain, as helper virus DNA sequences, non-limiting examples of Ad5 genes E2A, E4 and VA, controlled by their respective original promoters or by heterologous promoters.AAV helper plasmids may additionally contain expression cassettes for expressing marker proteins, such as fluorescent proteins, to allow simple detection of transfection of desired target cells.

[0105] The present disclosure provides a method for producing AAV particles, comprising transfecting a packaging cell line with any one of the AAV helper plasmids disclosed herein and any one of the AAV vectors disclosed herein.In an embodiment, the AAV helper plasmid and the AAV vector are co-transfected into the packaging cell line.In an embodiment, the cell line is a mammalian cell line, for example, a human embryonic kidney (HEK) 293 cell line.The present disclosure provides a cell comprising any one of the AAV vectors and / or AAV particles disclosed herein.

[0106] Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions comprising any one of the AAV vectors, AAV capsids, and / or AAV particles described herein. Typically, the AAV particles are administered for therapeutic purposes.

[0107] As described herein, pharmaceutical compositions may be formulated by any method known or developed in the art of pharmacology, including, but not limited to, contacting the active ingredient (e.g., viral particle or recombinant vector) with excipients or other accessory ingredients and dividing the product into dosage units or packaging. The viral particles of the present disclosure may be formulated with desired characteristics, such as increased stability, increased cell transfection, sustained or delayed release, biodistribution or targeting, regulated or enhanced translation of the encoded protein in vivo, and release profile of the encoded protein in vivo.

[0108] Thus, the pharmaceutical composition may further comprise saline, lipidoid, liposome, lipid nanoparticle, polymer, lipoplex, core-shell nanoparticle, peptide, protein, cell transfected with a viral vector (e.g., for implantation into a subject), nanoparticle mimic, or combinations thereof. In an embodiment, the pharmaceutical composition is formulated as a nanoparticle. In an embodiment, the nanoparticle is a self-assembled nucleic acid nanoparticle.

[0109] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as single unit doses, and / or as multiple single unit doses. The amount of active ingredient is generally equal to the dosage of active ingredient to be administered to a subject, and / or a convenient fraction of such a dosage (e.g., 1 / 2 or 1 / 3 of such a dosage, etc.). The formulations of the present invention may include one or more excipients, each in an amount that together increases the stability of the viral vector, increases cell transfection or transduction by the viral vector, increases the expression of the protein encoded by the viral vector, and / or modifies the release profile of the protein encoded by the viral vector. In an embodiment, the pharmaceutical composition includes an excipient. Non-limiting examples of excipients include solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, or combinations thereof.

[0110] In an embodiment, the pharmaceutical composition comprises a cryoprotectant. The term "cryoprotectant" refers to an agent capable of reducing or eliminating damage to a substance during freezing. Non-limiting examples of cryoprotectants include sucrose, trehalose, lactose, glycerol, dextrose, raffinose, and / or mannitol.

[0111] Treatment method The present disclosure provides a method for preventing or treating a disorder comprising, consisting essentially of, or consisting of administering to a subject a therapeutically effective amount of any one of the pharmaceutical compositions disclosed herein.

[0112] In embodiments, the disorder is a CNS disorder, a skin disorder, a lung disorder, a muscle disorder, a liver disorder, or an eye disease (or a retinal disease). In embodiments, the disorder is cystic fibrosis.

[0113] In embodiments, the disorder is hypophosphatasia, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), recessive dystrophic epidermolysis bullosa (RDEB), lysosomal storage disorders (including Duchenne muscular dystrophy, and Becker muscular dystrophy), juvenile Batten disease, infantile Batten disease, autosomal dominant disorders, muscular dystrophies, crystalline dystrophy of Vietti, retinoschisis (e.g., degenerative, hereditary disorders, , traction, exudative), hemophilia A, hemophilia B, multiple sclerosis, diabetes, Fabry disease, Pompe disease, neuronal ceroid lipofuscinosis 1 (CLN1), CLN3 disease (or juvenile neuronal ceroid lipofuscinosis), Gaucher disease, cancer, arthritis, muscle wasting, heart disease, intimal hyperplasia, Rett syndrome, epilepsy, Huntington's disease, Parkinson's disease, Alzheimer's disease, autoimmune diseases, cystic fibrosis, thalassemia, Hurler syndrome (MPS) IH), Sly syndrome, Scheie syndrome, Hurler-Scheie syndrome, Hunter syndrome, Sanfilippo syndrome A (mucopolysaccharidosis IIIA or MPSIIIA), Sanfilippo syndrome B (mucopolysaccharidosis IIIB or MPSIIIB), Sanfilippo syndrome C, Sanfilippo syndrome D, Morquio syndrome, Maroteaux-Lamy syndrome, Krabbe disease, phenylketonuria, spinal cerebral ataxia, LDL receptor deficiency, hyperammonemia, anemia, arthritis, or adenosine deaminase deficiency.

[0114] In addition to the specific transgenes disclosed herein, any known active enzyme sequence may be used as a transgene to deliver functional enzyme activity.

[0115] One of the problems for the treatment of cystic fibrosis is the size restriction in packaging CFTR gene in viral particles, and the difficulty of delivering viral particles to lung cells.The AAV particles of the present disclosure solve these problems by providing the CFTR transgene construct that is efficiently packaged and has better lung tropism.Therefore, in embodiments, the present disclosure provides compositions and methods for treating cystic fibrosis.

[0116] In an embodiment, the disorder is CLN3 disease.CLN3 disease or juvenile neuronal ceroid lipofuscinosis is a lysosomal storage disease caused by autosomal recessive genetic mutations in CLN3 gene.CLN3 disease is a progressive neurodegenerative disease that affects the central nervous system (CNS) and leads to behavioral problems, vision loss, and other cognitive disorders.

[0117] In an embodiment, the disorder is Fabry disease. Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency in the activity of alpha-galactosidase A (GLA) that leads to the accumulation of glycolipid products, globotriaosylceramide (Gb3), and lyso-Gb3 in lysosomes. Disease presentation is highly heterogeneous, but typically includes frequent attacks of peripheral neurotrophic pain, angiokeratoma, decreased sweating, corneal dystrophy, and gastrointestinal complications. As the disease progresses, patients suffer from cardiomyopathy, renal insufficiency, and cerebrovascular disease, all of which are major contributors to the shortened life span of Fabry disease patients. Men are the most severely affected patient population with mutations in the GLA gene, although it is becoming increasingly clear that female patients also exhibit symptoms but are often misdiagnosed. Enzyme replacement therapy (ERT) is currently the only FDA-approved therapy for treating Fabry disease, and requires biweekly injections of relatively large amounts of recombinant protein. Although ERT reduces the accumulation of Gb3 in the heart, kidneys, and vasculature, it is unable to completely treat all symptoms of Fabry disease, primarily due to its inability to efficiently enter the CNS. Gene therapy strategies are being investigated, and many have shown great promise in correcting glycolipid accumulation, but many are unable to efficiently enter the CNS and suffer from the immune response often seen during GLA supplementation.

[0118] In embodiments, the AAV viral vectors disclosed herein are used to treat Fabry disease in patients who do not respond to ERT or when ERT fails to address all symptoms. In embodiments, the AAV viral vectors disclosed herein are used to treat Fabry disease in patients who have already been administered ERT.

[0119] In an embodiment, the disorder is Pompe disease. Pompe disease is a lysosomal storage disorder caused by a deficiency in acid alpha glucosidase (GAA) activity, resulting in the accumulation of glycogen in lysosomes. The disease manifests itself primarily as a form of muscular dystrophy affecting both smooth and striated muscle tissues, as well as the central nervous system (CNS), with early death. Enzyme replacement therapy (ERT) is currently the only FDA-approved therapy for treating Pompe disease, and requires biweekly injections of relatively large amounts of recombinant protein. Although ERT significantly reduces the mortality rate of infantile Pompe disease patients, who typically die by age 2 without treatment, it is unable to completely ameliorate all symptoms of Pompe disease, primarily due to its inability to efficiently enter the CNS and resulting immune response against the GAA protein. Gene therapy strategies are being investigated, many of which have shown great promise in correcting glycogen accumulation and other symptoms of Pompe disease. Most suffer from the severe immune response seen during GAA replacement. Previous studies have demonstrated that liver-specific expression can render animals tolerant to the GAA protein and significantly reduce the humoral response.

[0120] In embodiments, the AAV viral vectors disclosed herein are used to treat Pompe disease in patients who have already been administered ERT, e.g., patients who do not respond to ERT, or when ERT fails to address all of the patient's symptoms.

[0121] In embodiments, the cancer is a solid cancer, e.g., bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, lip cancer, oral cavity cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung tumor, or thyroid.

[0122] In an embodiment, the disorder is an eye disease. The eye is an immune privileged tissue. Only a very small amount of virus is required for therapeutic benefit. In an embodiment, the eye disease affects photoreceptor and RPE cells. In some embodiments, the ocular disease is retinitis pigmentosa (e.g., autosomal recessive (SPATA7 gene, LRAT gene, TULP1 gene), autosomal dominant (AIPL1 gene), and X-linked (RPGR gene)), ocular disorders associated with mutations in the bestrophin-1 (BEST-1) gene (e.g., vitelliform macular dystrophy, age-related macular degeneration, autosomal dominant vitreoretinochoroidopathy, glaucoma, cataract), Leber congenital amaurosis (LCA; aryl-hydrocarbon interacting protein-like 1 (AIPL1) gene, cone-rod dystrophy (CRD; ABCA4 gene), Stargardt disease (ABCA4 gene), congenital choroideremia (CHM gene), Usher syndrome (MYO7A gene, CDH23 gene; USH2A gene; including, consisting essentially of, or consisting of, CLRN1 gene), retinoschisis (RS1 gene), crystalline dystrophy of Vietti (CYP4V2 gene) or color vision deficiency (CNGA3 gene, CNGB3 gene, GNAT2 gene, PDE6C gene, or PDE6H gene).

