Modified adeno-associated virus (AAV) viral capsid proteins and methods of use

Abstract

Provided herein are adeno-associated virus (AAV) particles comprising viral capsid proteins that preferentially transduce retinal cells. A modified AAV capsid protein disclosed herein comprises a framework protein and a peptide that is inserted into the framework protein. Also provided are nucleic acids encoding said modified AAV capsid protein. Also provided are methods of using the AAV particles, for example, for enhancing expression in retinal cells and for treating retinal disease.

Description

MODIFIED ADENO-ASSOCIATED VIRUS (AAV) VIRAL CAPSID PROTEINS AND METHODS OF USECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This International Patent Application claims priority to EP Application No.24386154.9, filed December 30, 2024; EP Application No. 25387129.7, filed September 8, 2025; and EP Application No. 25387146.1, filed October 3, 2025, all of which are hereby incorporated by reference in their entireties.REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing, which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on December 17, 2025, is named SeqList-162027-55076.xml and is 164,735 bytes in size.FIELD

[0003] The disclosure generally relates to the field of molecular biology and medicine. More particularly, the methods and compositions herein are useful for adeno-associated virus (AAV)-based gene therapy.BACKGROUND

[0004] Gene therapy holds great promise for the treatment or prevention of a variety of diseases caused by inherited and acquired genetic disorders, particular ocular diseases. The eye is one of few organs of the body for which gene therapy has received approval from the U.S. Food and Drug Administration. AAV vectors are widely used vectors for gene therapy due to their relatively low immunogenicity and ability to infect a variety of cell types. Vectors based on AAV are safe, since wild-type AAV is nonpathogenic and has no etiologic association with any known diseases. Furthermore, AAV vectors have enjoyed success in human clinical trials.

[0005] To date, numerous AAV serotypes in humans have been identified. Traditional AAV serotypes, however, often face limitations with respect to transduction efficiency and specificity of the target tissue / cell of interest, particularly in the complex environment of the eye.180489702.1

[0006] Accordingly, novel AAV capsid protein variants that transduce retinal cells with high efficiency and specificity are urgently needed.SUMMARY

[0007] Provided herein are modified AAV capsid proteins and methods of using said capsid proteins, including in, but not limited to, gene therapy.

[0008] Provided is a modified adeno-associated virus (AAV) viral protein (VP), wherein: (a) the modified AAV VP comprises a framework protein and a peptide that is inserted into the framework protein;(b) (i) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1 and the peptide is inserted into the framework protein at a position between residues 587 and 588, numbered relative to SEQ ID NO: 1,(ii) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2 and the peptide is inserted into the framework protein at a position between residues 450 and 451, numbered relative to SEQ ID NO:2; or(iii) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3 and the peptide is inserted into the framework protein at a position between residues 385 and 386, numbered relative to SEQ IDN0:3; and(c) the peptide comprises (i) a core sequence comprising a sequence that has 0, 1, 2, or 3 amino acid substitutions as compared to SEQ ID NO:4, SEQ ID NO: 17, SEQ ID NO:80, SEQ ID NO:97, SEQ ID NO: 110, SEQ ID NO: 131, or SEQ ID NO: 132 (ii) optionally a first linker sequence located at the N-terminus of the core sequence, and (iii) optionally a second linker sequence located at the C-terminus of the core sequence.

[0009] In some embodiments, provided is a modified AAV VP, wherein:(a) the modified AAV VP comprises a framework protein and a peptide that is inserted into the framework protein;(b) (i) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at 2180489702.1least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1 and the peptide is inserted into the framework protein at a position between residues 587 and 588, numbered relative to SEQ ID NO: 1,(ii) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2 and the peptide is inserted into the framework protein at a position between residues 450 and 451, numbered relative to SEQ ID NO:2; or(iii) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3 and the peptide is inserted into the framework protein at a position between residues 385 and 386, numbered relative to SEQ IDN0:3; and(c) the peptide comprises (i) a core sequence comprising a sequence that has 0, 1, 2, or 3 amino acid substitutions as compared to SEQ ID NO:4, SEQ ID NO: 17, SEQ ID NO:80, SEQ ID NO:97, SEQ ID NO: 110, SEQ ID NO: 131, or SEQ ID NO: 132 , (ii) a first linker sequence located at the N-terminus of the core sequence, and (iii) a second linker sequence located at the C-terminus of the core sequence.

[0010] In some embodiments, the peptide comprises a core sequence comprising SEQ ID NO:4, SEQ ID NO: 17, SEQ IDNO:80, SEQ IDNO:97, SEQ ID NO: 110, SEQ ID NO: 131, or SEQ ID NO: 132 .

[0011] In some embodiments, the first and / or the second linker predominantly comprises alanines, glycines, or serines. In some embodiments, the first and / or the second linker predominantly consists of alanines, glycines, or serines. In some embodiments, the first and / or the second linker predominantly consists of alanines. In one embodiment, the first linker comprises the sequence AAA and the second linker comprises the sequence AA.

[0012] In some embodiments, the peptide comprises a sequence that has 0, 1, 2, 3, 4, or 5 amino acid substitutions as compared to any one of SEQ ID NOs:4-29, SEQ ID NOs:80-92, or SEQ ID NOs:97-122. In some embodiments, the peptide comprises any one of SEQ ID NOs:4-29, SEQ ID NOs:80-92, or SEQ ID NOs:97-122. In some embodiments, the peptide comprises SEQ ID NO: 12, SEQ IDNO:25, or SEQ ID NO:88, SEQ ID NO: 105, or SEQ ID NO: 118.

[0013] In some embodiments, (a) the framework protein comprises SEQ ID NO:1 and the peptide is inserted into the framework protein at a position between residues 587 and 588, numbered relative to SEQ ID NO:1; (b) the framework protein comprises SEQ ID NO:2 and 3180489702.1the peptide is inserted into the framework protein at a position between residues 450 and 451, numbered relative to SEQ ID NO:2; or (c) the framework protein comprises SEQ ID NO:3 and the peptide is inserted into the framework protein at a position between residues 385 and 386, numbered relative to SEQ ID NO:3.

[0014] In some embodiments, the AAV VP comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:30-32. In some embodiments, the modified AAV VP comprises any one of SEQ ID NOs:30-32.

[0015] In some embodiments, the AAV VP comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:33-35. In some embodiments, the AAV VP comprises any one of SEQ ID NOs:33-35.

[0016] In some embodiments, the AAV VP comprises a sequence that is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:93-95. In some embodiments, the AAV VP comprises any one of SEQ ID NOs:93-95.

[0017] In some embodiments, the AAV VP comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 123 -125. In some embodiments, the AAV VP comprises any one of SEQ ID NOs: 123-125.

[0018] In some embodiments, the AAV VP comprises a sequence that is at least 80%, at least85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 126-128. In some embodiments, the AAV VP comprises any one of SEQ ID NOs: 126-128.

[0019] Provided is an AAV particle comprising one or more of a modified AAV VP disclosed herein. In one embodiment, the viral particle comprises an AAV genome. In one embodiment, the AAV genome comprises: (a) one or more inverted terminal repeats (ITR); (b) a promoter; and (c) a transgene encoding a gene product, wherein the transgene is operatively linked to the promoter. In one embodiment, the one or more ITRs are derived from AAV serotype AAV2.

[0020] In some embodiments, the promoter is selected from the group consisting of metabotropic glutamate receptor 6 (GRM6) promoter, cytomegalovirus (CMV) promoter, cytomegalovirus early enhancer / chicken b-actin (CAG) promoter, elongation factor 1 -alpha 1 (EFla) promoter, SV40 promoter, CBA (chicken beta-actin) promoter, rhodopsin (rho)4180489702.1promoter, neural retina-specific leucine zipper protein (NRL) promoter, phosphodiesterase 6B (PDE6B) promoter, human rhodopsin kinase (hRK) promoter, human L-opsin promoter or a promoter derived therefrom, human M-opsin promoter or a promoter derived therefrom, human S-opsin promoter or a promoter derived therefrom, retinoid isomerohydrolase (retinal pigment epithelium-specific 65 kDa protein, RPE65) promoter, bestrophin-1 (BEST1) promoter, membrane protein MLC1 (Mlcl) promoter, interphotoreceptor retinoid-binding protein (IRBP) promoter, and neurofilament heavy (NEFH) promoter. In some embodiments, the promoter is selected from the group consisting of CAG promoter, CMV promoter, RK promoter, L-opsin derived promoter, and M-opsin derived promoter.

[0021] In some embodiments, the transgene encodes a polypeptide or an RNA. In some embodiments, the RNA is selected from the group consisting of inhibitory antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA), a Piwi-interacting RNAs (piRNAs), a short-hairpin RNA (shRNA), small non-coding RNA (sncRNA), and a long non-coding RNA (IncRNA). In some embodiments, the polypeptide is selected from the group consisting of channel rhodopsin, halorhodopsin (halo), melanopsin (Opn4), rhodopsin (RHO), blue opsin, red opsin, halorhodopsin (NpHR), enhanced halorhodopsin (eNpHR), archaerhodopsin-3 (Arch), ArchT, leptosphaeria maculans (Mac), retinoid isomerohydrolase (retinal pigment epithelium-specific 65 kDa protein, RPE65), cyclic nucleotide-gated channel alpha-3 (CNGA3), cyclic nucleotide-gated channel beta-3 (CNGB3), cone cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha (PDE6C), Jaws (cruxhalorhodopsin), iClC2 (see Berndt et al, Structure-guided transformation of channelrhodopsin into a light-activated chloride channel, Science. 2014 Apr 25;344(6182):420-4), retinal cone rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma (PDE6H), G Protein Subunit Alpha Transducin 1 (GNAT1), GNAT2, KCNV2, voltage-dependent calcium channel subunit alpha-2 / delta-4 (CACNA2D4), R9AP (Rgs9-anchor protein or RGS9BP), iClC2, apolipoprotein E (APOE), HtrA serine peptidase 1 (HTRA1), complement 3 (C3), C3 modulator, C5, C5 modulator, rab escort protein-1 (REP1), retinal-specific phospholipid-transporting ATPase (ABCA4), cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6), retinoschisin 1 (RSI), nicotinamide adenine dinucleotide dehydrogenase subunit 4 (ND4), tyrosine-protein kinase Mer (MERTK), rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit beta (PDE6B), retinaldehyde-binding protein (RLBP1), retinal membrane guanylyl cyclase 1 (RetGCl / GUCY2D), inosine-5'-monophosphate dehydrogenase 1 (IMPDH1), peripherin-2 (Prph2 / RDS), complement factor H (CFH) modulator, complement factor I (CFI) modulator, and Retinol Dehydrogenase 125180489702.1(RDH12). In some embodiments, the polypeptide is selected from the group consisting of C3 modulator, C5 modulator, CFH modulator, CFI modulator, ABCA4, channel rhodopsin, halo, and Opn4. In some embodiments, the channel rhodopsin is selected from the group consisting of channel rhodopsin-1 (ChRl), channel rhodopsin-2 (ChR2), ChETA, (C123T / E134R), ChR2 Cl 28 A, ChR2 C128S, ChR2 C128T, and ChRl-ChR2 hybrids / chimera.

[0022] Provided is a method of increasing expression of a transgene in a retinal cell as compared to expression of the transgene in a non-retinal cell, the method comprising contacting the retinal cell with an AAV particle disclosed herein. Provided is a method of delivering a transgene to a retinal cell, the method comprising contacting the retinal cell with an AAV particle disclosed herein. In some embodiments, the retinal cell is selected from the group consisting of rod cell, cone cell, bipolar cell, Mueller glia cell, horizontal cell, retinal astrocyte, retinal ganglion cell, retinal pigment epithelium (RPE) cell, or amacrine cell.

[0023] Provided is a pharmaceutical composition comprising (a) an AAV particle disclosed herein and (b) a pharmaceutical acceptable excipient.

[0024] Provided is method of treating a retinal disease in a subject in needed thereof, the method comprising administering to the subject an AAV particle disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the retinal disease is a cone dysfunction or a rod dysfunction. In some embodiments, the retinal disease is selected from the group consisting of age related macular degeneration (AMD), Vitelliform macular dystrophy, or North Carolina macular dystrophy, Leber’s congenital amaurosis (LCA), choroideremia (CHM), achromatopsia (ACHM), usher syndrome (USH), X-linked retinoschisis, Stargardt disease, Retinitis pigmentosa (RP), diabetic macular edema, diabetic retinopathy, Leber hereditary optic neuropathy (LHON), Congenital stational night blindness, cone-dystrophy with supernormal rod response (CDSSR), glaucoma, acquired renal cystic disease (arCD), Best disease (vitelliform macular dystrophy (BVMD)), ocular angiogenesis, and autosomal dominant cone-rod dystrophy (adCRD). In some embodiments, the retinal disease is selected from the group consisting of AMD, Stargardt disease, glaucoma, and RP. In some embodiments, the AMD is selected from the group consisting of dry AMD, geographic atrophy (GA), and wet AMD. In some embodiments, the RP is selected from the group consisting of X-linked RP (XLRP), MERTK-associated RP, PDE6B-associated RP, RLBP1-associated RP, and non-syndromic RP.

[0025] In one embodiment, the subject is a human. In some embodiments, the AAV particle or the pharmaceutical composition is administered via direct retinal injection, subretinal6180489702.1injection, intravitreal injection, suprachoroidal injection, topical instillation, or intravenous injection.BRIEF DESCRIPTION OF THE FIGURES

[0026] Fig. 1 shows the immunohistochemistry analysis of green fluorescent protein (GFP) expression in transduced mouse eyes after intravitreal (IVT) injection. 1E10 viral genomes (VGs) were injected per eye via IVT. Five weeks post injection, cryosections from transduced retina were stained with Arrestin 3 (ARR3) (cone-specific), and DAPI. GFP expression was also assessed. AAV particles comprising 7m8 ( / .< ., control) and DE25 capsid proteins, respectively, showed a similar transduction profile, targeting the inner retina more prominently, followed by outer retina. In contrast, AAV particles comprising DE12 capsid proteins - in addition to inner retina transduction - also provided a higher degree of Muller glia transduction. Muller glia span the whole width of the retina. The transduction levels of these cells can be identified by reporter gene expression in the Muller glia cells’ filamentous processes (white asterisk).

[0027] Fig. 2 shows the results of qPCR analysis of GFP expression in transduced mouse retinas. Absolute quantification of GFP mRNA expression in mouse retinas three weeks post administration with intravitreal delivery. 1E10 VGs per eye, n=3-4 per vector. Data are plotted as mean + / - SEM (standard error of the mean).

[0028] Fig. 3 and Fig. 3B show the results from live GFP imaging of whole retinal organoids. Whole retinal organoids were imaged in brightfield overlayed with GFP. Week five post transduction (5E10VGs per organoid). n=7-8.

[0029] Fig. 4 shows the results of flow cytometric analysis of GFP expression in transduced retinal organoids. Flow cytometry data showing GFP expression in CD73+ photoreceptors three weeks post transduction. 5E10 VGs per organoid. Data are plotted as mean + / - SEM, n=5 organoids per vector. Raw % and median intensity data were analyzed by one way ANOVA and post hoc Tukey’s multiple comparison test.

[0030] Fig.5 shows the results of an immunohistochemistry analysis of GFP expression in transduced retinal organoids. Cryosections from transduced retinal organoids stained with ARR3 (cone-specific), Rhodopsin (rod-specific), and DAPI. OS indicates photoreceptor outer segments, ONL indicates the Outer Nuclear Layer, which consists of the photoreceptor nuclei, and INL indicates the Inner Nuclear Layer, which consists of the nuclei of secondary and supporting neurons like bipolar cells. GFP expression was also assessed. AAV particles comprising DE12 capsid proteins exhibited similar transduction efficiency and resulting7180489702.1transgene expression levels in photoreceptor cells compared to AAV particles comprising 7m8 capsid proteins. In contrast, human retinal organoids transduced with AAV particles comprising DE25 capsid proteins showed a marked increase in ONL expression levels compared to both expression levels promoted by AAV vectors comprising DE12 or 7m8 capsid proteins, respectively. All vectors showed higher transduction efficiency compared to AAV2. The increased levels of expression seen particularly with DE25 in the ONL suggest higher levels of transduction efficiency and expression levels in photoreceptor cells.

[0031] Figs. 6A and 6B show a quantitative comparison of transduction for all retinal cells (Fig. 6A) or across specific retinal cell types (Fig. 6B) using scRNAseq analysis. The percent of total cells infected by each serotype was quantified for all cells combined (Fig. 6A) or for each major cell type (Fig. 6B). Bars represent a mean of n=2 retinal organoids, and error bars represent ± SEM. Bars in Fig. 6B are from left to right as shown in the figure legend from top to bottom.

[0032] Figs. 7A, 7B, and 7C show that capsid protein variants DE18 and DE61 are particularly suitable for infecting RPE cells. Fig. 7A. EVOS imaging. Brightfield and GFP (white) overlaid images of mature, pigmented IPSC-RPE transduced with indicated capsids (Multiplicity of Infection (MOI) 1E4 VGs per cell) three weeks post transduction. Scale bar = 100pm. Note the increased GFP signal for DE18 and DE61 as compared to other capsids.Fig. 7B and Fig. 7C. Flow cytometry data showing GFP expression in IPSC-RPE cells transduced with the indicated capsids. IPSC-RPE cells were transduced in a 48 well format 8 weeks post seeding (MOI = 1E4 VGs per cell). Flow cytometry to measure GFP expression was performed on live cells 3 weeks post transduction. Dotted line = mean % GFP / intensity from AAV5 capsid. Fig. 7D and Fig. 7E. Flow cytometry data showing GFP expression in IPSC-RPE cells transduced with the indicated capsids at a range of MOIs. IPSC-RPE cells were transduced in a 48 well format, 12 weeks post seeding (MOI = 1E4, 1E3 and 1E2 VGs per cell). Flow cytometry to measure GFP expression was performed on live cells 3 weeks post transduction. Each point in the graph is derived from one well in two separate transduction experiments. Bars from left to right as shown in figure legend from top to bottom.DETAILED DESCRIPTION

[0033] Adeno-Associated Viruses (AAV)

[0034] AAV are small, single-stranded DNA viruses that use helper virus to facilitate efficient replication. The 4.7 kb genome of AAV is characterized by two inverted terminal repeats (ITR) and two open reading frames, which encode the Rep proteins and Cap proteins,8180489702.1respectively. AAV can infect both dividing and quiescent cells. Infection occurs by interaction of the capsid proteins with a cell-membrane receptor, followed by endocytosis of the AAV virion.

[0035] The Rep reading frame encodes four proteins having molecular weights of 78 kD, 68 kD, 52 kD, and 40 kD (regulatory proteins Rep78, Rep68, Rep52 and Rep40). These Rep proteins are involved in AAV genome replication, packaging, genomic integration and other processes.

[0036] The Cap reading frame encodes three structural viral proteins (VPs) having molecular weights of 85 kD (VP1), 72 kD (VP2), and 61 kD (VP3), respectively. These VPs form the virion capsid. The VP1, VP2, and VP3 proteins typically assemble in a ratio of 1 : 1 :8-10, respectively, to form the AAV capsid, although AAV capsids containing only VP3, or VP1 and VP3, or VP2 and VP3, have been produced.

[0037] Capsid proteins VP1, VP2, and VP3 are usually all encoded by a single cap open reading frame. The different VPs are generated through alternative splicing of the mRNA and use of an alternate translational start codon. As a result, the VP3 (59-61 kDa, 524-544 amino acids (aa), depending on the specific AAV serotype) sequence is shared among all VPs (it is also referred to as the VP3 common region). VP2 (64-67 kDa, 580-601 aa, depending on the AAV serotype) is approximately 57 aa longer than VP3. The VP2 N-terminal region (common to VP1 and VP2) is referred to as the VP1 / VP2 common region. VP1 (79-82 kDa, 713-738 aa, depending on the AAV serotype) is approximately 137 aa longer than VP2. This N-terminal region in VP1 is called the VP1 unique (VPlu) region. The VP3 common region assembles the icosahedral capsid. The VPlu contains an important phospholipase A2 (PLA2) enzyme, and VPlu and VP1 / VP2 common region contain nuclear localization sequences (NLSs). The N-terminal portions of VP1 and VP2 have been shown to be important for endosomal trafficking and escape, nuclear localization, and genome release.