[0123] In embodiments, the subject is a mammal, e.g., a human. In certain aspects, the human is a human infant, e.g., under 3 years of age, under 2 years of age, or under 1 year of age.

[0124] The treatment and prevention methods disclosed herein may be combined with suitable diagnostic techniques to identify and select patients for treatment or prevention.For example, the method of treating or preventing a disorder disclosed herein, such as cystic fibrosis, may further comprise carrying out a genetic test to identify the genetic mutation or deletion associated with the disorder in the subject.In an embodiment, the method of treating or preventing a disorder, such as cystic fibrosis, comprises administering to a subject previously identified as carrying a mutation associated with the disorder or at high risk of developing the disorder (e.g., based on genetic factors).

[0125] The present disclosure provides a method for increasing the level of a protein in a host cell, comprising contacting the host cell with any one of the AAV particles disclosed herein, the AAV particle comprising any one of the AAV vectors disclosed herein comprising an HNA sequence encoding the protein. In an embodiment, the protein is a therapeutic protein. In an embodiment, the host cell is in vitro, in vivo, or ex vivo. In an embodiment, the host cell is derived from a subject. In an embodiment, the subject suffers from a disorder that results in a decrease in the level and / or functionality of the protein compared to the level and / or functionality of the protein in a normal subject.

[0126] In embodiments, the level of protein is about 1×10 -7 ng, approximately 3 × 10 -7 ng, approximately 5 × 10 -7 ng, approximately 7 × 10 -7 ng, approximately 9 × 10 -7 ng, approximately 1 × 10 -6 ng, approximately 2 × 10 -6 ng, approximately 3 × 10 -6 ng, approximately 4 × 10 -6 ng, approximately 6 × 10 -6 ng, approximately 7 × 10 -6 ng, approximately 8 × 10 -6 ng, approximately 9 × 10 -6 ng, approximately 10 × 10 -6 ng, approximately 12 × 10 -6 ng, approximately 14 × 10 -6 ng, approximately 16 × 10 -6 ng, approximately 18 × 10 -6 ng, approximately 20 × 10 -6 ng, approximately 25 × 10 -6 ng, approximately 30 × 10 -6 ng, approximately 35 × 10 -6 ng, approximately 40 × 10 -6 ng, approximately 45 × 10 -6 ng, approximately 50 × 10 -6 ng, approximately 55 × 10 -6 ng, approximately 60 × 10 -6 ng, approximately 65 × 10 -6 ng, approximately 70 × 10 -6 ng, approximately 75 × 10-6 ng, approximately 80 × 10 -6 ng, approximately 85 × 10 -6 ng, approximately 90 × 10 -6 ng, approximately 95 × 10 -6 ng, approximately 10 × 10 -5 ng, approximately 20 × 10 -5 ng, approximately 30 × 10 -5 ng, approximately 40 × 10 -5 ng, approximately 50 × 10 -5 ng, approximately 60 × 10 -5 ng, approximately 70 × 10 -5 ng, approximately 80 × 10 -5 ng, or approximately 90x 10-5 increases to ng levels.

[0127] The present disclosure provides a method of introducing a gene of interest into a cell in a subject, comprising contacting the cell with an effective amount of any one of the AAV viral vector particles disclosed herein, the particle containing any one of the AAV vectors disclosed herein that includes the gene of interest.

[0128] Dosage and Administration Methods for determining the most effective means and dosage administration are known to those skilled in the art and will vary depending on the composition used for treatment, the purpose of the treatment, and the subject being treated. Single or multiple administrations can be carried out with the dosage level and pattern selected by the treating physician. It is noted that the dosage may be influenced by the route of administration. Suitable dosage forms and methods of administration of drugs are known in the art. A non-limiting example of such a suitable dosage is 10 mg / dose per administration. 9 From as few as 10 17 The vector genome can be as many as 1 or more.

[0129] In embodiments of the methods described herein, the number of viral particles (e.g., AAV) administered to a subject is about 10 9 ~about 10 17 Specifically, in some embodiments, the range is about 10 10 ~about 10 12, about 10 11 ~about 10 13 , about 10 11 ~about 10 12 , about 10 11 ~about 10 14 , about 5×l0 11 ~Approx. 5×10 12 , or about 10 12 ~about 10 13 For administration to the human eye, approximately 1 x 10 viral particles are administered to the subject. 10 A total dose of 5×10 vg / eye may be used, and for mouse eyes, 9 A total dose of vg / eye may be used. Non-invasive in vivo imaging techniques can be used to monitor efficacy / safety in animals, including, but not limited to, scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), multiphoton microscopy, and fluorescein angiography.

[0130] In embodiments, the AAV particles repair a genetic defect in a subject. In embodiments, the ratio of repaired target polynucleotides or polypeptides to unrepaired target polynucleotides or polypeptides in a successfully treated cell, tissue, organ, or subject is at least about 1.5:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 20:1, about 50:1, about 100:1, about 1000:1, about 10,000:1, or about 100,000:1, or about 1,000,000:1. The amount or proportion of repaired target polynucleotide or polypeptide can be determined by any method known in the art, including, but not limited to, Western blot, Northern blot, Southern blot, PCR, sequencing, mass spectrometry, flow cytometry, immunohistochemistry, immunofluorescence, fluorescent in situ hybridization, next generation sequencing, immunoblot, and ELISA.

[0131] In embodiments, the viral particles are introduced into the subject intravenously, intrathecally, intracerebrally, intraventricularly, intranasally, intratracheally, intraaurally, intraocularly or periocularly, orally, rectally, transmucosally, by inhalation, transdermal, parenterally, subcutaneously, intradermal, intramuscularly, intrapleurally, topically, intralymphatically, intravesically, and such introduction may also be intraarterially, intracardially, subventricularly, epidurally, intracerebrally, intraventricularly, subretinally, intravitreally, intraarticularly, intraperitoneally, intrauterinely, or any combination thereof. In embodiments, the viral particles are delivered to the desired target tissue, such as, for example, the lung, eye, or CNS, as non-limiting examples. In embodiments, the delivery of the viral particles is systemic. The intravesical administration route involves administering the drug directly into the cerebrospinal fluid of the ventricles. This can be done by direct injection into the cisterna magna or via a tube that is permanently located.

[0132] To treat eye diseases (or eye disorders) intraocularly, there are several administration methods known to those skilled in the art, including but not limited to lacrimal gland (LG) administration, topical eye drops, intrastromal administration to the cornea, intracameral administration (anterior chamber of the eye), intravitreal administration, subretinal administration, systemic administration, or combinations thereof. 80% of genetic eye disorders occur within the photoreceptors. Intravitreal delivery of small amounts of gene therapy can be performed in an outpatient clinic.

[0133] Administration of the AAV vector, AAV particle, or composition of the present disclosure can be performed in a single dose, continuously, or intermittently throughout the course of treatment. In embodiments, the AAV vector, AAV particle, or composition of the present disclosure is administered parenterally by injection, infusion, or implantation.

[0134] In embodiments, the AAV particles of the present disclosure exhibit enhanced tropism to the brain and cervical spine. In embodiments, the viral particles of the present disclosure can cross the blood-brain barrier (BBB). In embodiments, the AAV particles of the present disclosure exhibit high retinal tropism by subretinal and intravitreal injection. In embodiments, the AAV particles of the present disclosure target multiple ocular cell types, such as, for example, cones, rods, and retinal pigment epithelium (RPE). In embodiments, the AAV particles of the present disclosure evade neutralizing antibodies against native serotypes, thus allowing potential re-administration. In further aspects, the AAV particles and compositions of the present disclosure may be administered in combination with other known treatments for the disorder being treated.

[0135] kit The agents, vectors, or compositions described herein may, in embodiments, be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic, or research applications. In embodiments, the kits of the present disclosure include any one of the modified AAV capsid proteins, AAV vectors, AAV particles, host cells, isolated tissues, compositions, or pharmaceutical compositions as described herein.

[0136] In embodiments, the kit further includes instructions for use. Specifically, such kits may include one or more of the agents described herein with instructions describing the intended use and proper use of these agents. By way of example, in embodiments, the kit may include instructions for mixing one or more components of the kit and / or for isolating and mixing samples and applying to a subject. In embodiments, the agents in the kit are in pharmaceutical formulations and dosages appropriate for a particular use and method of administering the agents. Kits for research purposes may contain appropriate concentrations or amounts of components for carrying out various experiments.

[0137] The kit may be designed to facilitate the use of the methods described herein and may take many forms. Each of the compositions of the kit may be provided in liquid form (e.g., solution) or solid form (e.g., dry powder), as applicable. In certain cases, some of the compositions may be configurable or otherwise processable (e.g., active form), for example, by the addition of appropriate solvents or other species (e.g., water or cell culture medium), which may or may not be provided with the kit. In embodiments, the compositions may be provided in a preservation solution (e.g., a cryopreservation solution). Non-limiting examples of preservation solutions include DMSO, paraformaldehyde, and CryoStor® (Stem Cell Technologies, Vancouver, Canada). In embodiments, the preservation solution contains an amount of a metalloprotease inhibitor.