[0038] To illustrate the relationship for VP1, VP2, and VP3, provided herein are the sequences of VP1, VP2, and VP3 of AAV2. See Table 1. As shown in Table 1, the 587 / 588 insertion site in AAV2 VP1 corresponds to a 450 / 451 insertion site in AAV2 VP2 and to a 385 / 386 insertion site in AAV2 VP3.Table 1. Sequences of VP1, VP2, and VP3 of AAV2. The position in SEQ ID NO: 1 between residues 587 and 588 is marked with an *. The corresponding position in SEQ ID NO:2 between residues 450 and 451 is marked with an *. The corresponding position in SEQ ID NO:3 between residues 385 and 386 is marked with an *. See NCBI Reference Sequence:9180489702.1NC 001401.2. The sequence common to VP1 and VP2 is bold and in italics. The sequence common to VP1-VP3 is underlined.Protein SEQ Amino acid sequenceID NO AAV2 1 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRG VP1 LVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGD NPYL1<YNHADAEFQERL1<EDTSFGGNLGRAVFQAI<I<RVLEPLGL NPEPVKAAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQT GDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMAD^EGAD GVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSO SGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWORLINNNWGFR PKRLNFKLFNIOVKEVTONDGTTTIANNLTSTVOVFTDSEYQLPYV LGSAHQGCLPPFPADVFMVPOYGYLTLNNGSOAVGRSSFYCLEYF PSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDOYLY YLSRTNTPSGTTTOSRLOFSOAGASDIRDOSRNWLPGPCYROQRV SKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEE KFFPOSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYG SVSTNLORGN*ROAATADVNTOGVLPGMVWODRDVYLQGPIWA KIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKF ASFITOYSTGOVSVEIEWELOKENSKRWNPEIQYTSNYNKSVNVD FTVDTNGVYSEPRPIGTRYLTRNL AAV2 2 ^AAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADS VP2 VPDPQPLGQPPAAPSGLGTNTMAJGSGAPMADIANEGADGNG^SS GNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSOSGASND NH YFG YSTPWG YF DFN RFHC HF SPRD WORLI NNN WGF RPK RLNF KLFNIOVKEVTONDGTTTIANNLTSTVOVFTDSEYQLPYVLGSAH OGCLPPFPADVFMVPOYGYLTLNNGSOAVGRSSFYCLEYFPSQML RTGNNFTFSYTFEDVPFHSSYAHSOSLDRLMNPLIDQYLYYLSRTN TPSGTTTQSRLQFSOAGASDIRDOSRNWLPGPCYROORVSKTSAD NNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSG VLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEOYGSVSTNLO RGN*RQAATADVNTOGVLPGMVWODRDVYLQGPIWAKIPHTDG HFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITOY STGQVSVEIEWELOKENSKRWNPEIQYTSNYNKSVNVDFTVDTN GVYSEPRPIGTRYLTRNL AAV2 3 MATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTR VP3 TWALPTYNNHLYKQISSOSGASNDNHYFGYSTPWGYFDFNRFHC HFSPRDWORLINNNWGFRPI<RLNFI<LFNIOVI<EVTONDGTTTIAN NLTSTVOVFTDSEYOLPYVLGSAHOGCLPPFPADVFMVPOYGYLT LNNGSOAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSY AHSOSLDRLMNPLIDOYLYYLSRTNTPSGTTTOSRLOFSOAGASDI RDOSRNWLPGPCYROORVSKTSADNNNSEYSWTGATKYHLNGR DSLVNPGPAMASHKDDEEKFFPOSGVLIFGKOGSEKTNVDIEKVM ITDEEEIRTTNPVATEOYGSVSTNLORGN*ROAATADVNTOGVLP GMVWODRDVYLOGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPOI LIKNTPVPANPSTTFSAAKFASFITOYSTGOVSVEIEWELOKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL10180489702.1

[0039] The cap gene also encodes the assembly activating protein (AAP) from an alternative open reading frame. AAP supports capsid assembly, promoting targeting of the capsid proteins to the nucleolus and capsid formation. More recently, an X gene has been identified in the 3' end of the AAV2 genome (Cao et al., The X gene of adeno-associated virus 2 (AAV2) is involved in viral DNA replication, PLoS One. 2014 Aug 15;9(8):el04596, incorporated herein by reference in its entirety). The encoded X protein appears to be involved in the AAV life cycle, including DNA replication.

[0040] Flanking the rep and cap open reading frames at the 5' and 3' ends of the AAV genome are about 145 bp long ITRs. The two ITRs are the only cis elements essential for AAV replication, rescue, packaging, and integration of the AAV genome. The entire rep and cap domains can be excised and replaced with a therapeutic or reporter transgene (or any other transgene of interest to the person skilled in the art).

[0041] Modified AAV Capsid Proteins

[0042] Provided herein are modified AAV VP1, VP2, and VP3 capsid proteins that are particularly suitable for transducing retinal cells with high efficiency and / or specificity, particularly in the context of gene therapy. These modified AAV capsid proteins can be used for targeting genes to retinal cells for the treatment of ocular diseases, including, but not limited to, inherited retinal disorders and age-related macular degeneration. The use of the modified AAV capsid proteins disclosed herein can lead to high overall levels of retinal transduction and expression of the delivered transgene. Further, modified AAV capsid proteins disclosed herein that can transduce a broad range of retinal cell types with high efficiency allow for the treatment of complex retinal diseases that affect multiple cell layers. The terms “viral protein (VP),” “capsid protein” and “viral capsid protein” may be used interchangeably herein.

[0043] Framework Proteins

[0044] Provided herein are modified AAV capsid proteins that have been generated by inserting a peptide into a naturally occurring AAV framework protein (or a variant of a naturally occurring AAV framework capsid protein).

[0045] As used herein, an “AAV framework protein” is an AAV protein that is used as a starting point to produce a modified AAV capsid protein disclosed herein (z.e., by inserting a peptide into the AAV framework protein). As used herein, an “AAV VP1 framework protein” is an AAV VP1 protein that is used as a starting point to produce a modified AAV VP1 capsid protein disclosed herein (z.e., by inserting a peptide into the AAV VP1 framework protein). As used herein, an “AAV VP2 framework protein” is an AAV VP2 protein that is used as a starting 11180489702.1point to produce a modified AAV VP2 capsid protein disclosed herein (z.e., by inserting a peptide into the AAV VP2 framework protein). As used herein, an “AAV VP3 framework protein” is an AAV VP3 protein that is used as a starting point to produce a modified AAV VP3 capsid protein disclosed herein (z.e., by inserting a peptide into the AAV VP3 framework protein).

[0046] As used herein, an “AAV framework protein variant” refers to a variant of an AAV framework protein that comprises one or more alterations when compared to the parental AAV framework protein, including, but not limited to amino acid additions, substitutions, insertions, deletions, or posttranslational modifications, wherein the variant retains at least 10% of the biological activity of the parental AAV framework protein. For an AAV framework protein, the biological activity may relate to, for example, viral capsid assembly, binding to a target cell, entry into the host cell, endosomal trafficking, endosomal escape, nuclear trafficking, and / or genome release. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. An AAV framework protein variant may be a portion of the parental AAV framework protein that comprises a biologically active portion of the parental AAV framework protein. In some embodiments, the AAV framework protein variant comprises one or more conservative mutations as compared to its parental counterpart. In some embodiments, the AAV framework protein variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations as compared to its parental counterpart. In some embodiments, the AAV framework protein variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative mutations as compared to its parental counterpart. The terms “amino acid mutation” and “amino acid substitution” are used interchangeably herein.

[0047] As used herein, the terms “conservative amino acid substitutions” and “conservative modifications” refer to amino acid modifications that do not significantly affect or alter the function and / or activity of the presently disclosed proteins comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the proteins of this disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties such as charge and polarity. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge: positively-charged amino acids include lysine, arginine, histidine, negatively-charged amino acids include aspartic acid, glutamic acid, neutral charge amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine,12180489702.1phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine.

[0048] The AAV framework protein may be AAV VP1, VP2, or VP3.

[0049] In some embodiments, the framework protein is an AAV VP1, VP2, and / or VP3 derived from AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Hu.l, AAV-Hu.2, AAV-Hu.3, AAV-Hu.6, AAV-Hu.10, AAV-Hu.ll, AAV-Hu.13, AAV-Hu.15, AAV-Hu.16, AAV-Hu.17, AAV-Hu.18, AAV- Hu.19, AAV-Hu.20, AAV-Hu.37, AAV-Hu.45, AAV-Hu.47, AAV-Hu.48, AAV-Hu.49, AAV-Hu.52, AAV-Hu.58, AAVhu68, AAVhu69, AAVhu70, AAVhu71.74, AAVhu72, AAVhu73, AAVhu74.71, AAVhu75, AAVhu76, AAVhu77, AAVhu78.88, AAVhu79, AAVhu80, AAVhu81, AAVhu82, AAVhu83, AAVhu84, AAVhu86, AAVhu87, AAVhu88.78, AAVhu89, AAVhu90, AAVhu91, AAVhu92, hu.T17, hu.T32, hu.T40, hu.T41,Hu.S17, AAVv66, PAK56, Hu.LG15, hu.LvrOl, hu.LvrO2, hu.LvrO3, hu.LvrO4, hu.LvrO5, hu.LvrO6, hu.LvrO7, CVR l, CVR_2, CVR_3, CVR_4, CVR_5, CVR_6, CVR_7, JBL1,JBL2, JBL3, JBL4,JBL5, JBB1, JBB2, JBB3, JBB4, JBB6, JBB7, JBB8, JBB9, JBB11, JBB12, JBB13, CONB23, CONB36, CONB37, CONB39, CONS3, CONS6, CHC129, CHC163, CHC217, CHC367, CHC371, CHC387, CHC442, CHC471, CHC473, CHC508, CHC667, CHC668, CHC685, CHC704, CHC714, CHC767, CHC777, CHC790, CHC790, CHC877, CHC976, CHC985, CHC1010, CHC1017, CHC1020, CHC1024, CHC1024, CHC1158, CHC1260, CHC1273, CHC1286, CHC1286, CHC1343, CHC1350, CHC1449, CHC1449, CHC1534, CHC1570, CHC1591, CHC1602, CHC1704, CHC1919, CHC2040, CHC2087, CHC2102, CHC2107, CHC2112, CHC2128, CHC2141, CHC2206, CHC2208, CHC2320, CHC2497, CHC2557, CHC2731, CHC2806, CHC3013, CHC3086, CHC3142, CHC3511, CHC3765, AAVbb.l,AAVbb.2, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5,AAVcy.6., AAVpi.l, AAVpi.2, AAVpi.3., AAVrh.2,AAVrh.8, AAVrh.lO, AAVrh.12, AAVrh.13, AAVrh.14, AAVrh.16, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.32, AAVrh.32.33, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh75, AAVrh76, AAVrh77, AAVrh78, AAVrh79, AAVrh81, AAVrh82, AAVrh83, AAVrh84, AAVrh85, AAVrh86, AAVrh87, AAVrh89, AAVrh90, AAVrh91, AAVrh92, AAVrh93, AAVrh94, KNO1_S1, KN02_S2, KNO3_S3, KN04_S4,13180489702.1KN05_S5, KN06_S6, KN07_S7, KN08_S8, KN09_S9, KN10_S10, KN11_S11, KN12_S12, KN13_S13, KN14_S14, KN15_S15, KN16_S16, KN17_S17, KN18_S18, KN19_S19, KN20_S20, KN21_S21, KN22_S22, KN23_S23, KN24_S24, KN25_S25, KN26_S26, KN27_S27, KN28_S28, KN29_S29, KN30_S30, KN31_S31, KN32_S32, KN33_S33, KN34_S34, KN35_S35, KN36_S36, KN37_S37, KN38_S38, KN39_S39, KN40_S40, KN41_S41, KN42_S42, KN43_S43, KN44_S44, KN45_S45, KN46_S46, KN47_S47, KN48_S48, KN49_S49, KN50_S50, KN51_S51, KN52_S52, KN53_S53, KN54_S54, KN55_S55, KN56_S56, KN57_S57, KN58_S58, KN59_S59, KN60_S60, KN61_S61, KN62_S62, KN63_S63, KN64_S64, KN65_S65, KN66_S66, KN67_S67, KN68_S68, KN69_S69, KN70_S70, KN71_S71, KN72_S72, KN73_S73, KN74_S74, KN75_S75, KN76_S76, KN77_S77, KN78_S78, KN79_S79, KN80_S80, KN81_S81, KN82_S82, AAV-ra.l, stain YY.12, YY.25, YY.54, YY.78, YY.80, YY.93, XM.70, GZ.512, HD.16,HD.2O, HD.43, HD.94, MLP.6, MLP.26, AAVpol, AAVpo2.1, AAVpo4, AAVpo5, AAVpo6, AAVpo7, AAVpo8, AAV-Gol, VR-865, DA-1, YZ-1, ZN1,BR_DF12, RS / BR / 15 / 1R, GA / 1360 / 1994, 09YN, 1285, 10HB, 07YN, YNM, BAAV, BSRH, AAV2-HBKO, AAV2.7m8, AAV2.GL, AAV2.NN, AAV44.9, AAV44.9(E531D), AAV8BP2, AAV6-K531E-R576Q-K493S-K459S, AAV9.GL, AAV9.NN, ShHIO, or variants thereof.

[0050] In some embodiments, the framework protein is an AAV2 VP1, VP2, or VP3 protein.

[0051] In some embodiments, the AAV VP1 framework protein is an AAV2 VP1 capsid protein or a variant thereof. In some embodiments, the AAV VP 1 framework protein comprises SEQ ID NO:1 or a variant thereof. In some embodiments, the AAV VP1 framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1.

[0052] In some embodiments, the AAV VP2 framework protein is an AAV2 VP2 capsid protein or a variant thereof. In some embodiments, the AAV VP 1 framework protein comprises SEQ ID NO:2 or a variant thereof. In some embodiments, the AAV VP2 framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2.

[0053] In some embodiments, the AAV VP3 framework protein is an AAV2 VP3 capsid protein or a variant thereof. In some embodiments, the AAV VP3 framework protein comprises SEQ ID NO: 3 or a variant thereof. In some embodiments, the AAV VP3 framework protein 14180489702.1comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3.

[0054] Insertion Sites

[0055] As discussed herein, capsid proteins VP1, VP2, and VP3 are usually all encoded by a single cap open reading frame. The different VPs are generated through alternative splicing of the mRNA and use of an alternate translational start codon. VP1 encompasses the sequences of VP2 and VP3. VP2 encompasses VP3. See Table 1. In other words, the portions of the cap gene encoding for VP1 or VP2, respectively, also comprise the portion of the cap gene encoding VP3. As such, if the cap gene is modified in the region encoding for VP3, this modification would also affect the VP1 and VP2 proteins.

[0056] In some embodiments, a peptide is inserted between residues 587 and 588 (numbered relative to SEQ ID NO:1) of the AAV2 VP1 capsid protein or a variant thereof to produce a modified AAV VP1 capsid protein. In some embodiments, a peptide is inserted between residues 450 and 451 (numbered relative to SEQ ID NO:2) of the AAV2 VP2 capsid protein or a variant thereof to produce a modified AAV VP2 capsid protein. In some embodiments, a peptide is inserted between residues 385 and 386 (numbered relative to SEQ ID NO:3) of the AAV2 VP3 capsid protein or a variant thereof to produce a modified AAV VP3 capsid protein.

[0057] Residue 587 of AAV2 VP1 is part of a surface-exposed loop structure. In the AAV2 capsid, two arginines (R585 and R588) interact with each other and are part of the heparin-binding motif. Insertion of peptides between 587 / 588 significantly reduces binding of the viral particle to heparan proteoglycans. These glycans are ubiquitous on the cell surface of almost all tissues and serve as a primary receptor for AAV2 attachment and cell entry. Ablation of heparin binding can thus facilitate tissue-specific targeting. A person skilled in the art will appreciate that a peptide disclosed herein may also be inserted at a position that is adjacent to the 587 / 588 insertion site and that is part of the heparin binding loop. Likewise, a peptide disclosed herein may be inserted into the surface exposed loop of a VP protein of an AAV serotype other than AAV2 at a position that corresponds to the AAV2 loop that contains residue 587. See Table 2. For example, a peptide that - when inserted between 587 / 588 of AAV2 VP1 resulted in increased retinal transduction - can also lead to increased retinal transduction when inserted at the corresponding position in a VP1 capsid protein from a different AAV serotype (z.e., inserted after Q588 of the AAV9 VP1 capsid protein). See Khabou et al., Insight into the mechanisms of enhanced retinal transduction by the engineered AAV2 capsid variant -7m8,15180489702.1Biotechnol Bioeng. 2016 Dec;l 13(12):2712-2724, which is incorporated herein by reference in its entirety.Table 2. Amino acid sequences corresponding to amino acids 570-610 of AAV2 for different AAV serotypes. Insertion site corresponding to position 587 / 588 of AAV2 VP1 bold under under ined.Serotype Residues SEQ ID SequenceNO AAV2 570-611 71 PVATEQYGSVSTNLQRGNRQAATADVNTQGVLPG MVWQDRDV AAV1 571-612 72 PVATERFGTVAVNFQSSSTDPATGDVHAMGALPG MVWQDRDV AAV5 560-601 73 RVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPG SVWMERDV AAV6 571-612 74 P VATERFGT VAVNLQ S S STDPATGDVHVMGALPG MVWQDRDV AAV7 572-613 75 P VATEEYGIVS SNLQAANTAAQTQVVNNQGALPG MVWQNRDV AAV8 573-614 76 PVATEEYGIVADNLQQQNTAPQIGTVNSQGALPG MVWQNRDV AAV9 571-612 77 PVATESYGQVATNHQSAOAQAQTGWVQNQGILP GMVWQDRDV AAV 10 573-614 78 PVATEQYGVVADNLQQANTGPIVGNVNSQGALPGMVWQNRDV

[0058] A “serotype” is traditionally defined on the basis of a lack of cross-reactivity between antibodies to one virus as compared to another virus. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g. due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes). Under the traditional definition, a serotype means that the virus of interest has been tested against serum specific for all existing and characterized serotypes for neutralizing activity and no antibodies have been found that neutralize the virus of interest. As more naturally occurring virus isolates are discovered and capsid mutants generated, there may or may not be serological differences with any of the currently existing serotypes. Thus, in cases where the new AAV has no serological difference, this new AAV would be a subgroup or variant of the corresponding serotype. In many cases, serology testing for neutralizing activity has yet to be performed on mutant viruses with capsid sequence modifications to determine if they are of another serotype according to the traditional definition of serotype. Accordingly, for the sake of convenience and to avoid repetition, the term “serotype” broadly refers to both serologically distinct viruses (e.g. AAV)16180489702.1as well as viruses (e.g., AAV) that are not serologically distinct that may be within a subgroup or a variant of a given serotype.

[0059] In one embodiment, a peptide is inserted between residues 585 and 586 (numbered relative to SEQ ID NO:1) of the AAV2 VP1 capsid protein or a variant thereof to produce a modified AAV VP1 capsid protein. In some embodiments, a peptide is inserted between residues 448 and 449 (numbered relative to SEQ ID NO:2) of the AAV2 VP2 capsid protein or a variant thereof to produce a modified AAV VP2 capsid protein. In some embodiments, a peptide is inserted between residues 383 and 384 (numbered relative to SEQ ID NO:3) of the AAV2 VP3 capsid protein or a variant thereof to produce a modified AAV VP3 capsid protein.

[0060] In one embodiment, a peptide is inserted between residues 586 and 587 (numbered relative to SEQ ID NO:1) of the AAV2 VP1 capsid protein or a variant thereof to produce a modified AAV VP1 capsid protein. In some embodiments, a peptide is inserted between residues 449 and 450 (numbered relative to SEQ ID NO:2) of the AAV2 VP2 capsid protein or a variant thereof to produce a modified AAV VP2 capsid protein. In some embodiments, a peptide is inserted between residues 384 and 385 (numbered relative to SEQ ID NO:3) of the AAV2 VP3 capsid protein or a variant thereof to produce a modified AAV VP3 capsid protein.

[0061] In one embodiment, a peptide is inserted between residues 588 and 589 (numbered relative to SEQ ID NO:1) of the AAV2 VP1 capsid protein or a variant thereof to produce a modified AAV VP1 capsid protein. In some embodiments, a peptide is inserted between residues 451 and 452 (numbered relative to SEQ ID NO:2) of the AAV2 VP2 capsid protein or a variant thereof to produce a modified AAV VP2 capsid protein. In some embodiments, a peptide is inserted between residues 386 and 387 (numbered relative to SEQ ID NO:3) of the AAV2 VP3 capsid protein or a variant thereof to produce a modified AAV VP3 capsid protein.

[0062] Inserted Peptides

[0063] In some embodiments, the peptide that is inserted into the AAV framework protein comprises a core sequence comprising SEQ ID NO:4 (TSVGTIR), SEQ ID NO: 17 (TVNLVKA), SEQ ID NO:80 (FSSDRIK), SEQ ID NO: 97 (NVTNLLT), SEQ ID NO: 110 (NVTNVLT), or variations thereof. In some embodiments, the peptide that is inserted into the AAV framework protein comprises a core sequence comprising a sequence that has 1, 2, or 3 amino acid substitutions as compared to SEQ ID NO:4, SEQ ID NO: 17, SEQ ID NO:80, SEQ IDNO:97, or SEQ ID NO: 110.

[0064] In some embodiments, the peptide that is inserted into the AAV framework protein comprises a core sequence comprising NVTNXiLT, wherein Xi is A, I, L, M, F, W, Y, or V (SEQ ID NO: 131). In some embodiments, the peptide that is inserted into the AAV framework 17180489702.1protein comprises a core sequence comprising a sequence that has 1, 2, or 3 amino acid substitutions as compared to SEQ ID NO: 131.

[0065] In some embodiments, the peptide that is inserted into the AAV framework protein comprises a core sequence comprising NVTNX2LT, wherein X2 is A, I, L, M, or V (SEQ ID NO: 132). In some embodiments, the peptide that is inserted into the AAV framework protein comprises a core sequence comprising a sequence that has 1, 2, or 3 amino acid substitutions as compared to SEQ ID NO: 132.

[0066] In some embodiments, the peptide that is inserted into the AAV framework protein comprises a core sequence that is flanked by one or more linker residues. In some embodiments, the core sequence is flanked by one or more alanine, glycine, or serine residues. In some embodiments, the core sequence is flanked by at least one, at least two, or at least three alanine, glycine, or serine residues on one or both sides.

[0067] In one embodiment, the N-terminal end of the core sequence is linked to two alanine residues. In one embodiment, the N-terminal end of the core sequence is linked to three alanine residues. In one embodiment, the C-terminal end of the core sequence is linked to two alanine residues. In one embodiment, the C-terminal end of the core sequence is linked to three alanine residues.

[0068] In some embodiments, the peptide that is inserted into an AAV framework protein comprises any of the sequences in Table 3, or a variant thereof. Such a peptide variant may comprise one or more alterations when compared to any of the sequences shown in Table 3, including, but not limited to amino acid additions, substitutions, insertions, deletions, or posttranslational modifications, wherein the peptide variant retains at least 10% of the biological activity of the parental peptide. With respect to the inserted peptide, biological activity may refer to increasing transduction efficiency and / or specificity for one or more types of retinal cells when inserted into an AAV framework protein as compared to a control capsid protein.Table 3. Peptide sequences.SEQ ID NO Sequence4 (core sequence used in DE25) TSVGTIR5 TSVGTIRA6 ATSVGTIRA7 AATSVGTIRA8 AAATSVGTIRA9 TSVGTIRAA10 ATSVGTIRAA18180489702.1SEQ ID NO Sequence 11 AATSVGTIRAA 12 (core and linker sequences used in DE25) AAATSVGTIRAA 13 TSVGTIRAAA 14 ATSVGTIRAAA 15 AATSVGTIRAAA 16 AAATSVGTIRAAA 17 (core sequence used in DE 12) TVNLVKA18 TVNLVKAA19 ATVNLVKAA 20 AATVNLVKAA 21 AAATVNLVKAA 22 TVNLVKAAA 23 ATVNLVKAAA 24 AATVNLVKAAA 25 (core and linker sequences used in DE 12) AAATVNLVKAAA 26 TVNLVKAAAA 27 ATVNLVKAAAA 28 AATVNLVKAAAA 29 AAATVNLVKAAAA 80 (core sequence used in DE39) FSSDRIK81 FSSDRIK A82 AFSSDRIKA83 AAFSSDRIKA 84 AAAFSSDRIKA 85 FSSDRIKAA86 AFSSDRIKAA 87 AAFSSDRIKAA 88 (core and linker sequence used in DE39) AAAFSSDRIKAA 89 FSSDRIK AAA 90 AFSSDRIKAAA 91 AAFSSDRIKAAA 92 AAAFSSDRIKAAA 97 (core sequence used in DE 18) NVTNLLT98 NVTNLLTA99 ANVTNLLTA 100 AANVTNLLTA 101 AAANVTNLLTA 102 NVTNLLTAA 103 ANVTNLLTAA 104 AANVTNLLTAA 105 (core and linker sequences used in AAANVTNLLTAA DE 18)106 NVTNLLTAAA 107 ANVTNLLTAAA 108 AANVTNLLTAAA 109 AAANVTNLLTAAA110 (core sequence used in DE61) NVTNVLT19180489702.1SEQ ID NO Sequence111 NVTNVLTA112 ANVTNVLTA113 AANVTNVLTA114 AAANVTNVLTA115 NVTNVLTAA116 ANVTNVLTAA117 AANVTNVLTAA118 (core and linker sequences used in AAANVTNVLTAADE61)119 NVTNVLTAAA120 ANVTNVLTAAA121 AANVTNVLTAAA122 AAANVTNVLTAAA

[0069] Provided is a modified AAV VP1 capsid protein, wherein:(a) the modified AAV VP1 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1;(c) the peptide is inserted into the framework protein at a position between residues 587 and 588, numbered relative to SEQ ID NO:1; and(d) the peptide comprises (i) a core sequence comprising a sequence that has 0, 1, 2, or 3 amino acid substitutions as compared to SEQ ID NO:4, SEQ ID NO: 17, SEQ ID NO:80, SEQ ID NO:97, SEQ ID NO: 110, SEQ ID NO: 131, or SEQ ID NO: 132, (ii) optionally a first linker sequence located at the N-terminus of the core sequence, and (iii) optionally a second linker sequence located at the C-terminus of the core sequence.

[0070] As used herein, the term “identity” refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. For example, when a position in the compared nucleotide sequence is occupied by the same base, then the molecules are identical at that position. A degree identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at shared positions. For example, polypeptides having at least 85%, 90%, 95%, 98%, or 99% identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotides encoding such polypeptides, are contemplated. Methods and computer programs for determining both sequence identity and similarity are publicly available,20180489702.1including, but not limited to, the GCG program package (Devereux et al., A comprehensive set of sequence analysis programs for the VAX, Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387-95), BLASTP, BLASTN, FASTA (Altschul et al, Basic local alignment search tool, J Mol Biol.1990 Oct 5;215(3):403-10), and the ALIGN program (version 2.0). The well-known Smith Waterman algorithm may also be used to determine similarity. The BLAST program is publicly available from NCBI and other sources (BLAST Manual, Altschul, et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0 at http: / / www.ncbi.nlm.nih.gov / blast / ). In comparing sequences, these methods account for various substitutions, deletions, and other modifications.

[0071] Provided is a modified AAV VP1 capsid protein, wherein:(a) the modified AAV VP1 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1;(c) the peptide is inserted into the framework protein at a position between residues 587 and 588, numbered relative to SEQ ID NO:1; and(d) the peptide comprises (i) a core sequence comprising SEQ ID NO:4, SEQ ID NO: 17, SEQ ID NO:80, SEQ ID NO:97, SEQ ID NO: 110, SEQ ID NO: 131, or SEQ ID NO: 132, (ii) a first linker sequence located at the N-terminus of the core sequence, and (iii) a second linker sequence located at the C-terminus of the core sequence.

[0072] Provided is a modified AAV VP1 capsid protein, wherein:(a) the modified AAV VP1 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises SEQ ID NO:1;(c) the peptide is inserted into the framework protein at a position between residues 587 and 588, numbered relative to SEQ ID NO:1; and(d) the peptide comprises (i) a core sequence comprising SEQ ID NO:4, SEQ ID NO: 17, SEQ ID NO:80, SEQ ID NO:97, SEQ ID NO: 110, SEQ ID NO: 131, or SEQ ID NO: 132, (ii) a first linker sequence located at the N-terminus of the core sequence, and (iii) a second linker sequence located at the C-terminus of the core sequence.

[0073] Each linker sequence may predominantly comprise alanines, glycines, or serines. The linker sequences may consist entirely of alanines. Each linker sequence may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids long. The first and the second linker may be identical in21180489702.1sequence. The first and the second linker may differ in sequence. The first and the second linker may be identical in length. The first and the second linker may differ in length.

[0074] Provided is a modified AAV VP1 capsid protein, wherein:(a) the modified AAV VP1 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1;(c) the peptide is inserted into the framework protein at a position between residues 587 and 588, numbered relative to SEQ ID NO:1; and(d) the peptide comprises a sequence that has 0, 1, 2, 3, 4, or 5 amino acid substitutions as compared to any one of SEQ ID NOs:4-29, SEQ ID NOs:80-92, or SEQ ID NOs:97- 122.

[0075] Provided is a modified AAV VP1 capsid protein, wherein:(a) the modified AAV VP1 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1;(c) the peptide is inserted into the framework protein at a position between residues 587 and 588, numbered relative to SEQ ID NO:1; and(d) the peptide comprises any one of SEQ ID NOs:4-29, SEQ ID NOs:80-92, or SEQ ID NOs:97-122.

[0076] Provided is a modified AAV VP1 capsid protein, wherein:(a) the modified AAV VP1 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1;(c) the peptide is inserted into the framework protein at a position between residues 587 and 588, numbered relative to SEQ ID NO:1; and(d) the peptide comprises SEQ ID NO: 12, SEQ ID NO:25, or SEQ ID NO:88, SEQ ID NO: 105, or SEQ ID NO: 118.

[0077] Provided is a modified AAV VP1 capsid protein, wherein:22180489702.1(a) the modified AAV VP1 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises SEQ ID NO:1;(c) the peptide is inserted into the framework protein at a position between residues 587 and 588, numbered relative to SEQ ID NO:1; and(d) the peptide comprises SEQ ID NO: 12, SEQ ID NO:25, or SEQ ID NO:88, SEQ ID NO: 105, or SEQ ID NO: 118.

[0078] Provided is a modified AAV VP2 capsid protein, wherein:(a) the modified AAV VP1 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2;(c) the peptide is inserted into the framework protein at a position between residues 450 and 451, numbered relative to SEQ ID NO: 2; and(d) the peptide comprises (i) a core sequence comprising a sequence that has 0, 1, 2, or 3 amino acid substitutions as compared to SEQ ID NO:4, SEQ ID NO: 17, SEQ ID NO:80, SEQ ID NO:97, SEQ ID NO: 110, SEQ ID NO: 131, or SEQ ID NO: 132, (ii) optionally a first linker sequence located at the N-terminus of the core sequence, and (iii) optionally a second linker sequence located at the C-terminus of the core sequence.

[0079] Each linker sequence may predominantly comprise alanines, glycines, or serines. The linker sequences may consist entirely of alanines. Each linker sequence may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids long. The first and the second linker may be identical in sequence. The first and the second linker may differ in sequence. The first and the second linker may be identical in length. The first and the second linker may differ in length.

[0080] Provided is a modified AAV VP2 capsid protein, wherein:(a) the modified AAV VP2 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2;(c) the peptide is inserted into the framework protein at a position between residues 450 and 451, numbered relative to SEQ ID NO: 2; and23180489702.1(d) the peptide comprises (i) a core sequence comprising SEQ ID NO:4, SEQ ID NO: 17, SEQ ID NO:80, SEQ ID NO:97, SEQ ID NO: 110, SEQ ID NO: 131, or SEQ ID NO: 132, (ii) a first linker sequence located at the N-terminus of the core sequence, and (iii) a second linker sequence located at the C-terminus of the core sequence.

[0081] Provided is a modified AAV VP2 capsid protein, wherein:(a) the modified AAV VP2 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises SEQ ID NO:2;(c) the peptide is inserted into the framework protein at a position between residues 450 and 451, numbered relative to SEQ ID NO: 2; and(d) the peptide comprises (i) a core sequence comprising SEQ ID NO:4, SEQ ID NO: 17, SEQ ID NO:80, SEQ ID NO:97, SEQ ID NO: 110, SEQ ID NO: 131, or SEQ ID NO: 132, (ii) a first linker sequence located at the N-terminus of the core sequence, and (iii) a second linker sequence located at the C-terminus of the core sequence.

[0082] Provided is a modified AAV VP2 capsid protein, wherein:(a) the modified AAV VP2 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises a sequence that is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2;(c) the peptide is inserted into the framework protein at a position between residues 450 and 451, numbered relative to SEQ ID NO: 2; and(d) the peptide comprises a sequence that has 0, 1, 2, 3, 4, or 5 amino acid substitutions as compared to any one of SEQ ID NOs:4-29, SEQ ID NOs:80-92, or SEQ ID NOs:97- 122.

[0083] Provided is a modified AAV VP2 capsid protein, wherein:(a) the modified AAV VP2 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2;(c) the peptide is inserted into the framework protein at a position between residues 450 and 451, numbered relative to SEQ ID NO: 2; and24180489702.1(d) the peptide comprises any one of SEQ ID NOs:4-29, SEQ ID NOs:80-92, or SEQ ID NOs:97-122.

[0084] Provided is a modified AAV VP2 capsid protein, wherein:(a) the modified AAV VP2 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2;(c) the peptide is inserted into the framework protein at a position between residues 450 and 451, numbered relative to SEQ ID NO: 2; and(d) the peptide comprises SEQ ID NO: 12, SEQ ID NO:25, or SEQ ID NO:88, SEQ ID NO: 105, or SEQ ID NO: 118.

[0085] Provided is a modified AAV VP2 capsid protein, wherein:(a) the modified AAV VP2 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises SEQ ID NO:2;(c) the peptide is inserted into the framework protein at a position between residues 450 and 451, numbered relative to SEQ ID NO: 2; and(d) the peptide comprises SEQ ID NO: 12, SEQ ID NO:25, or SEQ ID NO:88, SEQ ID NO: 105, or SEQ ID NO: 118.

[0086] Provided is a modified AAV VP3 capsid protein, wherein:(a) the modified AAV VP3 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3;(c) the peptide is inserted into the framework protein at a position between residues 385 and 386, numbered relative to SEQ ID NO:3; and(d) the peptide comprises (i) a core sequence comprising a sequence that has 0, 1, 2, or 3 amino acid substitutions as compared to SEQ ID NO:4, SEQ ID NO: 17, SEQ ID NO:80, SEQ ID NO:97, SEQ ID NO: 110, SEQ ID NO: 131, or SEQ ID NO: 132, (ii) optionally a first linker sequence located at the N-terminus of the core sequence, and (iii) optionally a second linker sequence located at the C-terminus of the core sequence.

[0087] Provided is a modified AAV VP3 capsid protein, wherein:25180489702.1(a) the modified AAV VP3 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3;(c) the peptide is inserted into the framework protein at a position between residues 385 and 386, numbered relative to SEQ ID NO:3; and(d) the peptide comprises (i) a core sequence comprising SEQ ID NO:4, SEQ ID NO: 17, SEQ ID NO:80, SEQ ID NO:97, SEQ ID NO: 110, SEQ ID NO: 131, or SEQ ID NO: 132, (ii) a first linker sequence located at the N-terminus of the core sequence, and (iii) a second linker sequence located at the C-terminus of the core sequence.

[0088] Each linker sequence may predominantly comprise alanines, glycines, or serines. The linker sequences may consist entirely of alanines. Each linker sequence may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids long. The first and the second linker may be identical in sequence. The first and the second linker may differ in sequence. The first and the second linker may be identical in length. The first and the second linker may differ in length.

[0089] Provided is a modified AAV VP3 capsid protein, wherein:(a) the modified AAV VP3 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises SEQ ID NO:3;(c) the peptide is inserted into the framework protein at a position between residues 385 and 386, numbered relative to SEQ ID NO:3; and(d) the peptide comprises (i) a core sequence comprising SEQ ID NO:4, SEQ ID NO: 17, SEQ ID NO:80, SEQ ID NO:97, SEQ ID NO: 110, SEQ ID NO: 131, or SEQ ID NO: 132, (ii) a first linker sequence located at the N-terminus of the core sequence, and (iii) a second linker sequence located at the C-terminus of the core sequence.

[0090] Provided is a modified AAV VP3 capsid protein, wherein:(a) the modified AAV VP3 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3;(c) the peptide is inserted into the framework protein at a position between residues 385 and 386, numbered relative to SEQ ID NO:3; and26180489702.1(d) the peptide comprises a sequence that has 0, 1, 2, 3, 4, or 5 amino acid substitutions as compared to any one of SEQ ID NOs:4-29, SEQ ID NOs:80-92, or SEQ ID NOs:97- 122.

[0091] Provided is a modified AAV VP3 capsid protein, wherein:(a) the modified AAV VP3 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3;(c) the peptide is inserted into the framework protein at a position between residues 385 and 386, numbered relative to SEQ ID NO:3; and(d) the peptide comprises any one of SEQ ID NOs:4-29, SEQ ID NOs:80-92, or SEQ ID NOs:97-122.

[0092] Provided is a modified AAV VP3 capsid protein, wherein:(a) the modified AAV VP3 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3;(c) the peptide is inserted into the framework protein at a position between residues 385 and 386, numbered relative to SEQ ID NO:3; and(d) the peptide comprises SEQ ID NO: 12, SEQ ID NO:25, or SEQ ID NO:88, SEQ ID NO: 105, or SEQ ID NO: 118.

[0093] Provided is a modified AAV VP3 capsid protein, wherein:(a) the modified AAV VP3 capsid protein comprises a framework protein and a peptide that is inserted into the framework protein;(b) the framework protein comprises SEQ ID NO:3;(c) the peptide is inserted into the framework protein at a position between residues 385 and 386, numbered relative to SEQ ID NO:3; and(d) the peptide comprises SEQ ID NO: 12, SEQ ID NO:25, or SEQ ID NO:88, SEQ ID NO: 105, or SEQ ID NO: 118.

[0094] Provided is a modified AAV VP1 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:30.27180489702.1

[0095] Provided is a modified AAV VP1 capsid protein that comprises SEQ ID NO:30.

[0096] Provided is a modified AAV VP2 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 31.

[0097] Provided is a modified AAV VP2 capsid protein that comprises SEQ ID NO:31.

[0098] Provided is a modified AAV VP3 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:32.

[0099] Provided is a modified AAV VP3 capsid protein that comprises SEQ ID NO:32.

[0100] Provided is a modified AAV VP1 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:33.

[0101] Provided is a modified AAV VP1 capsid protein that comprises SEQ ID NO:33.

[0102] Provided is a modified AAV VP2 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:34.

[0103] Provided is a modified AAV VP2 capsid protein that comprises SEQ ID NO:34.

[0104] Provided is a modified AAV VP3 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:35.

[0105] Provided is a modified AAV VP3 capsid protein that comprises SEQ ID NO:35.

[0106] Provided is a modified AAV VP1 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:93.

[0107] Provided is a modified AAV VP1 capsid protein that comprises SEQ ID NO:93.

[0108] Provided is a modified AAV VP2 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:94.

[0109] Provided is a modified AAV VP2 capsid protein that comprises SEQ ID NO:94.

[0110] Provided is a modified AAV VP3 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 95.[OHl] Provided is a modified AAV VP3 capsid protein that comprises SEQ ID NO:95.28180489702.1

[0112] Provided is a modified AAV VP1 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 123.

[0113] Provided is a modified AAV VP1 capsid protein that comprises SEQ ID NO: 123.

[0114] Provided is a modified AAV VP2 capsid protein that is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 124.

[0115] Provided is a modified AAV VP2 capsid protein that comprises SEQ ID NO: 124.

[0116] Provided is a modified AAV VP3 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 125.

[0117] Provided is a modified AAV VP3 capsid protein that comprises SEQ ID NO: 125.

[0118] Provided is a modified AAV VP1 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 126.

[0119] Provided is a modified AAV VP1 capsid protein that comprises SEQ ID NO: 126.

[0120] Provided is a modified AAV VP2 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 127.

[0121] Provided is a modified AAV VP2 capsid protein that comprises SEQ ID NO: 127.

[0122] Provided is a modified AAV VP3 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128.

[0123] Provided is a modified AAV VP3 capsid protein that comprises SEQ ID NO: 128.