[0138] In an embodiment, the kit contains any one or more of the components described herein in one or more containers. Thus, in an embodiment, the kit may include a container that contains the agent described herein. The agent may be in liquid, gel, or solid (powder) form. The agent may be sterilely prepared, packaged in a syringe, and shipped refrigerated. Alternatively, they may be housed in a vial or other container for storage. A second container may have another agent that is sterilely prepared. Alternatively, the kit may contain an active agent that is premixed and shipped in a syringe, vial, tube, or other container. The kit may have one or more or all of the components required to administer the agent to a subject, such as a syringe, topical application device, or IV needle tube and bag.

[0139] While the invention has been described in conjunction with the above embodiments, it should be understood that the foregoing description and examples are intended to illustrate, and not to limit, the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0140] Additionally, when features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also thereby described in terms of any individual members or subgroups of members of the Markush group. EXAMPLES

[0141] Working Example [Example 1] [Capsid production platform] Some AAV capsid sequences provided herein (e.g., AAV204, AAV110) are generated using the AAV capsid generation platform shown in Figure 1. Briefly, the platform includes a DNase I fragmentation step, and an assembly and amplification step, ultimately resulting in the formation of a chimeric capsid library. Other AAV capsid sequences provided herein (e.g., AAV214) are generated by rational design.

[0142] The capsid proteins generated using these methodologies were analyzed by aligning them with the amino acid sequences of known AAV capsid proteins. A sequence alignment of the VP1 protein sequences from AAV204 (SEQ ID NO:2) and AAV6 (SEQ ID NO:63) is shown in Figure 20. A sequence alignment of the VP1 amino acid sequences of AAV214, AAV214A, AAV214e, AAV214e8, AAV214e9, AAV214e10, AAV214AB, and AAV ITB102_45 is provided in Figure 21. A sequence alignment of the VP2 amino acid sequences of AAV214, AAV214A, AAV214e, AAV214e8, AAV214e9, AAV214e10, AAV214AB, and AAV ITB102_45 is provided in Figure 22. A sequence alignment of the VP1 amino acid sequences of AAV214, AAV214A, AAV214e, AAV214e8, AAV214e9, AAV214e10, AAV214AB, and AAV ITB102_45 is provided in Figure 23.

[0143] Viral vectors can be produced using standard triple transfection methods known in the art. Briefly, three separate plasmids expressing viral capsid protein, helper protein (e.g., essential viral Rep and Cap proteins), and transgene of interest, respectively, are transfected into adherent or suspension 293 cells, and viral particles are then harvested using ultracentrifugation or chromatography, followed by diafiltration / ultrafiltration and terminal sterile filtration. See, e.g., Guo et al., Mol. Ther. Methods Clin. Dev., Vol. 13, pp. 40-46 at 44 (Nov. 2018), Wang et al., Human Gene Ther. Methods, Vol. 25, pp. 261-68 at 262, and Gao et al., Human Gene Ther. Methods, Vol. 11, pp. 2079-91 (each of which is incorporated by reference herein in its entirety and for all purposes).

[0144] [Example 2] [Characterization of AAV214 and AAV204 viral vectors] The transduction efficiency of AAV214 viral vectors or AAV204 vectors in different target tissues was assessed as described below.

[0145] The transduction efficiency of AAV214 viral vectors containing EGFP transgene (AAV214-GFP) and AAV9 viral vectors containing EGFP transgene (AAV9-GFP) was evaluated in vitro. HEK293 cells were seeded in 96-well plates at 50,000 cells per well. Cells were transduced with AAV214-GFP or AAV9-GFP at an MOI of 5E+5. Images taken 45 hours after transduction revealed that the transduction efficiency of AAV214-GFP was higher in HEK293 cells (Figure 2A). Please note that as used herein, unless otherwise specified, "GFP" refers to EGFP (see, for example, Zhang et al. (1996) Biochem. Biophys. Res. Commc'n. 227(3):707-11).

[0146] To test transduction efficiency in vivo, 10-week-old C57BL / 6 mice were administered 2E+11vg of AAV214-GFP or AAV9-GFP in 200μL of TMN200 (200mM Tris-HCl, 1mM MgCl2, 200mM NaCl, and 0.001% Pluronic F68) by intravenous (IV) injection. After 13 days, mice were euthanized, tissue samples of internal organs (brain, spinal cord (cervical and lumbar), sciatic nerve, eye, heart, kidney, liver, lung, testis, spleen, and muscle) were collected, and total DNA was isolated and analyzed using an absolute qPCR approach for GFP gene copy number estimation. The resulting AAV biodistribution data was plotted using Prism software (GraphPad Software) for statistical analysis. Unpaired t-tests performed on log-transformed data showed no statistically significant differences between transduction efficiencies of AAV9-GFP and AAV214-GFP in most tissues tested (p<0.05). However, in the case of sciatic nerve and muscle, the mean number of detected viral DNA copies per microgram of total DNA isolated from animals administered AAV214-GFP was higher and statistically significantly different from animals administered AAV9-GFP (sciatic nerve: 4.1-fold, p=0.0228; muscle: 3-fold, p=0.0125) (Figure 2B).

[0147] The same experiment was repeated by splitting the brain samples into two halves. One half was used for total DNA isolation followed by biodistribution analysis using absolute qPCR. The other half was used for total RNA isolation, DNase treatment, conversion to cDNA, and qPCR analysis to quantify EGFP gene expression levels. The resulting AAV biodistribution and transgene expression data were plotted using Prism software for statistical analysis. Unpaired t-tests performed on log-transformed data showed no statistically significant differences between AAV9-GFP and AAV214-GFP transduction efficiencies (p=0.7668) or expression levels (p=0.0709) in brain tissue (Figure 2C).

[0148] Wild-type C57BL / 6J mice were administered a set of AAV viral vectors, including AAV204-GFP, AAV110-GFP, and AAV214-GFP, by both subretinal (right eye) and intravitreal (left eye) injections. 1 μL of 5E+12vg / mL (5E+9vg / eye) of AAV vectors was injected with both administration methods, and animals were imaged 10 days later with an HRA2 Spectralis Scanning Laser Ophthalmoscope (Heidelberg Engineering, Carlsbad, CA, USA). Images in which cataracts prevented adequate observation were omitted from analysis. Funduscopy imaging revealed that all tested viruses had the ability to transfect retinal cells when administered into the subretinal space. However, only AAV204 and AAV110 showed enhanced transduction of retinal cells mediated by intravitreal delivery (Figure 3A). Immunohistochemical analysis of eyes from mice administered AAV204-GFP intravitreally demonstrated GFP expression in various types of retinal cells, including photoreceptors, RPE, Müller glial cells, retinal ganglion cells, and bipolar cells (Figure 3B).

[0149] AAV204-GFP and AAV9-GFP were each administered by intrathecal injection (1E+13vg) and / or intravitreal injection (1.5E+12vg) to 2.5-3 year old cynomolgus monkeys (Macaca fascicularis) weighing approximately 2 kg. After 4 weeks, animals were euthanized and GFP expression was assessed by RT-qPCR. Data analysis showed that AAV204-GFP-mediated delivery resulted in enhanced GFP expression in most of the tissues assayed, including specific areas of the brain and spinal cord. See Figure 4. Cynomolgus monkey eyes administered intravitreally (1.5E+12vg of AAV204-GFP per eye) were evaluated by scanning laser ophthalmoscopy (SLO), sectioned, and analyzed for GFP, rhodopsin, and genomic DNA using conventional immunochemical staining methods. As shown in Figure 5A and Figure 5B, administration of AAV204-GFP resulted in significant transduction of the vector in the peripheral retina and foveal regions of the eye. Enhanced expression of GFP delivered by AAV204 was seen in retinal cells including photoreceptors, RPE, bipolar cells, and ganglion cells (Figure 5B). Significant numbers of rods and cones were transduced within the macula (Figure 5C).

[0150] The AAV204 vector can also be combined with an RPE-specific promoter to specifically express proteins. Figures 5D-5F show expression of GFP from AAV204 driven by the VMD2 (vitelliform macular degeneration-2) promoter (SEQ ID NO: 159). 2.5 x 10 12 Viral genome (vg) vectors were administered intravitreally and expression was monitored on days 14 and 28 (sacrifice). Scanning laser ophthalmoscopy (SLO) imaging was performed on days 14 (FIG. 5D) and 28 (FIG. 5E). FIG. 5F shows GFP expression in the periphery and nucleus (DAPI) on day 28.

[0151] AAV204-GFP was also evaluated in non-human primate explant cultures. Cynomolgus monkey retinas were isolated from the eyes within 1 hour after the animals were humanely euthanized. Retinas were dissected into approximately 5 × 5 mm sections and cultured in transwell insert culture dishes. One day after isolation, explants were transduced with AAV204-GFP in culture medium and incubated for 1 week post-transduction. Explants were fixed, embedded, and sectioned for standard immunohistochemistry. Sections were stained for GFP (green) and rhodopsin (red) and imaged using a fluorescent microscope. Sections showed significant GFP expression in the photoreceptor layer following AAV204-GFP transduction (Figure 6).

[0152] The immunogenicity of the AAV204 vector was evaluated using a neutralizing antibody assay (Figure 7). Either the AAV9-Luc virus, consisting of the AAV9 capsid and a firefly luciferase expression cassette, or the AAV204-Luc virus, consisting of the AAV204 capsid and a firefly luciferase expression cassette, was incubated with various dilutions of serum from AAV9-treated human subjects (60 days after treatment) at an MOI of 25,000. After incubation, the virus / serum mixture was transferred to wells containing 20,000 Lec2 cells. The serum-treated cells were incubated for 24 hours, and then the emitted luminescence was measured and compared to control values ​​from cells transduced with untreated virus at the same MOI. The results showed that the AAV204 vector particles had reduced immunogenicity compared to the AAV9 vector particles (see Figure 8).

[0153] [Example 3] [Amelioration of defects caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene using a CFTR transgene delivered by an AAV204 viral vector] AAV204 vector particles containing nucleic acid encoding a codon-optimized CFTR transgene (i.e., a gene for CFTR containing a nucleic acid sequence set forth in SEQ ID NO:4 and encoding a protein lacking amino acids 708-759 of full-length CFTR) were prepared using the pA-CF3 plasmid (SEQ ID NO:89), which has, from 5' to 3', the 5' ITR, the mouse U1a promoter (SEQ ID NO:96), the CFTR transgene, a synthetic polyA sequence (49 bp), and the 3' ITR. The resulting particles were used to deliver the CFTR transgene into either cells or mice, as described below.

[0154] In vitro assay: AAV204 viral vector containing the CFTR transgene was used to transduce Lec2 cells. A FLIPR assay was used to measure the functionality of the CFTR transgene delivered by the AAV204-CFTR viral vector. The results show that stimulation with forskolin, a known CFTR chloride channel opener, restored human CFTR (hCFTR) ion channel function when cells were transduced with the AAV204-CFTR viral vector compared to cells transfected with a control AAV204 viral vector lacking the transgene. Furthermore, chloride-specific current signals were increased 3.5-fold compared to baseline in cells transduced with the AAV204-CFTR viral vector compared to the control. See Figure 10A, left panel. Preincubation with a selective inhibitor of CFTR, CFTRinh-172 (4-[[4-oxo-2-thioxo-3-[3 trifluoromethyl)phenyl]-5-thiazolidinylidene]methyl]benzoic acid) (Tcris Bioscience) (available at http: / / tocris.com / products / cftrinh-172_3430), prevents forskolin-induced changes in membrane potential in the presence of CFTRΔR. See FIG. 10B. These results demonstrate the functionality of the CFTRΔR expression cassette.

[0155] A membrane potential assay was performed to assess whether the functionality of the CFTR expression cassette was dependent on the amount of AAV204-CFTR virus used for transfection. See Figure 10B. The results show that the change in membrane potential in the presence of CFTR was indeed dependent on the dose of viral particles delivering the transgene. We also expressed full-length CFTR using AAV204 and obtained increased fluorescence in response to forskolin (Figure 10C). We also confirmed by Western blot that expression from AAV204 resulted in fully processed full-length CFTR and CFTR (data not shown). These data confirm that AAV204 delivery of either protein restores chloride channel function in vitro.

[0156] In vivo assay using mouse model: AAV204 viral vector containing luciferase transgene was administered intratracheally to mice. Bioluminescence imaging (BLI) was used to evaluate the ability of AAV204 viral vector to transduce lung cells reflected by luciferase expression compared to AAV6. The top panel of Figure 9 shows that luciferase expression mediated by AAV204 viral vector is about 3.5-fold higher compared to AAV6 viral vector. The bottom panel of Figure 9 illustrates the expression shown by ex vivo BLI in the left and right lungs (little or no expression in the liver or kidney). These results demonstrate that AAV204 viral vector has the ability to promote enhanced expression of reporter transgene in specific tissues of mice.

[0157] The efficacy of AAV204 vector particles containing the CFTRΔR transgene was tested in a mouse model of cystic fibrosis, termed "F508del." These mice carry a mutant CFTR gene that contains a deletion of the single amino acid F508, which is the most common CFTR mutation in humans and affects approximately 90% of CF patients (see, e.g., Park et al. (see, e.g., et al., PLoS One (Feb. 10, 2016) 11(2): e0149131). AAV204 vector particles containing the CFTRΔR transgene or the luciferase transgene were administered intranasally to wild-type and F508del mice. Nasal potential difference (NPD) was measured to determine functionality of the CFTRΔR transgene. As shown in FIG. 12A, mice administered AAV204-CFTR vector particles showed modified forskolin-stimulated currents compared to mice administered a control vector containing the luciferase transgene (AAV204-Luc).

[0158] Assay using human patient cells: The ability of AAV204 vector particles containing CFTRΔR transgene to mediate the delivery of hCFTR to human airway cells isolated from patients suffering from cystic fibrosis and to correct chloride transport in these cells was evaluated. When AAV204 vector particles containing GFP transgene were applied to the apical and basolateral compartments, AAV204 transduced human nasal epithelial and bronchial epithelial (HNE and HBE) cells isolated from cystic fibrosis patients and maintained in air-liquid interface culture. See Figure 11A. In these cells, CFTRΔR protein was membrane localized. See the left panel of Figure 11B. The right panel of Figure 11B shows a Western blot illustrating membrane localization.

[0159] The functionality of AAV204 vector particles containing the CFTRΔR transgene was evaluated as described below. The results show that CFTR currents were restored in explant cultures of human nasal epithelial cells derived from cystic fibrosis patients after transduction of AAV204 vectors containing the CFTRΔR transgene. See FIG. 11C. We also tested whether AAV particles could restore CFTR function in nasal and bronchial cells isolated from human cystic fibrosis patients by measuring changes in transmembrane conductance using Ussing chambers, known to those skilled in the art, to measure the movement of ions between the surfaces of polarized epithelia. Briefly, in Ussing chambers, the apical and basolateral surfaces of the epithelium face two separate chambers containing symmetrical salt solutions. Ion transport across the epithelium generates a potential difference between the two chambers. Diffusion forces that would otherwise create a potential difference would generate a short-circuit current (I ) across the epithelium. sc ), which actively counteracts this change in current (ΔI sc ) and allows the movement of ions by active transport after stimulation, as measured by calculation of the cystic fibrosis transmembrane conductance. See, e.g., Li et al., J. Cystic Fibrosis (July 2004) 3:123-126; Park et al., PLoS One (Feb. 10, 2016) 11(2):e0149131. As shown in Figure 12B, when CFTRΔR is transduced, forskolin-stimulated and CFTRinh-172-inhibited currents are 6-7 μA / cm compared to vehicle. 2 It recovered to.

[0160] Collectively, our results show that AAV204 mediates efficient delivery of highly expressed functional CFTR and further restores intracellular CFTR function in vitro in mouse models and in explant cultures of human patient cells. These results demonstrate the therapeutic potential of AAV204 particles containing the CFTRΔR transgene in cystic fibrosis.

[0161] [Example 4] [Amelioration of defects caused by CLCLN3 disease using an optimized CLN3 transgene delivered by an AAV214 vector] AAV capsid with enhanced tropism for CNS tissues after systemic administration (AAV214) and an optimized CLN3 (comprising the nucleic acid sequence of SEQ ID NO: 122) transgene cassette for improved biodistribution and expression in CNS and somatic tissues were developed and tested for functionality. AAV9 was used as a benchmark to evaluate the tropism of AAV214 and the biodistribution of the optimized CLN3 transgene cassette in a mouse model of juvenile neuronal ceroid lipofuscinosis that lacks a 1.02 kb segment spanning exon 7 and exon 8 of CLN3 (CLN3Δex7 / 8) in a CC57BL / 6 background. This CLN3 deletion occurs in approximately 85% of mutated CLN3 alleles and recapitulates many of the disease phenotypes associated with the human disease, including movement disorders, glial activation, and progressive accumulation of lysosomal storage material. [Table 6]

[0162] AAV9 and AAV214 viral vectors each carrying the CLN3 transgene (AAV9-CLN3 and AAV214-CLN3, respectively) were injected into wild-type mice at 2.0 × 10 13vg / kg (viral vector genomes / kilogram) intravenously. See Table 6. After 30 days, animals were humanely sacrificed and tissues were harvested for biodistribution analysis to assess delivery of vector particles to several different organs, including the primary regions of the CNS and spinal cord (cervical and lumbar). T-test analysis of log-transformed data showed no statistically significant differences between AAV214-CLN3 and AAV9-CLN3 in biodistribution values ​​for most tissues tested (see Figure 13). However, the sciatic nerve using AAV214-CLN3 demonstrated statistically significantly (p=0.0001) higher biodistribution (744%) compared to AAV9, while the spleen was better transduced with AAV9-CLN3 (p<0.0001). Current studies evaluating expression and dose response over a longer period of time indicate that AAV214-CLN3 can be used to effectively deliver a CLN3 expression cassette to CNS tissues via systemic administration.

[0163] Expression of the CLN3 transgene was assessed by RTqPCR using total RNA isolated from the left hemisphere of the brain. One-way ANOVA of log-transformed data showed no statistically significant difference in the mean CLN3 expression values ​​of AAV9-CLN3CLCLN3 vs. AAV214-CLN3 treated animals (p=0.4489). However, both tested viral vectors produced higher CLN3 expression levels than the control (p<0.0001; FIG. 14).

[0164] In summary, these results showed that the novel AAV214 viral vector containing an optimized CLN3 expression cassette demonstrated comparable tropism to AAV9 in most tissues, including the CNS, when administered via systemic administration in a mouse model of CLN3 disease. These results indicate that the AAV214 vector containing the optimized CLN3 transgene described herein can be used to prevent and treat CLN3 disease.