[0124] See Table 4 for illustrative modified VP capsid protein sequences.Table 4. Illustrative modified capsid proteins. Inserted peptide in bold. Core sequence bold and underlined. See also SEQ ID NO: 63 (DE 12); SEQ ID NO: 64 (DE25); SEQ ID NO: 96 (DE39); SEQ ID NO: 129 (DE18); and SEQ ID NO: 130 (DE61). _SEQ Protein Amino acid sequenceID NO30 AAV2 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSDE12 VP1 RGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLD29180489702.1SEQ Protein Amino acid sequenceID NO SGDNPYLI<YNHADAEFQERLI<EDTSFGGNLGRAVFQAI<I<RVL (SEQ ID EPLGL VEEP VKT APGKKRP VEHSP VEPD S S SGTGK AGQQP ARK NO:25 in RLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMA bold) DNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYN NHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTV QVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNG SQ AVGRS SF YCLEYFPSQMLRTGNNFTF S YTFED VPFHS S YAHS QSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIR DQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGR DSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKV MITDEEEIRTTNPVATEQYGSVSTNLQRGNAAATVNLVKAAAR QAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHP SPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTG QVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGV YSEPRPIGTRYLTRNL31 AAV2 MAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDA DE12 VP2 DSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGV GNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQS(SEQ ID GASNDNHYFGYSTPWGYFDFNRFHCHF SPRDWQRLINNNWGF NO:25 in RPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQL bold) PYVLGSAHQGCLPPFP AD VFMVPQYGYLTLNNGSQ AVGRS SFY CLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPL IDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGP CYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAM ASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTT NPVATEQYGSVSTNLQRGNAAATVNLVKAAARQAATADVNT QGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGL KHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWE LQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGT RYLTRNL32 AAV2 MATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTS DE12 VP3 TRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNR FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGT(SEQ ID TTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVP NO:25 in QYGYLTLNNGSQ AVGRS SF YCLEYFPSQMLRTGNNFTF SYTFE bold) DVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRL QFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWT GATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQG SEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNAAA TVNLVKAAARQAATADVNTQGVLPGMVWQDRDVYLQGPIW AKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSA AKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKS VNVDFTVDTNGVYSEPRPIGTRYLTRNL33 AAV2 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDS DE25 VP1 RGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLI<YNHADAEFQERLI<EDTSFGGNLGRAVFQAI<I<RVL30180489702.1SEQ Protein Amino acid sequenceID NO(SEQ ID EPLGL VEEP VKT APGKKRP VEHSP VEPD S S SGTGK AGQQP ARK NO: 12 in RLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMA bold) DNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYN NHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTV QVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNG SQ AVGRS SF YCLEYFPSQMLRTGNNFTF S YTFED VPFHS S YAHS QSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIR DQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGR DSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKV MITDEEEIRTTNPVATEQYGSVSTNLQRGNAAATSVGTIRAAR QAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHP SPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTG QVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGV YSEPRPIGTRYLTRNL34 AAV2 MAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDA DE25 VP2 DSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGV GNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQS(SEQ ID GASNDNHYFGYSTPWGYFDFNRFHCHF SPRDWQRLINNNWGF NO: 12 in RPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQL bold) PYVLGSAHQGCLPPFP AD VFMVPQYGYLTLNNGSQ AVGRS SFY CLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPL IDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGP CYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAM ASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTT NPVATEQYGSVSTNLQRGNAAATSVGTIRAARQAATADVNTQ GVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWEL QKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTR YLTRNL35 AAV2 MATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTS DE25 VP3 TRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNR FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGT(SEQ ID TTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVP NO: 12 in QYGYLTLNNGSQ AVGRS SF YCLEYFPSQMLRTGNNFTF SYTFE bold) DVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRL QFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWT GATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQG SEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNAAA TSVGTIRAARQAATADVNTQGVLPGMVWQDRDVYLQGPIWA KIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAA KFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSV NVDFTVDTNGVYSEPRPIGTR YLTRNL93 AAV2 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDS DE39 VP1 RGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLI<YNHADAEFQERLI<EDTSFGGNLGRAVFQAI<I<RVLEPLGL VEEP VKT APGKKRP VEHSP VEPD S S SGTGK AGQQP ARK31180489702.1SEQ Protein Amino acid sequenceID NO(SEQ ID RLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMA NO:88 in DNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYN bold) NHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTV QVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNG SQ AVGRS SF YCLEYFPSQMLRTGNNFTF S YTFED VPFHS S YAHS QSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIR DQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGR DSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKV MITDEEEIRTTNPVATEQYGSVSTNLQRGNAAAFSSDRIKAARQ AATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPS PLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTG QVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGV YSEPRPIGTRYLTRNL94 AAV2 TAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD DE39 VP2 SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVG NSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSG(SEQ ID ASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFR NO:88 in PKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLP bold) YVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYC LEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLI DQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPC YRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMA SHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTN PVATEQYGSVSTNLQRGNAAAFSSDRIKAARQAATADVNTQG VLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKH PPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQ KENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRY LTRNL95 AAV2 MATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTS DE39 VP3 TRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNR FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGT(SEQ ID TTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVP NO:88 in QYGYLTLNNGSQ AVGRS SF YCLEYFPSQMLRTGNNFTF SYTFE bold) DVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRL QFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWT GATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQG SEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNAAA FSSDRIKAARQAATADVNTQGVLPGMVWQDRDVYLQGPIWA KIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAA KFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSV NVDFTVDTNGVYSEPRPIGTRYLTRNL123 AAV2 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDS DE18 VP1 RGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLD SGDNPYLI<YNHADAEFQERLI<EDTSFGGNLGRAVFQAI<I<RVL EPLGL VEEP VKT APGKKRP VEHSP VEPD S S SGTGK AGQQP ARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMA32180489702.1SEQ Protein Amino acid sequenceID NO(SEQ ID DNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYN NO: 105 in NHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW bold) QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTV QVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNG SQ AVGRS SF YCLEYFPSQMLRTGNNFTF S YTFED VPFHS S YAHS QSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIR DQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGR DSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKV MITDEEEIRTTNPVATEQYGSVSTNLQRGNAAANVTNLLTAAR QAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHP SPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTG QVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGV YSEPRPIGTRYLTRNL124 AAV2 TAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD DEI 8 VP2 SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVG NSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSG(SEQ ID ASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFR NO: 105 in PKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLP bold) YVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYC LEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLI DQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPC YRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMA SHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTN PVATEQYGSVSTNLQRGNAAANVTNLLTAARQAATADVNTQ GVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWEL QKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTR YLTRNL125 AAV2 MATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTS DEI 8 VP3 TRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNR FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGT(SEQ ID TTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVP NO: 105 in QYGYLTLNNGSQ AVGRS SF YCLEYFPSQMLRTGNNFTF SYTFE bold) DVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRL QFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWT GATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQG SEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNAAA NVTNLLTAARQAATADVNTQGVLPGMVWQDRDVYLQGPIWA KIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAA KFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSV NVDFTVDTNGVYSEPRPIGTR YLTRNL126 AAV2 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDS DE61 VP1 RGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLD SGDNPYLI<YNHADAEFQERLI<EDTSFGGNLGRAVFQAI<I<RVL (SEQ ID EPLGL VEEP VKT APGKKRP VEHSP VEPD S S SGTGK AGQQP ARK NO: 118 in RLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMAbold) DNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYN33180489702.1SEQ Protein Amino acid sequenceID NO NHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTV QVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNG SQ AVGRS SF YCLEYFPSQMLRTGNNFTF S YTFED VPFHS S YAHS QSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIR DQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGR DSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKV MITDEEEIRTTNPVATEQYGSVSTNLQRGNAAANVTNVLTAAR QAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHP SPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTG QVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGV YSEPRPIGTRYLTRNL127 AAV2 TAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD DE61 VP2 SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVG NSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSG(SEQ ID ASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFR NO: 118 in PKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLP bold) YVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYC LEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLI DQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPC YRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMA SHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTN PVATEQYGSVSTNLQRGNAAANVTNVLTAARQAATADVNTQ GVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWEL QKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTR YLTRNL128 AAV2 MATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTS DE61 VP3 TRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNR FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGT(SEQ ID TTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVP NO: 118 in QYGYLTLNNGSQ AVGRS SF YCLEYFPSQMLRTGNNFTF SYTFE bold) DVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRL QFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWT GATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQG SEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNAAA NVTNVLTAARQAATADVNTQGVLPGMVWQDRDVYLQGPIWA KIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAA KFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTR YLTRNL

[0125] Viral Particles and Payloads

[0126] Provided herein is a viral particle comprising a modified AAV VP1, VP2, and / or VP3 capsid protein disclosed herein.

[0127] Viral Particles34180489702.1

[0128] As used herein, an “AAV virion” (also referred to as an “AAV particle”) refers to an AAV capsid comprising an AAV genome comprising a nucleic acid sequence encoding a gene product of interest. In one embodiment, the AAV virion is an AAV genome comprising (i) a nucleotide sequence encoding a gene product of interest ( / .< ., a transgene) and (ii) at least one AAV ITR sequence, wherein the AAV genome is encapsidated by capsid proteins. The nucleic acid sequence may be flanked by ITR sequences. The ITRs may be from a wild-type AAV serotype.

[0129] The AAV genome may have one or more wildtype AAV genes deleted but may still comprise functional ITR nucleic acid sequences. In embodiments, the AAV genome does not comprise any nucleotide sequences encoding viral proteins, such as the rep (replication) or cap (capsid) genes of AAV.

[0130] Provided is a viral particle comprising a modified AAV VP1, VP2, and / or VP3 capsid protein disclosed herein and a viral genome comprising nucleic acid encoding an RNA, peptide, or protein of interest.

[0131] In embodiments, viral particles comprising the modified AAV VP1, VP2, and / or VP3 capsid proteins disclosed herein have preferred properties as compared to a reference AAV VP1, VP2, and / or VP3 capsid protein.

[0132] To illustrate, a viral particle comprising a modified AAV VP1, VP2, and / or VP3 capsid protein disclosed herein may transduce retinal cells overall with a higher efficiency as compared to a viral particle comprising a reference AAV VP 1, VP2, and / or VP3 capsid protein lacking the modification. A viral particle comprising a modified AAV VP1, VP2, and / or VP3 capsid protein disclosed herein may transduce one or more types of retinal cells with a higher efficiency as compared to a viral particle comprising a reference AAV VP1, VP2, and / or VP3 capsid protein.

[0133] “ Transduction” can refer to the transfer of a transgene into a recipient host cell by a viral vector. Transduction of a target cell by an AAV virion comprising a modified AAV VP1, VP2, and / or VP3 capsid protein disclosed herein leads to transfer of the transgene contained in that AAV virion into the transduced cell. “Host cell” or “target cell” refers to the cell into which the DNA delivery takes place. AAV virions are able to transduce both dividing and nondividing cells.

[0134] Methods of determining the transduction efficiency of viral particles are known in the art. For example, one may use a viral vector comprising a nucleic acid encoding a reporter protein, including, but not limited to, a fluorescent protein. Target cells are then contacted with the viral vectors and expression of the reporter protein is determined (e.g., by measuring RNA 35180489702.1levels or protein levels). Reporter proteins suitable for the methods disclosed herein include, but are not limited to, GFP, YFP, and luciferase. The reporter protein expression is then normalized to the number of viral particles used. Higher reporter protein expression indicates higher transduction efficiency. In some embodiments, a viral particle comprising a modified AAV VP 1 capsid protein disclosed herein may transduct specific subtypes of retinal cells (e.g., rod cells, cone cells, bipolar cells, Mueller glia cells, horizontal cells, ganglion cells, retinal pigment epithelium (RPE) cells, and / or amacrine cells) with a higher efficiency as compared to a viral particle comprising a reference AAV VP1 capsid protein.

[0135] Viral particles comprising a modified AAV VP1, VP2, and / or VP3 capsid protein disclosed herein may also transduce retinal cells overall with a higher level of specificity as compared to a viral particle comprising a reference AAV VP1, VP2, and / or VP3 capsid protein. As used herein, “transduction specificity” may refer to the ability of a viral particle to promote transduction of e.g., retinal vs. non-retinal cells. In some embodiments, a viral particle comprising a modified AAV VP1, VP2, and / or VP3 capsid protein disclosed herein may transduce specific subtypes of retinal cells (e.g., rod cells, cone cells, bipolar cells, Mueller glia cells, horizontal cells, retinal astrocytes, retinal ganglion cells, RPE cells, amacrine cells) with a higher specificity as compared to a viral particle comprising a reference AAV VP1, VP2, and / or VP3 capsid protein. As such, “transduction specificity” may also refer to the ability of a viral particle to promote transduction of e.g., a specific type of cells vs. other cells (other retinal cells and / or non-retinal cells). Methods of determining the transduction specificity of viral particles are known in the art. For example, one may determine the transduction efficiency of a viral particle for retinal cells and divide that transduction efficiency by the transduction efficiency for the same viral particle for non-retinal cells.

[0136] In some embodiments, the modified AAV VP1, VP2, and / or VP3 capsid proteins disclosed herein provide for an at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, or at least 6 fold increase in expression in retinal cells as compared to a reference AAV VP1, VP2, and / or VP3 capsid protein.

[0137] In some embodiments, the modified AAV VP1, VP2, and / or VP3 capsid proteins disclosed herein provide for an at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, or at least 6 fold increase in expression in specific subtypes of retinal cells (e.g., rod cells, cone cells, bipolar cells, Mueller glia cells, horizontal cells, retinal astrocytes, retinal ganglion cells, RPE cells, and / or amacrine cells) as compared to a reference AAV VP1, VP2, and / or VP3 capsid protein.36180489702.1In some embodiments, the modified AAV VP1, VP2, and / or VP3 capsid proteins disclosed herein provide for an at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, or at least 6 fold increase in expression in one or more type of retinal cell as compared to a reference AAV VP1, VP2, and / or VP3 capsid protein.

[0138] In the context of treating disease, the recipient host cell into which the transgene is transduced preferably is a cell that is affected by the disease that is to be treated.

[0139] In some embodiments, the AAV particle comprising a modified AAV VP1, VP2, and / or VP3 capsid protein disclosed herein provides for similar or lower neutralizing antibody (nAb) titers as compared with an AAV particle comprising a reference AAV VP capsid protein.

[0140] In one embodiment, the reference AAV VP1, VP2, and / or VP3 capsid protein is a naturally occurring AAV VP1, VP2, and / or VP3 capsid protein. In one embodiment, the reference AAV VP1, VP2, and / or VP3 capsid protein is an artificial ( / .< ., not naturally occurring) AAV VP1, VP2, and / or VP3 capsid protein.

[0141] In one embodiment, the reference AAV VP1, VP2, and / or VP3 capsid protein is a capsid protein frequently used in the art for transducing the retina. In one embodiment, the reference AAV VP capsid protein is AAV2.7m8. See Khabou et al (2016). In AAV2.7m8, a peptide (LALGETTRPA, SEQ ID NO:36) is inserted at amino acid position 588 of the AAV2 VP1 capsid sequence. In some embodiments, the reference AAV VP capsid protein is derived from AAV2, AAVrh8, or AAVrhlO.

[0142] The viral particles of the disclosure may comprise a modified VP1 capsid protein disclosed herein, and unmodified VP2 and VP3 capsid proteins. The viral particles of the disclosure may comprise a modified VP2 capsid protein disclosed herein, and unmodified VP1 and VP3 capsid proteins. The viral particles of the disclosure may comprise a modified VP3 capsid protein disclosed herein, and unmodified VP1 and VP2 capsid proteins. The viral particles of the disclosure may comprise a modified VP1 and a modified VP2 capsid protein disclosed herein and an unmodified VP3 capsid protein. The viral particles of the disclosure may comprise a modified VP1 and a modified VP3 capsid protein disclosed herein and an unmodified VP2 capsid protein. The viral particles of the disclosure may comprise a modified VP2 and a modified VP3 capsid protein disclosed herein and an unmodified VP1 capsid protein. The viral particles of the disclosure may comprise a modified VP1, a modified VP2, and a modified VP3 capsid protein disclosed herein.

[0143] Provided is a viral particle comprising (a) modified AAV VP1 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,37180489702.1at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:30; (b) modified AAV VP2 capsid protein that is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:31; and / or (c) modified AAV VP3 capsid protein that is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:32.

[0144] Provided is a viral particle comprising (a) modified AAV VP1 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:30; (b) modified AAV VP2 capsid protein that is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:31; and (c) modified AAV VP3 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:32.

[0145] Provided is a viral particle comprising (a) modified AAV VP1 capsid protein comprising SEQ ID NO:30; (b) modified AAV VP2 capsid protein comprising SEQ ID NO:31; and (c) modified AAV VP3 capsid protein comprising SEQ ID NO:32.

[0146] Provided is a viral particle comprising (a) modified AAV VP1 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:33; (b) modified AAV VP2 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:34; and / or (c) modified AAV VP3 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:35.

[0147] Provided is a viral particle comprising (a) modified AAV VP1 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:33; (b) modified AAV VP2 capsid protein that is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:34; and (c) modified AAV VP338180489702.1capsid protein that is at least 8 %, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:35.

[0148] Provided is a viral particle comprising (a) modified AAV VP1 capsid protein comprising SEQ ID NO:33; (b) modified AAV VP2 capsid protein comprising SEQ ID NO:34; and (c) modified AAV VP3 capsid protein comprising SEQ ID NO:35.

[0149] Provided is a viral particle comprising (a) modified AAV VP1 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:93; (b) modified AAV VP2 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:94; and / or (c) modified AAV VP3 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:95.

[0150] Provided is a viral particle comprising (a) modified AAV VP1 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:93; (b) modified AAV VP2 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 94; and (c) modified AAV VP3 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:95.

[0151] Provided is a viral particle comprising (a) modified AAV VP1 capsid protein comprising SEQ ID NO:93; (b) modified AAV VP2 capsid protein comprising SEQ ID NO:94; and (c) modified AAV VP3 capsid protein comprising SEQ ID NO:95.

[0152] Provided is a viral particle comprising (a) modified AAV VP1 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 123; (b) modified AAV VP2 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 124; and / or (c) modified AAV VP3 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%,39180489702.1at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 125.

[0153] Provided is a viral particle comprising (a) modified AAV VP1 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 123; (b) modified AAV VP2 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 124; and (c) modified AAV VP3 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 125.

[0154] Provided is a viral particle comprising (a) modified AAV VP1 capsid protein comprising SEQ ID NO: 123; (b) modified AAV VP2 capsid protein comprising SEQ ID NO: 124; and (c) modified AAV VP3 capsid protein comprising SEQ ID NO: 125.

[0155] Provided is a viral particle comprising (a) modified AAV VP1 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 126; (b) modified AAV VP2 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 127; and / or (c) modified AAV VP3 capsid protein that is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128.

[0156] Provided is a viral particle comprising (a) modified AAV VP1 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 126; (b) modified AAV VP2 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 127; and (c) modified AAV VP3 capsid protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128.40180489702.1

[0157] Provided is a viral particle comprising (a) modified AAV VP1 capsid protein comprising SEQ ID NO: 126; (b) modified AAV VP2 capsid protein comprising SEQ ID NO: 127; and (c) modified AAV VP3 capsid protein comprising SEQ ID NO: 128.

[0158] Viral Genomes

[0159] Provided herein is an AAV virion comprising a modified AAV VP1, VP2, and / or VP3 capsid protein disclosed herein and an AAV genome comprising ITRs, a transgene, and / or additional regulatory elements.

[0160] Functional ITR sequences are useful for the replication, rescue, and packaging of AAV virions. The ITR sequences may be wild-type sequences or may have at least 80%, 85%, 90%, 95%, or 100% sequence identity with wild-type sequences or may be altered by, for example, insertion, mutation, deletion or substitution of nucleotides, as long as they remain functional. In this context, functionality refers to the ability to directly package the genome into the capsid shell and then allow for expression in the host cell to be transduced or target cell. The ITRs can be cloned from the AAV viral genome or excised from a vector comprising the AAV ITRs. The ITR nucleotide sequences can be either ligated at either end to a transgene as defined herein using standard molecular biology techniques, or the wild-type AAV sequence between the ITRs can be replaced with the desired nucleotide sequence. In some embodiments, the AAV genome comprises at least the nucleotide sequences of the ITR regions of one of the AAV serotypes, or nucleotide sequences substantially identical thereto, and at least one nucleotide sequence comprising a transgene (under control of a suitable regulatory element) inserted between the two ITRs.