[0165] [Example 5] [Amelioration of defects caused by Fabry disease using an optimized GLA transgene delivered by an AAV214 vector] AAV9 and AAV214 viral vectors containing the CBh promoter, CBA-MVM hybrid intron, native GLA transgene sequence, and TK65 polyA site were administered by IV injection to wild-type C57BL / 6 mice (see Table 7). The expected transgenic GLA protein size in plasma samples was confirmed by immunoblotting (Figure 15). GLA enzyme activity was assessed in plasma, brain, spinal cord, heart, kidney, liver, and eye. Statistical analysis performed on log-transformed GLA enzyme activity values ​​showed that all AAV214-GLA transduced samples had statistically significantly higher GLA activity compared to the control (p<0.0001). GLA enzyme activity was also statistically significantly higher in plasma, brain, and spinal cord tissues transduced in AAV214-GLA compared to AAV9-GLA (Figure 16). In summary, analysis of GLA enzyme activity indicates effective transduction of AAV214 constructs into multiple target tissues, particularly CNS tissues, demonstrating the therapeutic benefit of AAV214 vectors in subjects with Fabry disease. [Table 7]

[0166] After 10 days of testing in wild-type animals, no acute toxic effects were observed from systemic administration of the GLA transgene via AAV9 or AAV214 viral vectors. Animals treated in this study did not exhibit any adverse effects due to treatment. The effective delivery of AAV9 and AAV214 to target tissues, particularly the CNS, heart, and kidney, following systemic administration demonstrates their ability to safely transduce key target tissues relevant to Fabry disease.

[0167] [Example 6] [Amelioration of defects caused by Pompe disease using an optimized GAA transgene delivered by an AAV214 vector] AAV9-GAA and AAV214-GAA vectors containing the CBh promoter, CBA-MVM hybrid intron, codon-optimized GAA transgene sequence, and BGH polyA site were administered intravenously to wild-type C57BL / 6 mice (see Table 8). To determine whether the transgene was effectively delivered to the target tissues, GAA enzyme activity protein was tested in brain, spinal cord, diaphragm, biceps, liver, and plasma from treated mice. One-way ANOVA of log-transformed values ​​of GAA enzyme activity revealed that treated animals had statistically significantly higher GAA activity in all tested tissues compared to control animals (p<0.002). No statistically significant differences in enzyme activity were shown between tissues administered with AAV214 and tissues administered with AAV9, except for plasma (p=0.0018), where AAV9 had a slight advantage (Figure 19A-E). Analysis of GAA enzyme activity confirmed the ability to cross the blood-brain barrier and the effective transduction of the AAV214 construct into multiple target tissues, including the transduction into tissues important for the treatment of Pompe disease, such as the biceps and diaphragm (Figure 19). These results suggest that a single intravenous injection of the AAV214 viral vector containing the optimized GAA expression cassette described herein may be sufficient to achieve delivery of the corrected GAA transgene to target tissues. After 10 days of testing in wild-type animals, no acute toxic effects were observed from systemic administration of the GAA transgene via the AAV9 vector or the AAV2l4 vector.

[0168] FIG. 19F shows the repair of the underlying molecular pathology by AAV-delivered GAA. Glycogen analysis from gaa- / - mice treated intravenously with AAV capsids packaged with codon-optimized human GAA. Glycogen content was indirectly measured by the release of glucose after amyloglucosidase treatment. Free glucose was measured using Infinity Glucose Reagent and analyzed on a SpectraMax i3x. Data are presented as % of gaa- / - vehicle control treated animals. Data show the reduction in glycogen levels obtained by AAV-delivered GAA. Glycogen clearance was observed in all target tissues with AAV214 functioning as effectively as AAV9.

[0169] These data confirm that systemic delivery of AAV9 and AAV214, specifically with muscle and peripheral nervous system (PNS) expression, demonstrates both the ability to safely transduce key target tissues relevant to Pompe disease and the ability to restore GAA functionality. [Table 8]

[0170] [Example 7] [AAV110 vector particles exhibit highly specific muscle tropism] AAV110 particles were prepared using the pAAV110 plasmid (also called ITCord1.10 plasmid) encoding the AAV110 capsid protein. The AAV110-GFP viral vector, which contains the CBh promoter, CBA-MVM hybrid intron, EGFP transgene sequence, and BGH polyA site, was administered to C57Bl / 6 wild-type mice in a single injection into each leg (biceps femoris muscle) (a total of 1 × 10 11 vg, 5×10 12 vg / kg). Another group of animals received an equivalent amount of AAV9-GFP viral vector for comparison.

[0171] GFP expression was assessed by imaging leg muscles for fluorescence. Figure 24A. The data showed that both the left and right legs administered the AAV110-GFP viral vector expressed high levels of GFP, establishing muscle tropism for the AAV110 capsid. In contrast, AAV9-GFP vector particles provided substantially less muscle expression (Figure 24B).

[0172] To evaluate GFP transgene distribution in other tissues induced by intramuscular administration of AAV110-GFP or AAV9-GFP, transgene biodistribution (BD) was examined in a panel of organs. See Figure 25. The data confirm that AAV110 transduction occurs primarily in muscle, but also in the sciatic nerve and spleen. In contrast to AAV9, AAV110 shows little or no biodistribution in the brain, kidney, eye, lung, heart, liver, and testis. In all cases, BD was approximately 3% or less than that obtained with AAV9 intramuscular delivery of the transgene.

[0173] Immunohistochemical analysis of muscle tissue confirmed high levels of GFP expression in muscle for AAV110 and lower for AAV9 (Figure 26). This data confirms superior muscle tropism and expression by AAV110.

[0174] [Example 8] AAV214 vector particles provide high levels of expression in muscle following IM and IV administration. An AAV viral vector was generated that contained the AAV214 capsid protein and a luciferase expression cassette driven by the U1a promoter (AAV214-Luc). AAV214-Luc was injected into each muscle at 5 × 10 12The compound was administered to the right leg of adult wild type rats at a dose of 1000 mg / kg in a total volume of 0.1 mL per muscle. The left leg was untreated. To measure expression, the muscles were exposed to luciferin and the light emitted was measured. The data in the table below show that injected muscles, but not untreated muscles, show high expression 28 days after administration. Figure 27 shows luciferase activity in tissues, indicating the activity of the expressed enzyme. [Table 11]

[0175] Similar results were obtained using a different transgene, codon-optimized SMN-1 (survival of motor neuron 1), defective in spinal muscular atrophy (SMA), driven by the CBh promoter. The ability of intravenously administered AAV214-SMN1 viral vector particles was compared to AAV9-SMN1 vector particles to express SMN-1 when administered intravenously. Figure 28 shows the expression of SMN1 in tibialis anterior muscle tissue following infection of young wild-type mice. The data indicate that AAV214 vector particles provide improved expression with at least a 10-30% increase compared to vehicle and are suitable for muscle transduction when delivered intravenously.

[0176] Comparison of AAV214 and AAV9 viral vectors for transducing muscle tissue shows that IM-delivered AAV214 can transduce a larger muscle area than AAV9. Whole rat muscles (biceps femoris) were analyzed for GFP or mCherry expression by immunohistochemistry 10 days after IM injection. Fixed frozen sections were probed with GFP and mCherry pAbs. AAV214 shows a significantly larger transduced area compared to AAV9, which was mostly restricted to the upper part of the muscle, coinciding with the injection site. (Figure 26B).

[0177] [Example 9] [AAV vector particles containing AAV214-derived capsid proteins exhibit high expression in muscle after IV administration] The AAV214 VP1 protein was modified by exchanging its N-terminus with amino acid sequences from known AAV serotypes (AAV8, AAV9, AAVrh10), thereby producing the variants shown in the table below. AAV viral vector particles were then prepared with each of the newly derived capsid proteins essentially as described in Example 1, and evaluated for their ability to transduce muscle after intravenous administration. We found that each viral vector gave good muscle transduction in the leg and heart (Figure 29). One-way ANOVA of log-transformed biodistribution data revealed no statistically significant differences (p>0.05) in the mean biodistribution values ​​of the viral vectors tested. [Table 12]

[0178] [Example 10] [Capsid-induced cross-neutralizing antibody production] AAV204 and AAV214 have limited or very low cross-reactivity with AAV9 nAbs and are unlikely to induce cross-reactivity with AAV9 producing nAbs.

[0179] We tested the ability of animals administered either AAV9 or AAV214 by IM to produce neutralizing antibodies against AAV9. (Figure 31A). Analysis was performed by measuring the ability of animal sera to inhibit transduction of AAV9 luciferase vector into the permissive cell type Lec2. Three days after transduction, cells were assayed for luciferase activity. Each group consisted of two or three rats for either control, AAV9, or AAV214. Advantageously, animals injected with AAV214 by IM did not show a cross-reactive immune response to AAV9, which may allow for a larger patient population due to the inclusion of patients with pre-existing immunity to AAV9, either naturally occurring or from previous administration.

[0180] Similar data were obtained in non-human primates (NHPs). Both NHPs administered AAV9 intrathecally (IT) and intravenously (IV) expressed nAbs against AAV9 (Figure 31B). Sera from IT-treated animals showed much higher cross-reactivity to the other viruses tested (AAV204, AAV214, and AAV6). However, the route of administration does not appear to significantly affect the differential development of nAbs, since two animals were administered AAV204 intrathecally plus by the intravitreal route (IT+IV) and revealed similar differences in cross-reactivity (Figure 31C; see NHP-2 and NHP-3). Furthermore, animals administered AAV204 only intravitreally (IVT) (Figure 31C; see NHP-4) showed similar cross-reactivity to the IT+IVT-treated animals (NHP-3). The differences in expressed cross-reactivity may be explained by the identity of the AAV capsid protein epitopes against which the nAbs were generated. Serum samples from both AAV9-treated animals showed low reactivity to AAV204 (Fig. 31B), and two of three AAV204-treated animals demonstrated very low reactivity to AAV9 and AAV214, suggesting a high potential compatibility (Fig. 31C). In contrast, AAV6 revealed high cross-reactivity in all AAV204-treated animals (Fig. 31B and C).

[0181] Alternative Embodiments 1. Use of a polynucleotide encoding an adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 3, 30-34, 49, or 84, or a polynucleotide encoding an adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 1. 2. The polynucleotide of embodiment 1, wherein the amino acid sequence has at least 80% identity to SEQ ID NO: 3, 30-34, 49, or 84. 3. The polynucleotide of embodiment 1, wherein the amino acid sequence has at least 90% identity to SEQ ID NO: 3, 30-34, 49, or 84. 4. The polynucleotide of embodiment 1, wherein the amino acid sequence has at least 95% identity to SEQ ID NO: 3, 30-34, 49, or 84. 5. The polynucleotide of embodiment 1, wherein the amino acid sequence has at least 99% identity to SEQ ID NO: 3, 30-34, 49, or 84. 6. The polynucleotide of embodiment 1, wherein the amino acid sequence comprises SEQ ID NO: 3, 30-34, 49, or 84. 7. A polynucleotide according to any one of embodiments 1 to 6, wherein the amino acid sequence comprises the VP1, VP2, and VP3 portions of the AAV capsid protein, and the VP3 portion has the sequence of SEQ ID NO: 41. 8. The polynucleotide of embodiment 7, wherein the AAV capsid protein is at least 70%, 80%, 90%, or 99% identical to SEQ ID NO: 3, 30-34, 49, or 84. 9. The polynucleotide of any one of embodiments 1 to 8, wherein the polynucleotide is contained within a plasmid, a bacterial artificial chromosome, a yeast artificial chromosome, a phage, or a viral vector. 10. A host cell comprising a polynucleotide according to any one of embodiments 1 to 8. 11. An AAV capsid protein comprising an amino acid sequence having at least 70%, 80%, 90%, or 99% identity to SEQ ID NO: 3, 30-34, 49, or 84. 12. An AAV capsid protein comprising an amino acid sequence having the sequence of SEQ ID NO: 2, 3, 30-34, 49, or 84. 13. (i) an AAV capsid protein according to embodiment 11 or 12; (ii) An AAV viral vector, including an AAV vector. 14. The AAV viral vector of embodiment 13, wherein the AAV vector comprises a heterologous nucleic acid. 15. The AAV viral vector of embodiment 14, wherein the heterologous nucleic acid is a transgene. 16. The AAV viral vector of embodiments 13 to 15, wherein the transgene encodes cystic fibrosis transmembrane conductance regulator (CFTR), CLN3 protein, alpha-galactosidase A (GLA), or acid alpha-glucosidase (GAA). 17. The AAV viral vector of embodiment 15, wherein the transgene comprises a sequence having at least 70%, 80%, 90%, or 99% identity to any one of SEQ ID NOs: 5, 6, 7, 9, 10, 12, and 13. 18. The AAV viral vector of embodiment 15, wherein the transgene encodes a protein comprising an amino acid sequence having at least 70%, 80%, 90%, or 99% identity to any one of SEQ ID NOs: 4, 5, 8, 11, and 14. 19. An AAV viral vector according to any one of embodiments 13 to 18, wherein the heterologous nucleic acid is operably linked to a promoter. 20. The AAV viral vector of embodiment 19, wherein the promoter is a tissue-specific control promoter or a constitutive promoter. 21. The promoter is selected from the group consisting of Rous sarcoma virus (RSV) LTR promoter (optionally having an RSV enhancer), cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, beta actin promoter, phosphoglycerol kinase (PGK) promoter, U6 promoter, H1 promoter, CAG promoter, hybrid chicken beta actin promoter, MeCP2 promoter, EF1 promoter, ubiquitous chicken beta actin hybrid (CBh) promoter, U1a promoter, U1b promoter, Me The AAV viral vector of embodiment 20, wherein the constitutive promoter is a CP2 promoter, a MeP418 promoter, a MeP426 promoter, a minimal MeCP2 promoter, a VMD2 promoter, a mRho promoter, an EFla promoter, a Ubc promoter, a human beta-actin promoter, a TRE promoter, an Ac5 promoter, a polyhedrin promoter, a CaMKIIa promoter, a Gal1 promoter, a TEF1 promoter, a GDS promoter, an ADH1 promoter, a Ubi promoter, or an alpha-1-antitrypsin (hAAT) promoter. 22. The AAV viral vector of embodiment 10, wherein the promoter is a tissue-specific control promoter that is a central nervous system (CNS) cell-specific promoter, a lung-specific promoter, a skin-specific promoter, a muscle-specific promoter, a liver-specific promoter, or an eye-specific promoter. 23. The AAV viral vector of embodiment 14, wherein the heterologous nucleic acid encodes an mRNA, siRNA, gRNA, or microRNA. 24. The AAV viral vector of embodiment 14, wherein the heterologous nucleic acid encodes a polypeptide. 25. The AAV viral vector of embodiment 14, wherein the heterologous nucleic acid encodes cystic fibrosis transmembrane conductance regulator (CFTR), CLN3 protein, alpha-galactosidase A (GLA), or acid alpha-glucosidase (GAA). 26. The AAV viral vector of embodiment 25, wherein the heterologous nucleic acid encodes CFTR. 27. The AAV viral vector of embodiment 26, wherein CFTR comprises or consists of the amino acid sequence encoded by SEQ ID NO:4. 28. The AAV viral vector of embodiment 15, wherein the heterologous nucleic acid encodes a protein comprising an amino acid sequence having at least 70%, 80%, 90%, or 99% identity to any one of SEQ ID NOs: 5, 8, 11, and 14. 29. The AAV viral vector of embodiment 15, wherein the heterologous nucleic acid comprises a sequence having at least 70%, 80%, 90%, or 99% identity to any one of SEQ ID NOs: 4, 5, 6, 7, 9, 10, 12, and 13. 30. The AAV viral vector of embodiment 18, wherein the heterologous nucleic acid encodes a reporter protein. 31. A method for introducing a gene of interest into a cell in a subject, the method comprising contacting the cell with an effective amount of an AAV viral vector described in any one of embodiments 13 to 30. 32. The method of embodiment 31, wherein the AAV viral vector is introduced into the subject orally, rectally, transmucosally, by inhalation, transdermal, parenterally, intravenously, subcutaneously, intradermal, intramuscularly, intrapleurally, intracerebrally, intrathecally, intracerebrally, intraventricularly, intranasally, intraaurally, intraocularly, periocularly, topically, intralymphatically, intracapsularly, intrathecally, or intravitreally. 33. The method of embodiment 31 or 32, wherein the subject is a mammal. 34. The method of embodiment 31-33, wherein the subject is a human. 35. The method of any one of embodiments 31 to 34, wherein the cell is a somatic cell. 36. The method of embodiment 35, wherein the somatic cell is a neuronal cell, a retinal cell, a muscle cell, an epithelial cell, a lung cell, a liver cell, a stem cell, or a skin cell. 37. A pharmaceutical composition comprising a polynucleotide according to any one of embodiments 1 to 8, an AAV capsid protein according to embodiment 11 or 12, or an AAV viral vector according to any one of embodiments 13 to 30. 38. A method for treating a disorder, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition of embodiment 37. 39. The method of embodiment 38, wherein the disorder is a CNS disorder, a skin disorder, a lung disorder, a muscle disorder, a liver disorder, or a retinal disorder. 40. Disorders include amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Fabry disease, Pompe disease, CLN3 disease (or juvenile neuronal ceroid lipofuscinosis), recessive dystrophic epidermolysis bullosa (RDEB), juvenile Batten disease, autosomal dominant disorders, muscular dystrophy, hemophilia A, hemophilia B, multiple sclerosis, diabetes, Gaucher disease, cancer, arthritis, muscle wasting, heart disease, intimal hyperplasia, epilepsy, Huntington's disease, Parkinson's disease, Alzheimer's disease, cystic fibrosis, thalassemia, Hurler syndrome, Sly syndrome, Scheie syndrome, Hurler-Scheie syndrome 40. The method of embodiment 38 or 39, wherein the inflammatory disease is selected from the group consisting of inflammatory bowel disease, Hunter syndrome, Sanfilippo syndrome A (mucopolysaccharidosis IIIA or MPSIIIA), Sanfilippo syndrome B (mucopolysaccharidosis IIIB or MPSIIIB), Sanfilippo syndrome C, Sanfilippo syndrome D, Morquio syndrome, Maroteaux-Lamy syndrome, Krabbe disease, phenylketonuria, Batten disease, spinal cerebral ataxia, LDL receptor deficiency, hyperammonemia, arthritis, macular degeneration, retinitis pigmentosa, neuronal ceroid lipofuscinosis 1 (CLN1), or adenosine deaminase deficiency. 41. The method of embodiment 38, wherein the disorder is spinal muscular atrophy (SMA), recessive dystrophic epidermolysis bullosa (RDEB), Fabry disease, Pompe disease, CLN3 disease (or juvenile neuronal ceroid lipofuscinosis), MPS IIIA, MPS IIIB, juvenile Batten disease, and Duchenne muscular dystrophy (DMD), or Becker muscular dystrophy. 42. The method of embodiment 38, wherein the disorder is cancer, and the cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, lip cancer, oral cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, non-melanoma skin cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, or thyroid cancer. 43. The method of any one of embodiments 37 to 42, wherein the subject is a mammal. 44. The method of embodiment 43, wherein the subject is a human. 45. A kit comprising a polynucleotide according to any one of embodiments 1 to 8, a cell according to embodiment 10, an AAV capsid protein according to embodiment 12 or 13, and / or an AAV viral vector according to any one of embodiments 13 to 30. 46. ​​An AAV packaging system comprising a polynucleotide according to any one of embodiments 1 to 8, and a helper cell. 47. The AAV packaging system of embodiment 46, wherein the helper cell is a yeast cell, a mammalian cell, or an insect cell. 48. A nucleic acid encoding an AAV capsid protein, wherein the AAV capsid protein comprises a leucine residue at amino acid 129, an asparagine residue at amino acid 586, and a glutamic acid residue at amino acid 723, and wherein the amino acid positions within the AAV capsid protein are numbered relative to the amino acid positions within the amino acid sequence of SEQ ID NO:2. 49. The nucleic acid of embodiment 48, wherein the encoded AAV capsid amino acid sequence is at least 95% identical to the amino acid sequence of SEQ ID NO:2. 50. The nucleic acid of embodiment 48, wherein the encoded AAV capsid amino acid sequence is at least 99% identical to the amino acid sequence of SEQ ID NO:2. 51. The nucleic acid of embodiment 48, wherein the nucleic acid sequence is at least 99% identical to the nucleotide sequence of SEQ ID NO: 15. 52. The nucleic acid of embodiment 48, wherein the nucleic acid sequence is 100% identical to the nucleotide sequence of SEQ ID NO: 15. 53. A vector comprising a nucleic acid according to any one of embodiments 48 to 52. 54. An AAV capsid protein encoded by a nucleic acid according to embodiments 48 to 52. 55. The AAV capsid protein of embodiment 54, wherein the protein comprises the amino acid sequence of SEQ ID NO:2. 