[0161] In some embodiments, the ITR sequence(s) are derived from AAV serotype AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Hu.l, AAV-Hu.2, AAV-Hu.3, AAV-Hu.6, AAV-Hu.10, AAV-Hu.ll, AAV-Hu.13, AAV-Hu.15, AAV-Hu.16, AAV-Hu.17, AAV-Hu.18, AAV-Hu.19, AAV-Hu.20, AAV-Hu.37, AAV-Hu.45, AAV-Hu.47, AAV-Hu.48, AAV-Hu.49, AAV-Hu.52, AAV-Hu.58, AAVhu68, AAVhu69, AAVhu70, AAVhu71.74, AAVhu72, AAVhu73, AAVhu74.71, AAVhu75, AAVhu76, AAVhu77, AAVhu78.88, AAVhu79, AAVhu80, AAVhu81, AAVhu82, AAVhu83, AAVhu84, AAVhu86, AAVhu87, AAVhu88.78, AAVhu89, AAVhu90, AAVhu91, AAVhu92, hu.T17, hu.T32, hu.T40, hu.T41,Hu.S17, AAVv66, PAK56, Hu.LG15, hu.LvrOl, hu.LvrO2, hu.Lvr03, hu.LvrO4, hu.Lvr05, hu.LvrO6, hu.LvrO7, CVR l, CVR_2, CVR_3, CVR 4, CVR 5, CVR 6, CVR 7, JBL1,JBL2, JBL3, JBL4,JBL5, JBB1, JBB2, JBB3, JBB4, JBB6, JBB7, JBB8, JBB9, JBB11, JBB12, JBB13, CONB23,41180489702.1CONB36, CONB37, CONB39, CONS3, CONS6, CHC129, CHC163, CHC217, CHC367, CHC371, CHC387, CHC442, CHC471, CHC473, CHC508, CHC667, CHC668, CHC685, CHC704, CHC714, CHC767, CHC777, CHC790, CHC790, CHC877, CHC976, CHC985, CHC1010, CHC1017, CHC1020, CHC1024, CHC1024, CHC1158, CHC1260, CHC1273, CHC1286, CHC1286, CHC1343, CHC1350, CHC1449, CHC1449, CHC1534, CHC1570, CHC1591, CHC1602, CHC1704, CHC1919, CHC2040, CHC2087, CHC2102, CHC2107, CHC2112, CHC2128, CHC2141, CHC2206, CHC2208, CHC2320, CHC2497, CHC2557, CHC2731, CHC2806, CHC3013, CHC3086, CHC3142, CHC3511, CHC3765, AAVbb.l,AAVbb.2, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5,AAVcy.6., AAVpi.l, AAVpi.2, AAVpi.3., AAVrh.2,AAVrh.8, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.14, AAVrh.16, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.32, AAVrh.32.33, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh75, AAVrh76, AAVrh77, AAVrh78, AAVrh79, AAVrh81, AAVrh82, AAVrh83, AAVrh84, AAVrh85, AAVrh86, AAVrh87, AAVrh89, AAVrh90, AAVrh91, AAVrh92, AAVrh93, AAVrh94, KNO1_S1, KN02_S2, KN03_S3, KN04_S4, KN05_S5, KN06_S6, KN07_S7, KN08_S8, KN09_S9, KN10_S10, KN11_S11, KN12_S12, KN13_S13, KN14_S14, KN15_S15, KN16_S16, KN17_S17, KN18_S18, KN19_S19, KN20_S20, KN21_S21, KN22_S22, KN23_S23, KN24_S24, KN25_S25, KN26_S26, KN27_S27, KN28_S28, KN29_S29, KN30_S30, KN31_S31, KN32_S32, KN33_S33, KN34_S34, KN35_S35, KN36_S36, KN37_S37, KN38_S38, KN39_S39, KN40_S40, KN41_S41, KN42_S42, KN43_S43, KN44_S44, KN45_S45, KN46_S46, KN47_S47, KN48_S48, KN49_S49, KN50_S50, KN51_S51, KN52_S52, KN53_S53, KN54_S54, KN55_S55, KN56_S56, KN57_S57, KN58_S58, KN59_S59, KN60_S60, KN61_S61, KN62_S62, KN63_S63, KN64_S64, KN65_S65, KN66_S66, KN67_S67, KN68_S68, KN69_S69, KN70_S70, KN71_S71, KN72_S72, KN73_S73, KN74_S74, KN75_S75, KN76_S76, KN77_S77, KN78_S78, KN79_S79, KN80_S80, KN81_S81, KN82_S82, AAV-ra.l, stain YY.12, YY.25, YY.54, YY.78, YY.80, YY.93, XM.70, GZ.512, HD.16,HD.2O, HD.43, HD.94, MLP.6, MLP.26, AAVpol, AAVpo2.1, AAVpo4, AAVpo5, AAVpo6, AAVpo7, AAVpo8, AAV-Gol, VR-865, DA-1, YZ-1, ZN1,BR_DF12, RS / BR / 15 / 1R, GA / 1360 / 1994, 09YN, 1285, 10HB, 07YN, YNM, BAAV, BSRI1, AAV2-HBKO, AAV2.7m8, AAV2.GL, AAV2.NN, AAV44.9, AAV44.9(E531D), AAV8BP2, AAV6-K531E-R576Q-K493S-K459S, AAV9.GL, AAV9.NN, ShHIO, or variants thereof.

[0162] In one embodiment, the ITR sequence(s) are derived from AAV2.42180489702.1

[0163] An AAV genome can comprise single- stranded or double-stranded (self-complementary) DNA. The single-stranded nucleic acid molecule is either sense or antisense strand, as both polarities are equally capable of packaging into AAV capsids. Single-stranded AAV genomes may utilize the wild-type AAV2 ITR sequences, and double-stranded (self-complementary) AAV genomes may utilize a modified version of the ITRs.

[0164] In embodiments, the AAV genome further comprises an expression control sequence, including one that is operably linked to a transgene. As used herein, “operatively linked” refers to a first molecule joined to a second molecule, wherein the molecules are so arranged that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. For example, an expression control sequence is operatively linked to a transcribable polynucleotide molecule if the expression control sequence modulates transcription of the transcribable polynucleotide molecule of interest in a cell. Additionally, two portions of an expression control sequence are operatively linked to one another if they are joined such that the transcription-activating functionality of one portion is not adversely affected by the presence of the other portion. Two sequences may be operatively linked to one another by way of a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operatively linked to one another with no intervening nucleotides present.

[0165] Expression control sequences can include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency ( / .< ., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein processing and / or secretion. The AAV genome may also comprise additional elements, such as a 5' leader sequence, an intron, or a 3' non-translated sequence.

[0166] A great number of expression control sequences, e.g., native, constitutive, inducible and / or tissue-specific, are known in the art and may be utilized to drive expression of the transgene, depending upon the type of expression desired. For eukaryotic cells, expression control sequences commonly used include a promoter, an enhancer, and a polyadenylation sequence which may include splice donor and acceptor sites. The polyadenylation sequence generally is inserted following the transgene and before the 3' ITR sequence.

[0167] As used herein, the term “promoter” or “transcription regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the 43180489702.1transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.

[0168] A “constitutive” promoter is a promoter that is active ( / .< ., facilitates expression of the operatively linked transgene) in most tissues under most physiological and developmental conditions.

[0169] An “inducible” promoter is a promoter that is physiologically or developmentally regulated, e.g., by the presence or application of an inducer molecule.

[0170] A “tissue specific” promoter is preferentially active in specific types of tissues or cells and leads to increased expression in that tissue or cell as compared to in a control tissue or cell. A person skilled in the art may choose an appropriate control tissue or cell depending on the desired application. For example, expression in the control tissue or cell could refer to an average expression in all non-target tissues. Alternatively, the person skilled in the art may be specifically interested in achieving preferential expression in the target tissue as compared to a specific control tissue or a cell (e.g., because transfection or transduction of that control tissue or cell may lead to undesired biological effects).

[0171] In one embodiment, the AAV genome comprises a constitutive promoter. In one embodiment, the AAV genome comprises an inducible promoter. In one embodiment, the AAV genome comprises a promoter that allows preferential expression in rod cells, cone cells, bipolar cells, Mueller glia cells, horizontal cells, retinal astrocytes, retinal ganglion cells, RPE cells, and / or amacrine cells.

[0172] In one embodiment, the AAV genome comprises a promoter operatively linked to a transgene. In some embodiments, the promoter is a metabotropic glutamate receptor 6 (GRM6) promoter, cytomegalovirus (CMV) promoter, cytomegalovirus early enhancer / chicken b-actin (CAG) promoter, elongation factor 1 -alpha 1 (EFla) promoter, SV40 promoter, chicken betaactin (CBA) promoter, rhodopsin (rho) promoter, neural retina-specific leucine zipper protein (NRL) promoter, phosphodiesterase 6B (PDE6B) promoter, human rhodopsin kinase (hRK) promoter, human L-opsin promoter or a promoter derived therefrom, human M-opsin promoter or a promoter derived therefrom, human S-opsin promoter or a promoter derived therefrom, retinoid isomerohydrolase (retinal pigment epithelium-specific 65 kDa protein, RPE65) promoter, bestrophin-1 (BEST1) promoter, membrane protein MLC1 (Mlcl) promoter,44180489702.1interphotoreceptor retinoid-binding protein (IRBP) promoter, or a neurofilament heavy (NEFH) promoter. In some embodiments, the promoter is a CAG promoter, CMV promoter, RK promoter, L-opsin derived promoter, or M-opsin derived promoter. In some embodiments, the promoter is a human RK promoter, human L-opsin derived promoter, or human M-opsin derived promoter.

[0173] In some embodiments, the AAV genome comprises a nucleic acid regulatory element disclosed in International Patent Publication No. WO2024079665A1, entitled NUCLEIC ACID REGULATORY ELEMENTS FOR CONSTITUTIVE GENE EXPRESSION AND METHODS OF USE, incorporated herein by reference in its entirety.

[0174] In some embodiments, the AAV genome comprises a promoter disclosed in International Patent Publication No. WO2016128722A1, entitled OPTIMIZED RPE65 PROMOTER and coding sequences, which is incorporated herein by reference in its entirety. In some embodiments, the promoter essentially consists of (a) a sequence of no more than 800 contiguous nucleotides from SEQ ID NO:7 comprising nucleotides 12-761 of SEQ ID NO:65, or (b) a sequence having at least 90% sequence identity to said sequence of (a). In one embodiment, the promoter comprises the sequence of SEQ ID NO:65. In some embodiments, the promoter essentially consists of a sequence having at least 90% sequence identity to said sequence of SEQ ID NO: 65, wherein the sequence of the promoter is no longer than 800 nucleotides. In some embodiments, the promoter consists of (a) a sequence of no more than 800 contiguous nucleotides from SEQ ID NO:79 comprising nucleotides 12-761 of SEQ ID NO:65, or (b) a sequence having at least 90% sequence identity to said sequence of (a).

[0175] AGATCTTCGAAATACTCTCAGAGTGCCAAACATATACCAATGGACAAG AAGGTGAGGCAGAGAGCAGACAGGCATTAGTGACAAGCAAAGATATGCAGAAT TTCATTCTCAGCAAATCAAAAGTCCTCAACCTGGTTGGAAGAATATTGGCACTGA ATGGTATCAATAAGGTTGCTAGAGAGGGTTAGAGGTGCACAATGTGCTTCCATA ACATTTTATACTTCTCCAATCTTAGCACTAATCAAACATGGTTGAATACTTTGTTT ACTATAACTCTTACAGAGTTATAAGATCTGTGAAGACAGGGACAGGGACAATAC CCATCTCTGTCTGGTTCATAGGTGGTATGTAATAGATATTTTTAAAAATAAGTGA GTTAATGAATGAGGGTGAGAATGAAGGCACAGAGGTATTAGGGGGAGGTGGGC CCCAGAGAATGGTGCCAAGGTCCAGTGGGGTGACTGGGATCAGCTCAGGCCTGA CGCTGGCCACTCCCACCTAGCTCCTTTCTTTCTAATCTGTTCTCATTCTCCTTGGG AAGGATTGAGGTCTCTGGAAAACAGCCAAACAACTGTTATGGGAACAGCAAGCC CAAATAAAGCCAAGCATCAGGGGGATCTGAGAGCTGAAAGCAACTTCTGTTCCC CCTCCCTCAGCTGAAGGGGTGGGGAAGGGCTCCCAAAGCCATAACTCCTTTTAA45180489702.1GGGATTTAGAAGGCATAAAAAGGCCCCTGGCTGAGAACTTCCTTCTTCATTCTGC AGTTGG (SEQ ID NO:65). SEQ ID NO:65 corresponds to SEQ ID NO:2 in International Patent Publication No. WO2016128722A1.

[0176] TATTGTGCAAATAAGTGCTCACTCCAAATTAGTGGTATATTTATTGAAG TTTAATATTGTGTTTGTGATACAGAAGTATTTGCTTTAATTCTAAATAAAAATTTT ATGCTTTTATTGCTGGTTTAAGAAGATTTGGATTATCCTTGTACTTTGAGGAGAAG TTTCTTATTTGAAATATTTTGGAAACAGGTCTTTTAATGTGGAAAGATAGATATTA ATCTCCTCTTCTATTACTCTCCAAGATCCAACAAAAGTGATTATACCCCCCAAAA TATGATGGTAGTATCTTATACTACCATCATTTTATAGGCATAGGGCTCTTAGCTGC AAATAATGGAACTAACTCTAATAAAGCAGAACGCAAATATTGTAAATATTAGAG AGCTAACAATCTCTGGGATGGCTAAAGGATGGAGCTTGGAGGCTACCCAGCCAG TAACAATATTCCGGGCTCCACTGTTGAATGGAGACACTACAACTGCCTTGGATGG GCAGAGATATTATGGATGCTAAGCCCCAGGTGCTACCATTAGGACTTCTACCACT GTCCCTAACGGGTGGAGCCCATCACATGCCTATGCCCTCACTGTAAGGAAATGA AGCTACTGTTGTATATCTTGGGAAGCACTTGGATTAATTGTTATACAGTTTTGTTG AAGAAGACCCCTAGGGTAAGTAGCCATAACTGCACACTAAATTTAAAATTGTTA ATGAGTTTCTCAAAAAAAATGTTAAGGTTGTTAGCTGGTATAGTATATATCTTGC CTGTTTTCCAAGGACTTCTTTGGGCAGTACCTTGTCTGTGCTGGCAAGCAACTGA GACTTAATGAAAGAGTATTGGAGATATGAATGAATTGATGCTGTATACTCTCAGA GTGCCAAACATATACCAATGGACAAGAAGGTGAGGCAGAGAGCAGACAGGCAT TAGTGACAAGCAAAGATATGCAGAATTTCATTCTCAGCAAATCAAAAGTCCTCA ACCTGGTTGGAAGAATATTGGCACTGAATGGTATCAATAAGGTTGCTAGAGAGG GTTAGAGGTGCACAATGTGCTTCCATAACATTTTATACTTCTCCAATCTTAGCACT AATCAAACATGGTTGAATACTTTGTTTACTATAACTCTTACAGAGTTATAAGATC TGTGAAGACAGGGACAGGGACAATACCCATCTCTGTCTGGTTCATAGGTGGTAT GTAATAGATATTTTTAAAAATAAGTGAGTTAATGAATGAGGGTGAGAATGAAGG CACAGAGGTATTAGGGGGAGGTGGGCCCCAGAGAATGGTGCCAAGGTCCAGTGG GGTGACTGGGATCAGCTCAGGCCTGACGCTGGCCACTCCCACCTAGCTCCTTTCT TTCTAATCTGTTCTCATTCTCCTTGGGAAGGATTGAGGTCTCTGGAAAACAGCCA AACAACTGTTATGGGAACAGCAAGCCCAAATAAAGCCAAGCATCAGGGGGATCT GAGAGCTGAAAGCAACTTCTGTTCCCCCTCCCTCAGCTGAAGGGGTGGGGAAGG GCTCCCAAAGCCATAACTCCTTTTAAGGGATTTAGAAGGCATAAAAAGGCCCCT GGCTGAGAACTTCCTTCTTCATTCTGCAGTTGG (SEQ ID NO:79). SEQ ID NO:79 corresponds to SEQ ID NO:1 in International Patent Publication No. WO2016128722A1.46180489702.1

[0177] In some embodiments, the AAV genome comprises a transcriptional control unit (TCU) disclosed in International Patent Publication No. WO2019138250A1, entitled COMPOSITIONS AND METHODS FOR TREATING RETINAL DISORDER, which is incorporated herein by reference in its entirety. The TCU may comprise a fragment of the M / L opsin Locus Control Region (LCR). In one embodiment, the TCU comprises a fragment of the human M / L opsin LCR. In one embodiment, the TCU comprises a promoter region, such as the M opsin promoter or a fragment thereof. In one embodiment, the TCU comprises the human M opsin promoter or a fragment thereof. In some embodiments, the TCU comprises fragments and / or variants of the human M / L opsin LCR and the human M-opsin promoter or a fragment thereof, wherein the TCU has cone photoreceptor-specific promoter activity. In some embodiments, the TCU has no more than 2500 nucleotides in length and comprises in a 5' to 3' direction: (a) an LCR comprising (i) SEQ ID NO: 66; or (ii) a sequence having at least 90% sequence identity to said sequence (a)(i); and (b) a promoter element comprising SEQ ID NO: 67 and (i) at least the last 200 nucleotides but no more than 1100 nucleotides of SEQ ID NO: 68; or (ii) a sequence having at least 90% sequence identity to said sequence (b)(i). In one embodiment, the TCU comprises SEQ ID NO:69. In one embodiment, the TCU comprises (a) an LCR comprising SEQ ID NO: 66; and (b) a promoter element comprising SEQ ID NO: 70.

[0178] TAGGAATAGAAGGGTGGGTGCAGGAGGCTGAGGGGTGGGGAAAGGGC ATGGGTGTTTCATGAGGACAGAGCTTCCGTTTCATGCAATGAAAAGAGTTTGGAG ACGGATGGTGGTGACTGGACTATACACTTACACACGGTAGCGATGGTACACTTTG TATTATGTATATTTTACCACGATCTTTTTAAAGTGTCAAAGGCAAATGGCCAAAT GGTTCCTTGTCCTATAGCTGTAGCAGCCATCGGCTGTTAGTGACAAAGCCCCTGA GTCAAGATGACAGCAGCCCCCATAACTCCTAATCGGCTCTCCCGCGTGGAGTCAT TTAGGAGTAGTCGCATTAGAGACAAGTCCAACATCTAATCTTCCACCCTGGCCAG GGCCCCAGCTGGCAGCGAGGGTGGGAGACTCCGGGCAGAGCAGAGGGCGCTGA CATTGGGGCCCGGCCTGGCTTGGGTCCCTCTGGCCTTTCCCCAGGGGCCCTCTTTC CTTGGGGCTTTCTTGGGCCGCCACTGCTCCCGCTCCTCTCCCCCCATCCCACCCCC TCACCCCCTCGTTCTTCATATCCTTCTCTAGTGCTCCCTCCACTTTCATCCACCCTT CTGCAAGAGTGTGGGACCACAAATGAGTTTTCACCTGGCCTGGGGACACACGTG CCCCCACAGGTGCTGAGTGACTTTCTAGGACAGTAATCTGCTTTAGGCTAAAATG GGACTTGATCTTCTGTTAGCCCTAATCATCAATTAGCAGAGCCGGTGAAGGTGCA GAACCTACCGCCTTTCCAGGCCTCCTCCCACCTCTGCCACCTCCACTCTCCTTCCT GGGATGTGGGGGCTGGCACACGTGTGGCCCAGGGCATTGGTGGGATTGCACTGA GCTGGGTCATTAGCGTAATCCTGGACAAGGGCAGACAGGGCGAGCGGAGGGCCA47180489702.1GCTCCGGGGCTCAGGCAAGGCTGGGGGCTTCCCCCAGACACCCCACTCCTCCTCT GCTGGACCCCCACTTCATAGGGCACTTCGTGTTCTCAAAGGGCTTCCAAATAGCA TGGTGGCCTTGGATGCCCAGGGAAGCCTCAGAGTTGCTTATCTCCCTCTAGACAG AAGGGGAATCTCGGTCAAGAGGGAGAGGTCGCCCTGTTCAAGGCCACCCAGCCA GCTCATGGCGGTAATGGGACAAGGCTGGCCAGCCATCCCACCCTCAGAAGGGAC CCGGTGGGGCAGGTGATCTCAGAGGAGGCTCACTTCTGGGTCTCACATTCTTG(SEQ ID NO:66). SEQ ID NO:66 corresponds to SEQ ID NO:1 in International Patent Publication No. WO2019138250A1.

[0179] TCTAGA (SEQ ID NO:67). SEQ ID NO:67 corresponds to SEQ ID NO: 16 in International Patent Publication No. WO2019138250A1.