56. An AAV viral vector comprising an AAV capsid protein encoded by a nucleic acid according to embodiment 54 or 55, and an AAV vector comprising a heterologous nucleic acid. 57. The AAV viral vector of embodiment 56, wherein the heterologous nucleic acid is operably linked to a constitutive promoter. 58. The AAV viral vector of embodiment 56 or 57, wherein the heterologous nucleic acid encodes a polypeptide. 59. The AAV viral vector of embodiment 56 or 57, wherein the heterologous nucleic acid encodes an antisense RNA, a microRNA, or an RNAi. 60. The AAV viral vector of embodiment 56, wherein the AAV capsid protein comprises the amino acid sequence of SEQ ID NO:2. 61. A nucleic acid encoding an AAV capsid protein comprising a VP1 portion, a VP2 portion, and a VP3 portion, wherein the VP3 portion comprises variable regions (VR) I-IX; (a) VR-II comprises the amino acid sequence DNNGVK (SEQ ID NO:54); (b) VR-III comprises the amino acid sequence NDGS (SEQ ID NO:55); (c) VR-IV comprises the amino acid sequence INGSGQNQQT (SEQ ID NO:56); (d) VR-V comprises the amino acid sequence RVSTTTGQNNNSNFAWTA (SEQ ID NO:57); (e) VR-VI comprises the amino acid sequence HKEGEDRFFPLSG (SEQ ID NO:58); (f) VR-VII comprises the amino acid sequence KQNAARDNADYSDV (SEQ ID NO:59); (g) VR-VIII comprises the amino acid sequence ADNLQQQNTAPQI (SEQ ID NO: 60); and (h) VR-IX is a nucleic acid comprising the amino acid sequence NYYKSTSVDF (sequence number 61). 62. The nucleic acid of embodiment 61, wherein the VR-I region comprises SASTGAS (sequence number 52). 63. The nucleic acid of embodiment 61, wherein the VR-I region comprises NSTSGGSS (SEQ ID NO: 53) or SSTSGGSS (SEQ ID NO: 87). 64.VP3 part, (i) asparagine (N) at amino acid 223; (ii) an alanine (A) residue at amino acid 224; (iii) a threonine (T) residue at amino acid 410; (iv) a histidine residue at amino acid 724, and (v) a proline (P) residue at amino acid 734; The nucleic acid of embodiments 61 to 63, wherein the amino acid positions within the AAV capsid protein are numbered relative to the amino acid positions within the amino acid sequence of SEQ ID NO:3. 65. The nucleic acid of embodiments 61 to 64, wherein the encoded AAV capsid amino acid sequence is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, 30, 31, 32, 33, 34, 49, or 84. 66. The nucleic acid of embodiments 61 to 65, wherein the encoded AAV capsid amino acid sequence is at least 99% identical to the amino acid sequence of SEQ ID NO: 3, 30, 31, 32, 33, 34, 49, or 84. 67. The nucleic acid according to embodiments 61 to 66, wherein the nucleic acid sequence is at least 99% identical to a nucleotide sequence selected from SEQ ID NOs: 18, 19, 20, 21, 22, 23, 47, 82, or 98. 68. The nucleic acid of embodiment 61, wherein the nucleic acid sequence is 100% identical to a nucleotide sequence selected from SEQ ID NOs: 18, 19, 20, 21, 22, 23, 47, 82, or 98. 69. A vector comprising a nucleic acid according to any one of embodiments 61 to 68. 70. An AAV capsid protein encoded by a nucleic acid according to embodiments 61 to 68. 71. The AAV capsid protein of embodiment 70, wherein the protein comprises the amino acid sequence of SEQ ID NO: 3, 30, 31, 32, 33, 34, 49, or 84. 72. An AAV viral vector comprising an AAV capsid protein encoded by a nucleic acid described in embodiments 61 to 68 and an AAV vector, wherein the AAV vector comprises a heterologous nucleic acid. 73. The AAV viral vector of embodiment 72, wherein the heterologous nucleic acid is operably linked to a constitutive promoter. 74. The AAV viral vector of embodiment 72 or 73, wherein the heterologous nucleic acid encodes a polypeptide. 75. The AAV viral vector of embodiment 72 or 73, wherein the heterologous nucleic acid encodes an antisense RNA, a microRNA, or an RNAi. 76. The AAV viral vector of embodiments 72 to 75, wherein the AAV capsid protein comprises the amino acid sequence of SEQ ID NO: 3, 30, 31, 32, 33, 34, 49, or 84. 77. A nucleic acid encoding an AAV capsid protein comprising a VP1 portion, a VPVP2 portion, and a VP3 portion, wherein the VP3 portion comprises variable regions (VR) I-IX; (a) VR-II comprises the amino acid sequence SASTGAS (SEQ ID NO:52); (b) VR-II comprises the amino acid sequence DNNNGVK (SEQ ID NO:54); (c) VR-III comprises the amino acid sequence NDGS (SEQ ID NO:55); (d) VR-IV comprises the amino acid sequence INGSGQNQQT (SEQ ID NO:56); (e) VR-V comprises the amino acid sequence RVSTTTGQNNNSNFAWTA (SEQ ID NO:57); (f) VR-VI comprises the amino acid sequence HKEGEDRFFPLSG (SEQ ID NO:58); (g) VR-VII comprises the amino acid sequence KQNAARDNADYSDV (SEQ ID NO:59); (h) VR-VIII comprises the amino acid sequence ADNLQQQNTAPQI (SEQ ID NO: 60); and (i) VR-IX is a nucleic acid comprising the amino acid sequence NYYKSTSVDF (sequence number 61). 78.VP3 part, (i) asparagine (N) at amino acid 223; (ii) an alanine (A) residue at amino acid 224; (iii) a threonine (T) residue at amino acid 410; (iv) a histidine residue at amino acid 724, and (v) a proline (P) residue at amino acid 734; The nucleic acid of embodiment 77, wherein the amino acid positions within the AAV capsid protein are numbered relative to the amino acid positions within the amino acid sequence of SEQ ID NO:3. 79. The nucleic acid according to embodiment 77 or 78, wherein the VP3 portion has the sequence of SEQ ID NO: 41. 80. The nucleic acid of embodiments 77-79, wherein the VP1 and VP2 portions of the encoded AAV capsid amino acid sequence are at least 95% identical to the amino acid sequences of the VP1 and VP2 portions of SEQ ID NO: 3, 31, 32, 33, or 34. 81. The nucleic acid of embodiments 77-80, wherein the encoded AAV capsid amino acid sequence is at least 99% identical to the amino acid sequence of SEQ ID NO: 3, 31, 32, 33, or 34. 82. The nucleic acid of embodiments 77-81, wherein the nucleic acid sequence is at least 99% identical to a nucleotide sequence selected from SEQ ID NOs: 18, 20, 21, 22, or 23. 83. The nucleic acid according to embodiments 77-82, wherein the nucleic acid sequence is 100% identical to a nucleotide sequence selected from SEQ ID NOs: 18, 20, 21, 22, or 23. 84. A vector comprising a nucleic acid according to any one of embodiments 57 to 83. 85. An AAV capsid protein encoded by a nucleic acid according to embodiments 77 to 83. 86. The AAV capsid protein of embodiment 85, wherein the protein comprises the amino acid sequence of SEQ ID NO: 3, 31, 32, 33, or 34. 87. An AAV viral vector comprising an AAV capsid protein encoded by the nucleic acid of embodiment 88 and an AAV vector comprising a heterologous nucleic acid. 88. The AAV viral vector of embodiment 87, wherein the heterologous nucleic acid is operably linked to a constitutive promoter. 89. The AAV viral vector of embodiment 87 or 88, wherein the heterologous nucleic acid encodes a polypeptide. 90. The AAV viral vector of embodiment 87 or 88, wherein the heterologous nucleic acid encodes an antisense RNA, a microRNA, or an RNAi. 91. An AAV viral vector according to embodiments 87 to 90, wherein the AAV capsid protein comprises the amino acid sequence of SEQ ID NO: 3, 31, 32, 33, or 34. 92. A nucleic acid encoding an AAV capsid protein comprising a VP1 portion, a VP2 portion, and a VP3 portion, wherein the VP1 portion comprises a leucine (L) residue at amino acid 129, the VP2 portion comprises a threonine (T) or asparagine (N) residue at amino acid 157, and a lysine (K) or serine (S) residue at amino acid 162, and the VP3 portion comprises an asparagine (N) residue at amino acid 223, an alanine (A) residue at amino acid 224, a histidine (H) residue at amino acid 272, a threonine (T) residue at amino acid 410, a histidine (H) residue at amino acid 724, and a proline (P) residue at amino acid 734, wherein the amino acid positions within the AAV capsid protein are numbered relative to the amino acid positions within the amino acid sequence of SEQ ID NO:3. 93. The VP3 portion comprises variable regions (VR) I to IX, (a) VR-II comprises the amino acid sequence SASTGAS (SEQ ID NO:52); (b) VR-II comprises the amino acid sequence DNNNGVK (SEQ ID NO:54); (c) VR-III comprises the amino acid sequence NDGS (SEQ ID NO:55); (d) VR-IV comprises the amino acid sequence INGSGQNQQT (SEQ ID NO:56); (e) VR-V comprises the amino acid sequence RVSTTTGQNNNSNFAWTA (SEQ ID NO:57); (f) VR-VI comprises the amino acid sequence HKEGEDRFFPLSG (SEQ ID NO:58); (g) VR-VII comprises the amino acid sequence KQNAARDNADYSDV (SEQ ID NO:59); (h) VR-VIII comprises the amino acid sequence ADNLQQQNTAPQI (SEQ ID NO: 60); and 93. The nucleic acid of embodiment 92, wherein (i) VR-IX comprises the amino acid sequence NYYKSTSVDF (sequence number 61). 94. The nucleic acid of embodiment 92 or 93, wherein the VP1 portion further comprises an aspartic acid (D) or alanine (A) residue at amino acid 24, and the amino acid positions within the AAV capsid protein are numbered relative to the amino acid positions within the amino acid sequence of SEQ ID NO: 3. 95.VP2 part, (i) a proline (P) residue at amino acid 148; (ii) an inserted arginine (R) residue at amino acid 152; (iii) an arginine (R) residue at amino acid 168; (iv) an isoleucine (I) residue at amino acid 189, and (v) a serine (S) residue at amino acid 200; The nucleic acid of embodiments 92 to 94, wherein the amino acid positions within the AAV capsid protein are numbered relative to the amino acid positions within the amino acid sequence of SEQ ID NO:3. 96. The nucleic acid of embodiments 92-96, wherein the encoded AAV capsid amino acid sequence is at least 95% identical to the amino acid sequence of SEQ ID NO: 31, 32, 33, or 34. 97. The nucleic acid of embodiment 92, wherein the encoded AAV capsid amino acid sequence is at least 99% identical to the amino acid sequence of SEQ ID NO: 31, 32, 33, or 34. 98. The nucleic acid of embodiment 92, wherein the nucleic acid sequence is at least 99% identical to a nucleotide sequence selected from SEQ ID NOs: 20, 21, 22, or 23. 99. The nucleic acid of embodiment 98, wherein the nucleic acid sequence is 100% identical to a nucleotide sequence selected from SEQ ID NOs: 20, 21, 22, or 23. 100. A vector comprising a nucleic acid according to any one of embodiments 92 to 99. 101. An AAV capsid protein encoded by a nucleic acid according to embodiments 92 to 99. 102. The AAV capsid protein of embodiment 101, wherein the protein comprises the amino acid sequence of SEQ ID NO: 31, 32, 33, or 34. 103. An AAV viral vector comprising an AAV capsid protein encoded by a nucleic acid according to embodiment 101 or 102, and an AAV vector comprising a heterologous nucleic acid. 104. The AAV viral vector of embodiment 103, wherein the heterologous nucleic acid is operably linked to a constitutive promoter. 105. The AAV viral vector of embodiment 103 or 104, wherein the heterologous nucleic acid encodes a polypeptide, an antisense RNA, a microRNA, or an RNAi.

Claims

1. A nucleic acid encoding an AAV capsid protein having the amino acid sequence of SEQ ID NO:

2.

2. The nucleic acid described in claim 1, whose nucleic acid sequence is at least 95% identical to the nucleotide sequence of SEQ ID NO:

15.

3. A vector comprising a nucleic acid described in any one of claims 1 to 2.

4. An AAV capsid protein comprising the amino acid sequence of SEQ ID NO:

2. (i) an AAV capsid protein comprising the amino acid sequence of SEQ ID NO:2; and (ii) an AAV vector comprising, in the 5' to 3' direction: (a) a first AAV inverted terminal repeat, (b) a promoter, (c) a heterologous nucleic acid; (d) a polyA tail, and (e) a second AAV inverted terminal repeat An AAV vector comprising An AAV viral vector comprising:

6. The AAV viral vector described in claim 5, wherein the promoter is a constitutive promoter.

7. The promoter is selected from the group consisting of a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a beta-actin promoter, a phosphoglycerol kinase (PGK) promoter, a U6 promoter, an H1 promoter, a CAG promoter, a hybrid chicken beta-actin promoter, a MeCP2 promoter, an EF1 promoter, a ubiquitous chicken beta-actin hybrid (CBh) promoter, a U1a promoter, a U1b promoter, a MeCP2 6. The AAV viral vector of claim 5, wherein the promoter is a nucleotide sequence selected from the group consisting of a nucleotide sequence of the ...

8. The AAV viral vector described in claim 5, wherein the promoter is a tissue-specific control promoter, and the tissue-specific control promoter is an eye-specific promoter.

9. The AAV viral vector described in claim 8, wherein the eye-specific promoter is a VMD2 promoter, a PDE6b promoter, or an mRho promoter.

10. An AAV viral vector described in any one of claims 5 to 9, wherein the heterologous nucleic acid encodes mRNA, siRNA, gRNA, or microRNA.

11. An AAV viral vector described in any one of claims 5 to 9, wherein the heterologous nucleic acid encodes a polypeptide.

12. The AAV viral vector described in claim 11, wherein the polypeptide is retinoschisin 1 (RS1) or phosphodiesterase 6B (PDE6B).

13. An AAV viral vector described in claim 11 or 12, wherein the polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to any one of SEQ ID NOs: 8, 14, 134-146, and 148-151.

14. The AAV viral vector described in claim 13, wherein the heterologous nucleic acid sequence comprises a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to any one of SEQ ID NOs: 99 to 133.

15. A composition comprising an AAV viral vector described in any one of claims 5 to 12.

16. The composition of claim 15, wherein the composition is for oral, rectal, transmucosal, inhalation, transdermal, parenteral, intravenous, subcutaneous, intradermal, intramuscular, intrapleural, intracerebral, intrathecal, intraventricular, intranasal, intraaural, intraocular, periocular, topical, intralymphatic, intracapsular, subretinal, or intravitreal administration.

17. A composition described in claim 15 or 16 for use in the treatment of a disease or disorder.

18. The disease or disorder is selected from the group consisting of amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Fabry disease, Pompe disease, juvenile neuronal ceroid lipofuscinosis, recessive dystrophic epidermolysis bullosa (RDEB), juvenile Batten disease, autosomal dominant disorders, muscular dystrophy, hemophilia A, hemophilia B, multiple sclerosis, diabetes, Gaucher disease, cancer, arthritis, muscle wasting, heart disease, intimal hyperplasia, epilepsy, Huntington's disease, Parkinson's disease, Alzheimer's disease, cystic fibrosis, thalassemia, Hurler syndrome, Sly syndrome, Scheie syndrome, Hurler-Scheie syndrome, Haas syndrome, 18. The composition for use of claim 17, wherein the disease is selected from the group consisting of: Enter's syndrome, Sanfilippo syndrome A (mucopolysaccharidosis IIIA or MPSIIIA), Sanfilippo syndrome B (mucopolysaccharidosis IIIB or MPSIIIB), Sanfilippo syndrome C, Sanfilippo syndrome D, Morquio syndrome, Maroteaux-Lamy syndrome, Krabbe disease, phenylketonuria, Batten disease, spinal cerebral ataxia, LDL receptor deficiency, hyperammonemia, macular degeneration, retinitis pigmentosa, neuronal ceroid lipofuscinosis 1 (CLN1), or adenosine deaminase deficiency.

19. The composition for use according to claim 17, wherein the disease or disorder is an eye disease or disorder.

20. The composition for use according to claim 19, wherein the eye disease or disorder is macular degeneration, Leber's congenital amaurosis, cone-rod dystrophy, Stargardt disease, congenital choroideremia, Usher syndrome, retinoschisis, Vietti's crystalline dystrophy, or color vision deficiency.

21. The composition for use according to claim 19, wherein the eye disease or disorder is retinitis pigmentosa.

22. The composition for use according to claim 21, wherein the retinitis pigmentosa is autosomal recessive retinitis pigmentosa, autosomal dominant retinitis pigmentosa, or X-linked retinitis pigmentosa.

23. The composition for use according to claim 19, wherein the eye disease or disorder is vitelliform macular dystrophy, age-related macular degeneration, autosomal dominant vitreochoroidopathy, glaucoma, or cataract.

24. The composition for use according to claim 19 or 23, wherein the eye disease or disorder is caused by a mutation in the bestrophin-1 (BEST-1) gene.

25. The composition for use according to claim 19, wherein the eye disease or disorder is retinoschisis.

26. The composition for use according to any one of claims 19 to 25, wherein the composition is for intraocular, periocular, subretinal, or intravitreal administration.

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