[0180] TAAAAAGCAAGTCTTGCCAGGGCAGTGGTGTGCACCTGTGGTCCCAGC TACTCAGGATGCTGAGGCAGGAGGATTACTTGTGCCCAGCAAGTAGAGGCTGCA GTGACCTGTGACTGTGCTACTGCCCTCCAACCTGGGTGACAGAGTGAGACCTTGT CTCAAAAAAAAAAGAGCGGGGGGGGGGGGCCGGGCCGGGCGTGGTGGCTCACA GCTGTAATCCCAGCACTTTGGGAAGCCAAGGCGGGTGGATCACTTGAGGTCAGG AGTTTGAGACCATCATGGTCAACACTGCGAAACACTGTCCCTACTAAAAATACA AAAATTAGCCGGGCATGGTGGCACACACCTGTAATCCCAGCTACTGGGGAGGCT GAGGCAGGAGAATTGCTTGAGCCGGGGAGACGGAGGTTGCAGTGAGCCGAGAC TGCGCCACTGCACTCCAGCCTGACTGACAAGAGTGAGATTGTCTCAAAAAAAAA AAAAAAGTAATCACTAGAAAAGAAGCTACATATGTACATAACATCCAAATAACC AAGAGGAGAAAAAAATGGGACTTGATTAATCAAAACAAAAACAAAAAAGAAAG AAAGAAAGGGGGAGAAAATAAAACAAGGGCTGGGTGTGCTGGCTCATGCCTGT AATCCCAGCACTTTGGAAGCCAAGGTGGGTGGATCTCTTGAGCTCAGGAGGTCA AGACCAGCCTGGGCAACATGGCGAAACCCCGTCTCTATTAAAAAAAAAATTAAT ACAACAATTATCCTGGAGTGGTGGTGCACACCTGTAGTCCCAGCTACCCAGGAC GCTGAGACGGGAGGATCGCTTGATCCCGGGGATGTCGAGGCTGCCGTGATCGCA CCACTGCCCTCCAGCCAGGGTGGCAGACTGAGACCCCATCTCAAAAAATAAATA AATAAAAGCAAACAAGAAAAAAAAAGGCTTGAAACATATCTGATAGATAAAGG GCTAATCAACACAATATATAAAGAACTGCAAATCAGTAAACTAAGAGCAAATAA CCCAATATAAAGACATTAAAGGGTAGCCACGGACATCTCAGACGACGAAAAACA AAAGACAGTAAACGTATAATAAAACATGTAATTGCAAGGTGATCCGGGAATAGT AAGCGAAAAGCAACAATTAAATACTATTTTCTCATCCACCAGAACGCCAAAAAT TAAAAAGCCTAACAATGTCCAGGGCTGGCGAGAATGTGGCAGAAGGTGATGTCA CATACCCTGCAAGTGGGAATCTAAACAGATTCAGGGTTTTGGTTTTTTTTTAATCG48180489702.1CAATTAGGTGGCCTGTTAAATTTTTTTTCTTGAGACAGAGTTTTGCTCTTGTTGCC CAGGCTGGAGTGCAATGGCTCGATCTTGGCTCACCGCAACCTCGACCTCCCAGGT ACAAGCGATTCTCCTGTCTCAGCCTCCCAAGTAGCTGGGAGTACAGGTATTTGCC ACTAAGCCCAGCTAATTGTTTTTTATTTAGTAGAAACGGGGTTTCACCATGTTAGT CAGGCTGGTCGGGAACTCCTGACCTCAGGAGATCTACCCGCCTTGGCCTCCCAAA GTGCTGGGATTACAGGCGTGTGCCACTGTGCCCAGCCACTTTTTTTTAGACAGAG TCTTGGTCTGTTGCCCAGGCTAGAGTTCAGTGGCGCCATCTCAGCTCACTGCAAC CTCCGCCTCCCAGATTCAAGCGATTCTCCTGCCTCGACCTCCCAGTAGCTGGGAT TACAGGTTTCCAGCAAATCCCTCTGAGCCGCCCCCGGGGGCTCGCCTCAGGAGCA AGGAAGCAAGGGGTGGGAGGAGGAGGTCTAAGTCCCAGGCCCAATTAAGAGAT CAGATGGTGTAGGATTTGGGAGCTTTTAAGGTGAAGAGGCCCGGGCTGATCCCA CTGGCCGGTATAAAGCACCGTGACCCTCAGGTGACGCACCATCTAGAGCTGCCG TCGGGGACAGGGCTTTCCATAGCC (SEQ ID NO:68). SEQ ID NO:68 corresponds to SEQ ID NO: 17 in International Patent Publication No. WO2019138250A1.

[0181] TAGGAATAGAAGGGTGGGTGCAGGAGGCTGAGGGGTGGGGAAAGGGC ATGGGTGTTTCATGAGGACAGAGCTTCCGTTTCATGCAATGAAAAGAGTTTGGAG ACGGATGGTGGTGACTGGACTATACACTTACACACGGTAGCGATGGTACACTTTG TATTATGTATATTTTACCACGATCTTTTTAAAGTGTCAAAGGCAAATGGCCAAAT GGTTCCTTGTCCTATAGCTGTAGCAGCCATCGGCTGTTAGTGACAAAGCCCCTGA GTCAAGATGACAGCAGCCCCCATAACTCCTAATCGGCTCTCCCGCGTGGAGTCAT TTAGGAGTAGTCGCATTAGAGACAAGTCCAACATCTAATCTTCCACCCTGGCCAG GGCCCCAGCTGGCAGCGAGGGTGGGAGACTCCGGGCAGAGCAGAGGGCGCTGA CATTGGGGCCCGGCCTGGCTTGGGTCCCTCTGGCCTTTCCCCAGGGGCCCTCTTTC CTTGGGGCTTTCTTGGGCCGCCACTGCTCCCGCTCCTCTCCCCCCATCCCACCCCC TCACCCCCTCGTTCTTCATATCCTTCTCTAGTGCTCCCTCCACTTTCATCCACCCTT CTGCAAGAGTGTGGGACCACAAATGAGTTTTCACCTGGCCTGGGGACACACGTG CCCCCACAGGTGCTGAGTGACTTTCTAGGACAGTAATCTGCTTTAGGCTAAAATG GGACTTGATCTTCTGTTAGCCCTAATCATCAATTAGCAGAGCCGGTGAAGGTGCA GAACCTACCGCCTTTCCAGGCCTCCTCCCACCTCTGCCACCTCCACTCTCCTTCCT GGGATGTGGGGGCTGGCACACGTGTGGCCCAGGGCATTGGTGGGATTGCACTGA GCTGGGTCATTAGCGTAATCCTGGACAAGGGCAGACAGGGCGAGCGGAGGGCCA GCTCCGGGGCTCAGGCAAGGCTGGGGGCTTCCCCCAGACACCCCACTCCTCCTCT GCTGGACCCCCACTTCATAGGGCACTTCGTGTTCTCAAAGGGCTTCCAAATAGCA TGGTGGCCTTGGATGCCCAGGGAAGCCTCAGAGTTGCTTATCTCCCTCTAGACAG49180489702.1AAGGGGAATCTCGGTCAAGAGGGAGAGGTCGCCCTGTTCAAGGCCACCCAGCCA GCTCATGGCGGTAATGGGACAAGGCTGGCCAGCCATCCCACCCTCAGAAGGGAC CCGGTGGGGCAGGTGATCTCAGAGGAGGCTCACTTCTGGGTCTCACATTCTTGGA TCACAGGTATTTGCCACTAAGCCCAGCTAATTGTTTTTTATTTAGTAGAAACGGG GTTTCACCATGTTAGTCAGGCTGGTCGGGAACTCCTGACCTCAGGAGATCTACCC GCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCGTGTGCCACTGTGCCCAGCCACTTTTTTTTAGACAGAGTCTTGGTCTGTTGCCCAGGCTAGAGTTCAGTGGCGCCATCTCAGCTCACTGCAACCTCCGCCTCCCAGATTCAAGCGATTCTCCTGCCTCGACCT CCCAGTAGCTGGGATTACAGGTTTCCAGCAAATCCCTCTGAGCCGCCCCCGGGGG CTCGCCTCAGGAGCAAGGAAGCAAGGGGTGGGAGGAGGAGGTCTAAGTCCCAG GCCCAATTAAGAGATCAGATGGTGTAGGATTTGGGAGCTTTTAAGGTGAAGAGG CCCGGGCTGATCCCACTGGCCGGTATAAAGCACCGTGACCCTCAGGTGACGCAC CATCTAGAGCTGCCGTCGGGGACAGGGCTTTCCATAGCC (SEQ ID NO: 69). SEQ ID NO: 69 corresponds to SEQ ID NO: 15 in International Patent Publication No. WO2019138250A1.

[0182] ACAGGTATTTGCCACTAAGCCCAGCTAATTGTTTTTTATTTAGTAGAAA CGGGGTTTCACCATGTTAGTCAGGCTGGTCGGGAACTCCTGACCTCAGGAGATCT ACCCGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCGTGTGCCACTGTGCCCAG CCACTTTTTTTTAGACAGAGTCTTGGTCTGTTGCCCAGGCTAGAGTTCAGTGGCG CCATCTCAGCTCACTGCAACCTCCGCCTCCCAGATTCAAGCGATTCTCCTGCCTC GACCTCCCAGTAGCTGGGATTACAGGTTTCCAGCAAATCCCTCTGAGCCGCCCCC GGGGGCTCGCCTCAGGAGCAAGGAAGCAAGGGGTGGGAGGAGGAGGTCTAAGT CCCAGGCCCAATTAAGAGATCAGATGGTGTAGGATTTGGGAGCTTTTAAGGTGA AGAGGCCCGGGCTGATCCCACTGGCCGGTATAAAGCACCGTGACCCTCAGGTGA CGCACCATCTAGAGCTGCCGTCGGGGACAGGGCTTTCCATAGCC (SEQ ID NO:70). SEQ ID NO:70 corresponds to SEQ ID NO:3 in International Patent Publication No. WO2019138250A1.

[0183] In some embodiments, the promoter comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to a rod-specific promoter of any one of SEQ ID NOs:37-62. In some embodiments, the promoter comprises any one of SEQ ID NOs:37-62. See Table 5.Table 5. Illustrative rod-specific promoter sequences disclosed in U.S. Provisional Application No. 63 / 709,883, entitled PROMOTERS FOR DRIVING GENE EXPRESSION50180489702.1IN ROD PHOTORECEPTORS AND USES THEREOF, which is incorporated herein by reference in its entirety.SEQ Promoter SequenceID NO37 Component AAACCAGAAAGTCTCTAGCTGTCCAGAGGACATAGCACAG B AGGCCCATGGTCCCTATTTCAAACCCAGGCCACCAGACTG AGCTGGGACCTTGGGACAGACAAGTCATGCAGAAGTTAG GGGACCTTCTCCTCCCTTTTCCTGGATCCTGAGTACCTCTC CTCCCTGACCTCAGGCTTCCTCCTAGTGTCACCTTGGCCCC TCTTAGAAGCCAATTAGGCCCTCAGTTTCTGCAGCGGGGA TTAATATGATTATGAACACCCCCAATCTCCCAGATGCTGAT TCAGCCAGGAGCTTAGGAGGGGGAGGTCACTTTATAAGGG TCTGGGGGGGTCAGAACCCAGAGTCATC38 Component ACAGAGTGGAGAACCGAAGCACACTCTCGGGAGGGCGGG Al GTAGCTGGCTGCACCCAGGCTGTGTAGCCTCAGTCCAGAT GTAAGGGTGGGTGGAAAAGAGCCTTGCCCAATGAGGGAG AACAGTGAAACCAAGGCCATAGGGTCTAAAGATTCACGA ACCAGGCTCTCATGGAGAAAGCAGGTGAGGTTTACTGTAT AGATGGGTGTGCCCCTACCCCACACTGAGGCTTCCTCGTCT GAGCAAACTGAGGCCCAGAGAGGGGAAGGAAGCAGGACT ACCATGGTGACTCAAAGACCAGCTAGAATCCAGCCTCCTC TCCTCGAGGCTTCCACTGCCCCACGCCAGGCCTGTGTGACT CAGTCTAGGGCCTTTCCATTACCCCAGCTAAACCTTTCTTT AGTCATTTATACCATGGTGTGAATGGCTGGCTGGTCTTTCC TGAGAGCTATCTTTGATGAGGGGAGGGAGGCATAGCCAG GTTTGGGAAGCTGATACCCCAGGAAGCCCAGTTGACTGTG TGGGTTATAGCCCAGGCTGTCACTGATTTGTAACGGGACCT39 Component CTATAATCCTGGCACTTTGGGAGGCCGAGGAGGGTGGATC A2 GCTTGAGCCAGGAGTTCAGGACCAGCCTGGGCAACATAGC GAGACTCCACCCCTACAAAAAATACAAAAACTAGTGGTGT GCACTTGTGGTCCCAGCTACTCAGGAGGCTGAGGTGAGAG GATCGCTTGAGCCCAGGAGGCAGAGGCTACAGTGAGCTAT GATTGTGGCACTGCACTCCAGCCTGGGCGACAGAGACCTT GTCTCAAAACTTTTTTTTTCTTCGTCAAGCTTTACAGAATA AAGAGCACTGTCACCTCAGTGATGGCTGTTAGTTCCCCAT CACCAGGGCTCCATGAGGTTGCAATTGTGAAACTCACAAA GGAGGAACCTGAGACAGAGAGGGGAAGTACTGAGATCAT CTAGGTCCATTCCCCCACTCACTCGTTCATTCAACAAATAT TCAGGAGCACCTTCTAGGTGCCAGGCCCTGGAGACACATC AGTGAACAAAACAGACATCATCCCACCTCTTTCCACTACA GGCCAAGCACCATGCTGGTCTCTGGGAACCCTGTTGTGAG CAAGACAGACCCAGGCTTACCCTTGTGGACTCATGTTACA GGCAGGGAGACGGGCACAAAACACAAATAAAAAGCTTCC ATGCTGTCAGAAGCACTATGCAAAAAGCAAGATGCTGAG GTACTGCTAAGCTGTGTGGGATGGGGGCTCAGCCCGGCCA GGGA40 Component CAATGATGGAGACCGGGGCGGGGTCCAGAGGTAACTTGTC GCCCCGCCCCGATAGCCTAGGGCCCAGCGGGGATTAAGTT51180489702.1SEQ Promoter SequenceID NOTTTTTTTTTTTTCTTTTGAGGAATCTTACTCCAATGATGGCGCCTGCTGAATTCGAGGAATCTTACTAGACCGGGGCGGGGC CACATAAGGTTGCAAATCCCACAGAGAGCCTGCGCCTGGG GTTGTTTTCTGTAGGCGCCTGTGCCCCCGCCCCTGCCACCA ATGATGGCGCCTGCTGAATTC41 Component GAAGGGTTGGGGGGGCTGAGCTGGATTCACCTGTCCTTGT D CTCTGATTGGCTCTTGGACACCCCTAGCCCCCAAATCCCAC TAAGCAGCCCCACCAGGGATTGCACAGGTCCGTAGAGAGC CAGTTGATTGCAGGTCCTCCTGGGGCCAGAAGGGTGCCTG GGAGGCCAGGTTCTGGGGATCCCCTCCATCCAGAAGAACC ACCTGCTCACTCTGTCCCTTCGCCTGCTGCTGGGAC42 Component CCAATGATGGAGACCGGGGCGGGGTCCAGAGGTAACTTGT E GTTCTCTCTCTGTCTCTCTTCTGCATTTATGTCGCATGAGGAGAGTCCCTCTAAGCCTGCCGAGAGTAGTATATGAGGCTTGCCTGCTCAATTCGAGGAATCTTACTAGACCGGGGCGGGGC CACATAAGGTTGCAAATCCCACAGAGAGCCTGCGCCTGGG GTTGTTTTCTGTAGGCGCCTGTGCCCCCGCCCCTGCCACCA ATGATGGCGCCTGCTCAATTCCTATGTGTCTGGCACCAGA AACGGAAGCTGCAGGTTGCAGCCCCTGCCCTCATGGAGCT CCTCCTGTCAGAGGAGTGTGGGGACTGGATGACTCCAGAG GTAACTTGTGGGGGAACGAACAGGTAAGGGGCTGTGTGA CGAGATGAGAGACTGGGAGAAT43 Component AAACCAGAAAGTCTCTAGCTGTCCAGAGGACATAGCACAG F AGGCCCATGGTCCCTATTTCAAACCCAGGCCACCAGACTG AGCTGGGACCTTGGGACAGACAAGTCATGCAGAAGTTAG GGGACCTTCTCCTCCCTTTTCCTGGATACTGAGTACCTCTC CTCCCTGACCTCAGGCTTCCTCCTAGTGTCACCTTGGCCCC TCTTAGAAGCCAATTAGGCCCTCAGTTTCTGCAGCGGGGA TTAATATGATTATGAACACCCCCAATCTCCCAGATGCTGAT TCAGCCAGGAGCTTAGGAGGGG44 Component GAGGTCACTTTATAAGGGTCTGGGGGGGTCAGAACCCCTA G GTTCATCCAGCTGGAGCGAGCCGAACGGTCGTTGAGCCAA GAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTA ATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTT TCAGGTTGGCCGCGGATCCGCCACC45 Component CGGCGCCTATATAAGGTGTGGGCGGGTGTGGCACAGCTAG H TTCCGTCGCAGCCGGCGAGCCGAACGGTCGTTGAGCCAAG AGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAA TGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTT CAGGTTGGCCGCGGATCCGCCACC46 Component TGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAC I ATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACA TTGATTATTGACTAGAATTCGCTAGCAAGATCCAAGCTCA GATCTCGATCGAGTTGGGCCCCAGAAGCCTGGTGGTTGTT TGTCCTTCTCAGGGGAAAAGTGAGGCGGCCCCTTGGAGGA AGGGGCCGGGCAGAATGATCTAATCGGATTCCAAGCAGCTCAGGGGATTGTCTTTTTCTAGCACCTTCTTGCCACTCCTAA 52180489702.1SEQ Promoter SequenceID NO GCGTCCTCCGTGACCCCGGCTGGGATTTAGCCTGGTGCTGT GTCAGCCCCGGTCTCCCAGGGGCTTCCCAGTGGTCCCCAG GAACCCTCGACAGGGCCCGGTCTCTCTCGTCCAGCAAGGG CAGGGACGGGCCACAGGCCAAGGGCCCTCGATCGAGGAA CTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTAGACCG GGGCGGGGTCCAGAGGTAACTTGTGCCCCGCCCCGATAGC CTAGGGCCCAGCGGGGATTAAGTTTTTTTTTTTTTTCTTTTG AGGAATCTTACTCCAATGATGGCGCCTGCTCAATTCGAGG AATCTTACTAGACCGGGGCGGGGCCACATAAGGTTGCAAA TCCCACAGAGAGCCTGCGCCTGGGGTTGTTTTCTGTAGGC GCCTGTGCCCCCGCCCCTGCCACCAATGATGGCGCCTGCT CAATTCCTATGTGTCTGGCACCAGAAACGGAAGCTGCAGG TTGCAGCCCCTGCCCTCATGGAGCTCCTCCTGTCAGAGGA GTGTGGGGACTGGATGACTCCAGAGGTAACTTGTGGGGGA ACGAACGCGTAAGGGGCTGTGTGACGAGATGAGAGACTG GGAGAAT47 Component GAGGTCACTTTATAAGGGTCTGGGGGGGTCAGAACCCAGA J GTCATCCAGCTGGAGCCCTGAGTGGCTGAGCTCAGGCCTT CGCAGCATTCTTGGGTGGGAGCAGCCACGGGTCAGCCACA AGGGCCACAGCCCTTTTTGTCTTTTATTTCAGGTCCCGGAT CCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGAT TTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTTCTG CTCTAAAAGCTGCGGAATTGTACCCGCGGCCGC48 1 CCATCCTCAAGGCCCAGGCCTGGGACACAGCCTTTCTGGC AGAGCCCAAGGCAGGGCTGCTGAGACCCTGCCCAGAGAA GACTCTAAGCCAGAAAGAACCTGCCTGCGTCCTGGCCTGG GGGCTGGAGAGTTGGGGTACACCTCTTGCCACCGCTGAGT GGGAGCTGACAAGCCCCTGCTACCATGAGGAGTCTCCAGG CATCACATGGGAACTCCAAGCTGTTATTCTCCCATCAGCC ACTCCGTGTCTTGGCCAAGGTGTATGGGAAGCCAGGCCCC TGGTAAGCCGGGCAGGTCCCCTGGCAACCCATGGTGCTCC AAACCTGGGCCATGCAGGAGCCCTTCCCTGGGGCCCACAT CCTTGCTGGACCCTGGGAGTTGGGAGGTGCTGCTGGTTTC CTGGGCATCAACAGCCTCTTTTCTCCCCCTCCCCCATTCAG CACCTGCTAATCCTTTGCACACGGCCCAGCTACTGCCAATT AATCTTCCTTGTGTCTGAGGGGCCAGCTACGAGTGGCAGC AAGAAGGCAATTCCTGGCTGGCGGTTGGCATCTAAGCAG49 2 TCTGTGCAGACATCTCTTCTTTGCTCTTACTAGACCAAGGT GAAAGAAAACTCTCACCTTCTCCCATCTGGCCCCACAGCA TCTGGAACACACTGATCCTCATAATCCTTGTTCTTGAGAAA TATTAATGACTTAATCTCCCAAGCTTGCTCCCTCTCCTGTG CAGGCCATCTCAGTATGTTTTGCAGACAAGACCCAGAGAA GTCCA50 3 CCAATGATGGAGACCGGGGCGGGGTCCAGAGGTAACTTGT GTTCTCTCTCTGTCTCTCTTCTGCATTTATGTCGCATGAGGAGAGTCCCTCTAAGCCTGCCGAGAGTAGTATATGAGGCTTGCCTGCTGAATTCGAGGAATCTTACTAGACCGGGGCGGGGC 53180489702.1SEQ Promoter SequenceID NO CACATAAGGTTGCAAATCCCACAGAGAGCCTGCGCCTGGG GTTGTTTTCTGTAGGCGCCTGTGCCCCCGCCCCTGCCACCA ATGATGGCGCCTGCTGAATTCCTATGTGTCTGGCACCAGA AACGGAAGCTGCAGGTTGCAGCCCCTGCCCTCATGGAGCT CCTCCTGTCAGAGGAGTGTGGGGACTGGATGACTCCAGAG GTAACTTGTGGGGGAACGAACAGGTAAGGGGCTGTGTGA CGAGATGAGAGACTGGGAGAATAAACCAGAAAGTCTCTA GCTGTCCAGAGGACATAGCACAGAGGCCCATGGTCCCTAT TTCAAACCCAGGCCACCAGACTGAGCTGGGACCTTGGGAC AGACAAGTCATGCAGAAGTTAGGGGACCTTCTCCTCCCTT TTCCTGGATCCTGAGTACCTCTCCTCCCTGACCTCAGGCTT CCTCCTAGTGTCACCTTGGCCCCTCTTAGAAGCCAATTAGG CCCTCAGTTTCTGCAGCGGGGATTAATATGATTATGAACA CCCCCAATCTCCCAGATGCTGATTCAGCCAGGAGCTTAGG AGGGGGAGGTCACTTTATAAGGGTCTGGGGGGGTCAGAA CCCAGAGTCATCCAGCTGGAGCCCTGAGTGGCTGAGCTCA GGCCTTCGCAGCATTCTTGGGTGGGAGCAGCCACGGGTCA GCCACAAGGGCCACAGCC51 4 TCCCTGCAGGTCATAAAATCCCAGTCCAGAGTCACCAGCC CTTCTTAACCACTTCCTACTGTGTGACCCTTTCAGCCTTTA CTTCCTCATCAGTAAAATGAGGCTGATGATATGGGCATCC ATACTCCAGGGCCAGTGTGAGCTTACAACAAGATAAGGAG TGGTGCTGAGCCTGGTGCCGGGCAGGCAGCAGGCATGTTT CTCCCAATTATGCCCTCTCACTGCCAGCCCCACCTCCATTG TCCTCACCCCCAGGGCTCAAGGTTCTGCCTTCCCCTTTCTC AGCCCTGACCCTACTGAACATGTCTCCCCACTCCCAGGCA GTGCCAGGGCCTCTCCTGGAGGGTTGCGGGGACAGAAGG ACAGCCGGAGTGCAGAGTCAGCGGTTGAGGGATTGGGGC TATGCCAGCTAATCCGAAGGGTTGGGGGGGCTGAGCTGGA TTCACCTGTCCTTGTCTCTGATTGGCTCTTGGACACCCCTA GCCCCCAAATCCCACTAAGCAGCCCCACCAGGGATTGCAC AGGTCCGTAGAGAGCCAGTTGATTGCAGGTCCTCCTGGGG CCAGAAGGGTGCCTGGGAGGCCAGGTTCTGGGGATCCCCT CCATCCAGAAGAACCACCTGCTCACTCTGTCCCTTCGCCTG CTGCTGGGACCGCGGCCGC52 5 GCTGCCCAGGCCACAGCCGCCTTTGGGTTCCATCTTGCTAA TAAACACTGGCTCTGGGACTAGACTTTGGCTTCTCTCCTTG GTCAGGGGATTTAGAGCCACACGTGGTGGGCCTGGGGAGC CAGGTGGGGGGCTGGGAAGGGAGTAGATTGGCTCCATTTA AAGAAACCCAACATGTAGAAACCACATTTAGTTTGACAAA CCTGGGCTGCTGCACCACCCCAAACACTTGGGTAGTGCGA AGGGCTGTAGAGACCCCTCAGGGCCCACTCACTCCTCAGG GTCAGGGAGGGTGGGTGGAGCCTGAAGGCTGAAAGTGGC AGAAATGCAACGGCCTCACTCCTGGACAGATCCCCATCAT CCAACAGGGTGGGTGTAGAAGCTGCCAGCCTGTGGTAGGA GCCAGCATTCACTTCCCACGCATACTATTTAGAGACAGCCGTTAGTGCTCAGTGTCTGGTGCCCCGGCACCCCCTTGTGGT54180489702.1SEQ Promoter SequenceID NO CTCTTGAGTCTGTGGCAGAGTTGAGTGCTGAAAGGCAGCC AGGTTCCAGGGAAGAGTCCAGCCAGGAGGCTCAGGGTGC CTGGAGGAGCCTTTGAGAGCCCAAGATTTGGGTTTTCCTA TGGAAGTTGAGGGTGGGAAGTCTCTAGAACCTGGGAGCTC AACCTGAACAATTTCTGCCCGGCAGTGAGCCCTGCACCTG AACAGAGCACCAGACCCCCTAAGCTGAGGCAGGCAGGCC CTGGAGATAATCTTAGGCAGGCCTCAGAGCACCCACAGCA GACAGAGCAGGAGCAGGATAGGTGAGCCCCATTCCTCCAT ACCCCAGGGAACACTACACCTGCTCTGGGATGCAATTTCT CCACCTGGCAACAGGCCTGCAAAACATTCTCTGGAAGAGC CTGGCCACTTCTGCAGATTAGAGACTTTGTTGCACCCAAAT TCAAGTGTACATATAGGCACAGGCGCCTGCAAGCATGCAT GCACACACACAGTCATACACAGGCAGAGATGGACATGCA CAGGCACACATATGCTCAGGTACAAACAGGCATGTTTACA CCCCTGCGTATTCCCTGCAGGTCATAAAATCCCAGTCCAG AGTCACCAGCCCTTCTTAACCACTTCCTACTGTGTGACCCT TTCAGCCTTTACTTCCTCATCAGTAAAATGAGGCTGATGAT ATGGGCATCCATACTCCAGGGCCAGTGTGAGCTTACAACA AGATAAGGAGTGGTGCTGAGCCTGGTGCCGGGCAGGCAG CAGGCATGTTTCTCCCAATTATGCCCTCTCACTGCCAGCCC CACCTCCATTGTCCTCACCCCCAGGGCTCAAGGTTCTGCCT TCCCCTTTCTCAGCCCTGACCCTACTGAACATGTCTCCCCA CTCCCAGGCAGTGCCAGGGCCTCTCCTGGAGGGTTGCGGG GACAGAAGGACAGCCGGAGTGCAGAGTCAGCGGTTGAGG GATTGGGGCTATGCCAGCTAATCCGAAGGGTTGGGGGGGC TGAGCTGGATTCACCTGTCCTTGTCTCTGATTGGCTCTTGG ACACCCCTAGCCCCCAAATCCCACTAAGCAGCCCCACCAG GGATTGCACAGGTCCGTAGAGAGCCAGTTGATTGCAGGTC CTCCTGGGGCCAGAAGGGTGCCTGGGAGGCCAGGTTCTGG GGATCCCCTCCATCCAGAAGAACCACCTGCTCACTCTGTC CCTTCGCCTGCTGCTGGGACCGCGGCCGC53 6 ACAGAGTGGAGAACCGAAGCACACTCTCGGGAGGGCGGG GTAGCTGGCTGCACCCAGGCTGTGTAGCCTCAGTCCAGAT GTAAGGGTGGGTGGAAAAGAGCCTTGCCCAATGAGGGAG AACAGTGAAACCAAGGCCATAGGGTCTAAAGATTCACGA ACCAGGCTCTCATGGAGAAAGCAGGTGAGGTTTACTGTAT AGATGGGTGTGCCCCTACCCCACACTGAGGCTTCCTCGTCT GAGCAAACTGAGGCCCAGAGAGGGGAAGGAAGCAGGACT ACCATGGTGACTCAAAGACCAGCTAGAATCCAGCCTCCTC TCCTCGAGGCTTCCACTGCCCCACGCCAGGCCTGTGTGACT CAGTCTAGGGCCTTTCCATTACCCCAGCTAAACCTTTCTTT AGTCATTTATACCATGGTGTGAATGGCTGGCTGGTCTTTCC TGAGAGCTATCTTTGATGAGGGGAGGGAGGCATAGCCAG GTTTGGGAAGCTGATACCCCAGGAAGCCCAGTTGACTGTG TGGGTTATAGCCCAGGCTGTCACTGATTTGTAACGGGACC TAAACCAGAAAGTCTCTAGCTGTCCAGAGGACATAGCACAGAGGCCCATGGTCCCTATTTCAAACCCAGGCCACCAGACT55180489702.1SEQ Promoter SequenceID NO GAGCTGGGACCTTGGGACAGACAAGTCATGCAGAAGTTA GGGGACCTTCTCCTCCCTTTTCCTGGATCCTGAGTACCTCT CCTCCCTGACCTCAGGCTTCCTCCTAGTGTCACCTTGGCCC CTCTTAGAAGCCAATTAGGCCCTCAGTTTCTGCAGCGGGG ATTAATATGATTATGAACACCCCCAATCTCCCAGATGCTG ATTCAGCCAGGAGCTTAGGAGGGGGAGGTCACTTTATAAG GGTCTGGGGGGGTCAGAACCCAGAGTCATC54 7 CTATGTGTCTGGCACCAGAAACGGAAGCTGCAGGTTGCAG CCCCTGCCCTCATGGAGCTCCTCCTGTCAGAGGAGTGTGG GGACTGGATGACTCCAGAGGTAACTTGTGGGGGAACGAA CAGGTAAGGGGCTGTGTGACGAGATGAGAGACTGGGAGA ATAAACCAGAAAGTCTCTAGCTGTCCAGAGGACATAGCAC AGAGGCCCATGGTCCCTATTTCAAACCCAGGCCACCAGAC TGAGCTGGGACCTTGGGACAGACAAGTCATGCAGAAGTTA GGGGACCTTCTCCTCCCTTTTCCTGGATCCTGAGTACCTCT CCTCCCTGACCTCAGGCTTCCTCCTAGTGTCACCTTGGCCC CTCTTAGAAGCCAATTAGGCCCTCAGTTTCTGCAGCGGGG ATTAATATGATTATGAACACCCCCAATCTCCCAGATGCTG ATTCAGCCAGGAGCTTAGGAGGGGGAGGTCACTTTATAAG GGTCTGGGGGGGTCAGAACCCAGAGTCATCCAGCTGGAGC CCTGAGTGGCTGAGCTCAGGCCTTCGCAGCATTCTTGGGT GGGAGCAGCCACGGGTCAGCCACAAGGGCCACAGCC55 8 AGAAGCCAATTAGGCCCTCAGTTTCTGCAGCGGGGATTAA TATGATTATGAACACCCCCAATCTCCCAGATGCTGATTCA GCCAGGAGCTTAGGAGGGG56 9 CTATAATCCTGGCACTTTGGGAGGCCGAGGAGGGTGGATC GCTTGAGCCAGGAGTTCAGGACCAGCCTGGGCAACATAGC GAGACTCCACCCCTACAAAAAATACAAAAACTAGTGGTGT GCACTTGTGGTCCCAGCTACTCAGGAGGCTGAGGTGAGAG GATCGCTTGAGCCCAGGAGGCAGAGGCTACAGTGAGCTAT GATTGTGGCACTGCACTCCAGCCTGGGCGACAGAGACCTT GTCTCAAAACTTTTTTTTTCTTCGTCAAGCTTTACAGAATA AAGAGCACTGTCACCTCAGTGATGGCTGTTAGTTCCCCAT CACCAGGGCTCCATGAGGTTGCAATTGTGAAACTCACAAA GGAGGAACCTGAGACAGAGAGGGGAAGTACTGAGATCAT CTAGGTCCATTCCCCCACTCACTCGTTCATTCAACAAATAT TCAGGAGCACCTTCTAGGTGCCAGGCCCTGGAGACACATC AGTGAACAAAACAGACATCATCCCACCTCTTTCCACTACA GGCCAAGCACCATGCTGGTCTCTGGGAACCCTGTTGTGAG CAAGACAGACCCAGGCTTACCCTTGTGGACTCATGTTACA GGCAGGGAGACGGGCACAAAACACAAATAAAAAGCTTCC ATGCTGTCAGAAGCACTATGCAAAAAGCAAGATGCTGAG GTACTGCTAAGCTGTGTGGGATGGGGGCTCAGCCCGGCCA GGGAAAACCAGAAAGTCTCTAGCTGTCCAGAGGACATAG CACAGAGGCCCATGGTCCCTATTTCAAACCCAGGCCACCA GACTGAGCTGGGACCTTGGGACAGACAAGTCATGCAGAAGTTAGGGGACCTTCTCCTCCCTTTTCCTGGATCCTGAGTAC56180489702.1SEQ Promoter SequenceID NO CTCTCCTCCCTGACCTCAGGCTTCCTCCTAGTGTCACCTTG GCCCCTCTTAGAAGCCAATTAGGCCCTCAGTTTCTGCAGC GGGGATTAATATGATTATGAACACCCCCAATCTCCCAGAT GCTGATTCAGCCAGGAGCTTAGGAGGGGGAGGTCACTTTA TAAGGGTCTGGGGGGGTCAGAACCCAGAGTCATC57 10 AGTCAGCGGTTGAGGGATTGGGGCTATGCCAGCCCGATTA GAAGGGTTGGGGGGGCTGAGCTGGATTCACCTGTCCTTGT CTCTGATTGGCTCTTGGACACCCCTAGCCCCCAAATCCCAC TAAGCAGCCCCACCAGGGATTGCACAGGTCCGTAGAGAGC CAGTTGATTGCAGGTCCTCCTGGGGCCAGAAGGGTGCCTG GGAGGCCAGGTTCTGGGGATCCCCTCCATCCAGAAGAACC ACCTGCTCACTCTGTCCCTTCGCCTGCTGCTGGGAC58 V12 (Enhl- ACAGAGTGGAGAACCGAAGCACACTCTCGGGAGGGCGGG TFBS- GTAGCTGGCTGCACCCAGGCTGTGTAGCCTCAGTCCAGAT Rho(MD)- GTAAGGGTGGGTGGAAAAGAGCCTTGCCCAATGAGGGAG MVMi) AACAGTGAAACCAAGGCCATAGGGTCTAAAGATTCACGA ACCAGGCTCTCATGGAGAAAGCAGGTGAGGTTTACTGTAT AGATGGGTGTGCCCCTACCCCACACTGAGGCTTCCTCGTCT GAGCAAACTGAGGCCCAGAGAGGGGAAGGAAGCAGGACT ACCATGGTGACTCAAAGACCAGCTAGAATCCAGCCTCCTC TCCTCGAGGCTTCCACTGCCCCACGCCAGGCCTGTGTGACT CAGTCTAGGGCCTTTCCATTACCCCAGCTAAACCTTTCTTT AGTCATTTATACCATGGTGTGAATGGCTGGCTGGTCTTTCC TGAGAGCTATCTTTGATGAGGGGAGGGAGGCATAGCCAG GTTTGGGAAGCTGATACCCCAGGAAGCCCAGTTGACTGTG TGGGTTATAGCCCAGGCTGTCACTGATTTGTAACGGGACC TCCAATGATGGAGACCGGGGCGGGGTCCAGAGGTAACTTG TTGTTCTCTCTCTGTCTCTCTCTTGCATTTATGTCGCATGAGGAGAGTCCCTCTAAGCCTGCCGAGAGTAGTATATGAGGCT GCCTGCTCAATTCGAGGAATCTTACTAGACCGGGGCGGGG CCACATAAGGTTGCAAATCCCACAGAGAGCCTGCGCCTGG GGTTGTTTTCTGTAGGCGCCTGTGCCCCCGCCCCTGCCACC AATGATGGCGCCTGCTCAATTCCTATGTGTCTGGCACCAG AAACGGAAGCTGCAGGTTGCAGCCCCTGCCCTCATGGAGC TCCTCCTGTCAGAGGAGTGTGGGGACTGGATGACTCCAGA GGTAACTTGTGGGGGAACGAACAGGTAAGGGGCTGTGTG ACGAGATGAGAGACTGGGAGAATAAACCAGAAAGTCTCT AGCTGTCCAGAGGACATAGCACAGAGGCCCATGGTCCCTA TTTCAAACCCAGGCCACCAGACTGAGCTGGGACCTTGGGA CAGACAAGTCATGCAGAAGTTAGGGGACCTTCTCCTCCCT TTTCCTGGATACTGAGTACCTCTCCTCCCTGACCTCAGGCT TCCTCCTAGTGTCACCTTGGCCCCTCTTAGAAGCCAATTAG GCCCTCAGTTTCTGCAGCGGGGATTAATATGATTATGAAC ACCCCCAATCTCCCAGATGCTGATTCAGCCAGGAGCTTAG GAGGGGGAGGTCACTTTATAAGGGTCTGGGGGGGTCAGA ACCCCTAGTTCATCCAGCTGGAGCGAGCCGAACGGTCGTTGAGCCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGG57180489702.1SEQ Promoter SequenceID NO GGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATC ACTTTTTTTCAGGTTGGCCGCGGATCCGCCACC59 VI 3 (Enh2- CTATAATCCTGGCACTTTGGGAGGCCGAGGAGGGTGGATC TFBS- GCTTGAGCCAGGAGTTCAGGACCAGCCTGGGCAACATAGC Rho(MD)- GAGACTCCACCCCTACAAAAAATACAAAAACTAGTGGTGT MVMi) GCACTTGTGGTCCCAGCTACTCAGGAGGCTGAGGTGAGAG GATCGCTTGAGCCCAGGAGGCAGAGGCTACAGTGAGCTAT GATTGTGGCACTGCACTCCAGCCTGGGCGACAGAGACCTT GTCTCAAAACTTTTTTTTTCTTCGTCAAGCTTTACAGAATA AAGAGCACTGTCACCTCAGTGATGGCTGTTAGTTCCCCAT CACCAGGGCTCCATGAGGTTGCAATTGTGAAACTCACAAA GGAGGAACCTGAGACAGAGAGGGGAAGTACTGAGATCAT CTAGGTCCATTCCCCCACTCACTCGTTCATTCAACAAATAT TCAGGAGCACCTTCTAGGTGCCAGGCCCTGGAGACACATC AGTGAACAAAACAGACATCATCCCACCTCTTTCCACTACA GGCCAAGCACCATGCTGGTCTCTGGGAACCCTGTTGTGAG CAAGACAGACCCAGGCTTACCCTTGTGGACTCATGTTACA GGCAGGGAGACGGGCACAAAACACAAATAAAAAGCTTCC ATGCTGTCAGAAGCACTATGCAAAAAGCAAGATGCTGAG GTACTGCTAAGCTGTGTGGGATGGGGGCTCAGCCCGGCCA GGGACCAATGATGGAGACCGGGGCGGGGTCCAGAGGTAA CTTGTGCCCCGCCCCGATAGCCTAGGGCCCAGCGGGGATT AAGTTTTTTTTTTTTTTCTTTTGAGGAATCTTACTCCAATGA TGGCGCCTGCTCAATTCGAGGAATCTTACTAGACCGGGGC GGGGCCACATAAGGTTGCAAATCCCACAGAGAGCCTGCGC CTGGGGTTGTTTTCTGTAGGCGCCTGTGCCCCCGCCCCTGC CACCAATGATGGCGCCTGCTCAATTCCTATGTGTCTGGCAC CAGAAACGGAAGCTGCAGGTTGCAGCCCCTGCCCTCATGG AGCTCCTCCTGTCAGAGGAGTGTGGGGACTGGATGACTCC AGAGGTAACTTGTGGGGGAACGAACAGGTAAGGGGCTGT GTGACGAGATGAGAGACTGGGAGAATAAACCAGAAAGTC TCTAGCTGTCCAGAGGACATAGCACAGAGGCCCATGGTCC CTATTTCAAACCCAGGCCACCAGACTGAGCTGGGACCTTG GGACAGACAAGTCATGCAGAAGTTAGGGGACCTTCTCCTC CCTTTTCCTGGATACTGAGTACCTCTCCTCCCTGACCTCAG GCTTCCTCCTAGTGTCACCTTGGCCCCTCTTAGAAGCCAAT TAGGCCCTCAGTTTCTGCAGCGGGGATTAATATGATTATG AACACCCCCAATCTCCCAGATGCTGATTCAGCCAGGAGCT TAGGAGGGGGAGGTCACTTTATAAGGGTCTGGGGGGGTCA GAACCCCTAGTTCATCCAGCTGGAGCGAGCCGAACGGTCG TTGAGCCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGT GGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAA TCACTTTTTTTCAGGTTGGCCGCGGATCCGCCACC60 V14 (Enh2- CTATAATCCTGGCACTTTGGGAGGCCGAGGAGGGTGGATC TFBS- GCTTGAGCCAGGAGTTCAGGACCAGCCTGGGCAACATAGC Rho(BTIm GAGACTCCACCCCTACAAAAAATACAAAAACTAGTGGTGTd2)-MVMi) GCACTTGTGGTCCCAGCTACTCAGGAGGCTGAGGTGAGAG58180489702.1SEQ Promoter SequenceID NO GATCGCTTGAGCCCAGGAGGCAGAGGCTACAGTGAGCTAT GATTGTGGCACTGCACTCCAGCCTGGGCGACAGAGACCTT GTCTCAAAACTTTTTTTTTCTTCGTCAAGCTTTACAGAATA AAGAGCACTGTCACCTCAGTGATGGCTGTTAGTTCCCCAT CACCAGGGCTCCATGAGGTTGCAATTGTGAAACTCACAAA GGAGGAACCTGAGACAGAGAGGGGAAGTACTGAGATCAT CTAGGTCCATTCCCCCACTCACTCGTTCATTCAACAAATAT TCAGGAGCACCTTCTAGGTGCCAGGCCCTGGAGACACATC AGTGAACAAAACAGACATCATCCCACCTCTTTCCACTACA GGCCAAGCACCATGCTGGTCTCTGGGAACCCTGTTGTGAG CAAGACAGACCCAGGCTTACCCTTGTGGACTCATGTTACA GGCAGGGAGACGGGCACAAAACACAAATAAAAAGCTTCC ATGCTGTCAGAAGCACTATGCAAAAAGCAAGATGCTGAG GTACTGCTAAGCTGTGTGGGATGGGGGCTCAGCCCGGCCA GGGACCAATGATGGAGACCGGGGCGGGGTCCAGAGGTAA CTTGTGCCCCGCCCCGATAGCCTAGGGCCCAGCGGGGATT AAGTTTTTTTTTTTTTTCTTTTGAGGAATCTTACTCCAATGA TGGCGCCTGCTCAATTCGAGGAATCTTACTAGACCGGGGC GGGGCCACATAAGGTTGCAAATCCCACAGAGAGCCTGCGC CTGGGGTTGTTTTCTGTAGGCGCCTGTGCCCCCGCCCCTGC CACCAATGATGGCGCCTGCTCAATTCCTATGTGTCTGGCAC CAGAAACGGAAGCTGCAGGTTGCAGCCCCTGCCCTCATGG AGCTCCTCCTGTCAGAGGAGTGTGGGGACTGGATGACTCC AGAGGTAACTTGTGGGGGAACGAACAGGTAAGGGGCTGT GTGACGAGATGAGAGACTGGGAGAATAAACCAGAAAGTC TCTAGCTGTCCAGAGGACATAGCACAGAGGCCCATGGTCC CTATTTCAAACCCAGGCCACCAGACTGAGCTGGGACCTTG GGACAGACAAGTCATGCAGAAGTTAGGGGACCTTCTCCTC CCTTTTCCTGGATACTGAGTACCTCTCCTCCCTGACCTCAG GCTTCCTCCTAGTGTCACCTTGGCCCCTCTTAGAAGCCAAT TAGGCCCTCAGTTTCTGCAGCGGGGATTAATATGATTATG AACACCCCCAATCTCCCAGATGCTGATTCAGCCAGGAGCT TAGGAGGGGCGGCGCCTATATAAGGTGTGGGCGGGTGTG GCACAGCTAGTTCCGTCGCAGCCGGCGAGCCGAACGGTCG TTGAGCCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGT GGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAA TCACTTTTTTTCAGGTTGGCCGCGGATCCGCCACC61 VI 5 (Enh2- CTATAATCCTGGCACTTTGGGAGGCCGAGGAGGGTGGATC RHOpatent GCTTGAGCCAGGAGTTCAGGACCAGCCTGGGCAACATAGC (MD)- GAGACTCCACCCCTACAAAAAATACAAAAACTAGTGGTGT MVMi) GCACTTGTGGTCCCAGCTACTCAGGAGGCTGAGGTGAGAG GATCGCTTGAGCCCAGGAGGCAGAGGCTACAGTGAGCTAT GATTGTGGCACTGCACTCCAGCCTGGGCGACAGAGACCTT GTCTCAAAACTTTTTTTTTCTTCGTCAAGCTTTACAGAATA AAGAGCACTGTCACCTCAGTGATGGCTGTTAGTTCCCCAT CACCAGGGCTCCATGAGGTTGCAATTGTGAAACTCACAAAGGAGGAACCTGAGACAGAGAGGGGAAGTACTGAGATCAT59180489702.1SEQ Promoter SequenceID NO CTAGGTCCATTCCCCCACTCACTCGTTCATTCAACAAATAT TCAGGAGCACCTTCTAGGTGCCAGGCCCTGGAGACACATC AGTGAACAAAACAGACATCATCCCACCTCTTTCCACTACA GGCCAAGCACCATGCTGGTCTCTGGGAACCCTGTTGTGAG CAAGACAGACCCAGGCTTACCCTTGTGGACTCATGTTACA GGCAGGGAGACGGGCACAAAACACAAATAAAAAGCTTCC ATGCTGTCAGAAGCACTATGCAAAAAGCAAGATGCTGAG GTACTGCTAAGCTGTGTGGGATGGGGGCTCAGCCCGGCCA GGGAAAACCAGAAAGTCTCTAGCTGTCCAGAGGACATAG CACAGAGGCCCATGGTCCCTATTTCAAACCCAGGCCACCA GACTGAGCTGGGACCTTGGGACAGACAAGTCATGCAGAA GTTAGGGGACCTTCTCCTCCCTTTTCCTGGATACTGAGTAC CTCTCCTCCCTGACCTCAGGCTTCCTCCTAGTGTCACCTTG GCCCCTCTTAGAAGCCAATTAGGCCCTCAGTTTCTGCAGC GGGGATTAATATGATTATGAACACCCCCAATCTCCCAGAT GCTGATTCAGCCAGGAGCTTAGGAGGGGGAGGTCACTTTA TAAGGGTCTGGGGGGGTCAGAACCCCTAGTTCATCCAGCT GGAGCGAGCCGAACGGTCGTTGAGCCAAGAGGTAAGGGT TTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTAC CTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGCCG CGGATCCGCCACC62 VI 6 (hRK- TGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAC TFBSRho) ATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACA TTGATTATTGACTAGAATTCGCTAGCAAGATCCAAGCTCA GATCTCGATCGAGTTGGGCCCCAGAAGCCTGGTGGTTGTT TGTCCTTCTCAGGGGAAAAGTGAGGCGGCCCCTTGGAGGA AGGGGCCGGGCAGAATGATCTAATCGGATTCCAAGCAGCT CAGGGGATTGTCTTTTTCTAGCACCTTCTTGCCACTCCTAA GCGTCCTCCGTGACCCCGGCTGGGATTTAGCCTGGTGCTGT GTCAGCCCCGGTCTCCCAGGGGCTTCCCAGTGGTCCCCAG GAACCCTCGACAGGGCCCGGTCTCTCTCGTCCAGCAAGGG CAGGGACGGGCCACAGGCCAAGGGCCCTCGATCGAGGAA CTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTAGACCG GGGCGGGGTCCAGAGGTAACTTGTGCCCCGCCCCGATAGC CTAGGGCCCAGCGGGGATTAAGTTTTTTTTTTTTTTCTTTTG AGGAATCTTACTCCAATGATGGCGCCTGCTCAATTCGAGG AATCTTACTAGACCGGGGCGGGGCCACATAAGGTTGCAAA TCCCACAGAGAGCCTGCGCCTGGGGTTGTTTTCTGTAGGC GCCTGTGCCCCCGCCCCTGCCACCAATGATGGCGCCTGCT CAATTCCTATGTGTCTGGCACCAGAAACGGAAGCTGCAGG TTGCAGCCCCTGCCCTCATGGAGCTCCTCCTGTCAGAGGA GTGTGGGGACTGGATGACTCCAGAGGTAACTTGTGGGGGA ACGAACGCGTAAGGGGCTGTGTGACGAGATGAGAGACTG GGAGAATAAACCAGAAAGTCTCTAGCTGTCCAGAGGACAT AGCACAGAGGCCCATGGTCCCTATTTCAAACCCAGGCCAC CAGACTGAGCTGGGACCTTGGGACAGACAAGTCATGCAGAAGTTAGGGGACCTTCTCCTCCCTTTTCCTGGATACTGAGT60180489702.1SEQ Promoter SequenceID NO ACCTCTCCTCCCTGACCTCAGGCTTCCTCCTAGTGTCACCT TGGCCCCTCTTAGAAGCCAATTAGGCCCTCAGTTTCTGCAG CGGGGATTAATATGATTATGAACACCCCCAATCTCCCAGA TGCTGATTCAGCCAGGAGCTTAGGAGGGGGAGGTCACTTT ATAAGGGTCTGGGGGGGTCAGAACCCAGAGTCATCCAGCT GGAGCCCTGAGTGGCTGAGCTCAGGCCTTCGCAGCATTCT TGGGTGGGAGCAGCCACGGGTCAGCCACAAGGGCCACAG CCCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGG TGCAAATCAAAGAACTGCTCCTCAGTGGATTTTGCCTTTAC TTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGC

[0184] Transgenes

[0185] Provided herein is a viral particle comprising a modified AAV VP1, VP2, and / or VP3 capsid protein disclosed herein and an AAV genome comprising a transgene. As used herein, a “transgene” or a “nucleic acid sequence encoding a gene product of interest” (used interchangeably herein) refers to a non-native nucleic acid with respect to the AAV nucleic acid sequence. The term transgene refers to a polynucleotide that can be introduced into a cell or organism. Gene products of interest include any polynucleotide, such as a gene that encodes a polypeptide or protein, a polynucleotide that is transcribed into an inhibitory polynucleotide, or a polynucleotide that is not transcribed (e.g., lacks an expression control element, such as a promoter that drives transcription). The gene product of interest may be an RNA. The RNA may be an inhibitory antisense oligonucleotide (ASO), a small interfering RNA (siRNA), a microRNA (miRNA), a Piwi-interacting RNAs (piRNAs), a short-hairpin RNA (shRNA), small non-coding RNA (sncRNA), or a long non-coding RNA (IncRNA).

[0186] The AAV genome may comprise two nucleotide sequences (or more), each being different or encoding different transgenes. The different nucleotide sequences may be linked by an IRES (internal ribosome entry site) element, providing, for example, a bicistronic transcript under control of a single promoter. Suitable IRES elements are described in e.g., Hsieh et al., Improved gene expression by a modified bicistronic retroviral vector, Biochem Biophys Res Commun. 1995 Sep 25;214(3):910-7, incorporated herein by reference in its entirety. Furthermore, the different nucleotide sequences encoding different gene products may be linked by a viral 2A sequence to allow for efficient expression of both transgenes from a single promoter. Examples of 2A sequences include those from foot and mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1 (Kim et al., High61180489702.1cleavage efficiency of a 2 A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice, PLoS One. 2011;6(4):el8556, incorporated herein by reference in its entirety).

[0187] The transgene is preferably inserted within the AAV genome between ITR sequences.

[0188] The transgene may be a functional mutant allele that replaces or supplements a defective one. In some embodiments, the transgene is inhibitory in nature, / .< ., expression of the transgene inhibits, decreases, or reduces expression, activity or function of an endogenous or exogenous gene or protein, such as an undesirable or aberrant (e.g., pathogenic) gene or protein.

[0189] The transgene may be exogenous. An exogenous molecule or sequence is understood to be a molecule or sequence not normally occurring in the cell, tissue and / or individual to be treated.

[0190] In one embodiment, the transgene encodes a “therapeutic polypeptide” or “therapeutic protein,” which are understood herein as a polypeptide or protein that can have a beneficial effect on an individual. Preferably said individual is a human, more preferably said human suffers from a disease.

[0191] The transgene may be codon-optimized. The transgene may additionally encode for a signal sequence.

[0192] Examples of transgenes that can be used with the compositions and methods disclosed herein include, but are not limited to, genes for channel rhodopsin-1 (ChRl), channel rhodopsin-2 (ChR2), ChETA (ChR2 with a double point mutation, C123TZE134R), ChR2 C128A, ChR2 C128S, ChR2 C128T, ChRl-ChR2 hybrids / chimeras, halorhodopsin (halo), melanopsin (Opn4), rhodopsin (RHO), blue opsin, red opsin, halorhodopsin (NpHR), enhanced halorhodopsin (eNpHR), archaerhodopsin-3 (Arch), ArchT, leptosphaeria maculans (Mac), retinoid isomerohydrolase (retinal pigment epithelium-specific 65 kDa protein, RPE65), cyclic nucleotide-gated channel alpha-3 (CNGA3), cyclic nucleotide-gated channel beta-3 (CNGB3), cone cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha (PDE6C), Jaws (cruxhalorhodopsin), iClC2 (see Berndt et al, Structure-guided transformation of channelrhodopsin into a light-activated chloride channel, Science. 2014 Apr 25;344(6182):420-4), retinal cone rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma (PDE6H), G Protein Subunit Alpha Transducin 1 (GNAT1), GNAT2, KCNV2, voltage-dependent calcium channel subunit alpha-2 / delta-4 (CACNA2D4), R9AP (Rgs9-anchor protein or RGS9BP), iC!C2, apolipoprotein E (APOE), HtrA serine peptidase 162180489702.1(HTRA1), complement 3 (C3), C3 modulators (activators and inhibitors), C5, C5 modulators (activators and inhibitors), rab escort protein- 1 (REP1), retinal-specific phospholipidtransporting ATPase (ABCA4), cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6), retinoschisin 1 (RSI), nicotinamide adenine dinucleotide dehydrogenase subunit 4 (ND4), tyrosine-protein kinase Mer (MERTK), rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit beta (PDE6B), retinaldehyde-binding protein (RLBP1), retinal membrane guanylyl cyclase 1 (RetGCl / GUCY2D), inosine-5'-monophosphate dehydrogenase 1 (IMPDH1), peripherin-2 (Prph2 / RDS), complement factor H (CFH) modulators (activators and inhibitors), complement factor I (CFI) modulators (activators and inhibitors), and Retinol Dehydrogenase 12 (RDH12). Additional examples of transgenes include genes encoding therapeutic antibodies or antigen-binding fragments thereof, including, but not limited to anti-VEGF or anti-VEGFR antibodies or antigen-binding fragments thereof. In some embodiments, the transgene encodes for a chimeric VEGF -binding protein or antigen-binding fragments thereof disclosed in International Patent Publication No. WO2013028541A1, entitled VEGF-BINDING PROTEIN FOR BLOCKADE OF ANGIOGENESIS, incorporated herein by reference in its entirety. In some embodiments, the transgene encodes for an anti-VEGFR2 antibody or antigen-binding fragments thereof disclosed in International Patent Publication No. WO2014055998A1, entitled Human anti-vegfr-2 / kdr antibodies, incorporated herein by reference in its entirety.

[0193] Expression of a transgene may be measured in ways known in the art. For example, a target cell may be infected in vitro, and the number of copies of the transgene in the cell monitored by Southern blotting or quantitative polymerase chain reaction (PCR). The level of RNA expression may be monitored by Northern blotting or quantitative reverse transcriptase (RT)-PCR; and the level of protein expression may be monitored by Western blotting, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or by the specific methods detailed in the Examples.

[0194] The AAV genome may further comprise a marker or reporter gene, such as a gene for example encoding an antibiotic resistance gene, a fluorescent protein (e.g., GFP) or a gene encoding a chemically, enzymatically or otherwise detectable and / or selectable product (e.g., lacZ, alkaline phosphatase (AP), SEAP, Luc, Neo, Bia, etc.) known in the art.

[0195] Methods of Making Viral Particles

[0196] AAV virions can be produced using methods known in the art, for example using a mammalian AAV production system or an insect cell AAV production system. Methods known in the art are for example described in Pan et al., Disease-inducible transgene expression from 63180489702.1a recombinant adeno-associated virus vector in a rat arthritis model, J Virol. 1999 Apr;73(4):3410-7; Clark et al., Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses, Hum Gene Ther. 1999 Apr 10; 10(6): 1031-9; Wang et al., Production and purification of recombinant adeno-associated vectors, Methods Mol Biol. 2011;807:361-404; Urabe et al., Insect cells as a factory to produce adeno-associated virus type 2 vectors, Hum Gene Ther. 2002 Nov 1; 13(16): 1935-43; Kohlbrenner et al., Successful production of pseudotyped rAAV vectors using a modified baculovirus expression system, Mol Ther. 2005 Dec; 12(6): 1217-25; International Patent Publication No. WO 2007 / 046703, International Patent Publication No. WO 2007 / 148971, International Patent publication No. WO 2011 / 112089, International Patent Publication No. WO 2013 / 036118; and US Patent No. 6,723,551, all of which are incorporated herein by reference in their entireties.

[0197] Many methods involve (a) the introduction of the AAV genome construct into a host cell, (b) the introduction of an AAV helper construct into the host cell, wherein the helper construct comprises the viral functions missing from the wild-type AAV genome and (c) introducing a helper virus construct into the host cell. All functions for AAV vector replication and packaging should be present to achieve replication and packaging of the AAV genome into AAV capsids. The AAV vectors disclosed herein may also be referred to as recombinant AAV (rAAV) vectors since they are not naturally occurring.

[0198] Briefly, in order to package the rAAV genome into a rAAV virion, a host cell is used that contains sequences necessary to express AAV rep and AAV cap or functional fragments thereof as well as helper genes essential for AAV production. The AAV rep and cap sequences are obtained from an AAV source. The AAV rep and cap sequences may be introduced into the host cell in any manner known to one in the art, including, without limitation, transfection, transduction, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection, and protoplast fusion. In one embodiment, the rep and cap sequences may be transfected or transduced into the host cell by one or more nucleic acid molecules and exist stably in the cell as an episome. In another embodiment, the rep and cap sequences are stably integrated into the genome of the cell. Another embodiment has the rep and cap sequences transiently expressed in the host cell. For example, a useful nucleic acid molecule for such transfection or transduction comprises, from 5' to 3', a promoter, an optional spacer interposed between the promoter and the start site of the rep gene sequence, an AAV rep gene sequence, and an AAV cap gene sequence.64180489702.1

[0199] The rep and cap sequences, along with their expression control sequences, may be supplied on a single vector, or each sequence may be supplied on its own vector. Preferably, the rep and cap sequences are supplied on the same vector. Alternatively, the rep and cap sequences may be supplied on a vector that contains other DNA sequences that are to be introduced into the host cells. Preferably, the promoter used in this construct may be any suitable constitutive, inducible or native promoters known to one of skill in the art. The molecule providing the rep and cap proteins may be in any form which transfers these components to the host cell. Desirably, this molecule is in the form of a plasmid, which may contain other non-viral sequences, such as those for marker genes. This molecule does not contain the AAV ITRs and generally does not contain the AAV packaging sequences. To avoid the occurrence of homologous recombination, other virus sequences, particularly those of adenovirus, are avoided in this plasmid. This plasmid is desirably constructed so that it may be stably transfected or transduced into a cell.

[0200] Although the molecule providing rep and cap may be transiently transfected or transduced into the host cell, it is preferred that the host cell be stably transformed with sequences necessary to express functional rep / cap proteins in the host cell, e.g., as an episome or by integration into the chromosome of the host cell. Depending upon the promoter controlling expression of such stably transfected host cell, the rep / cap proteins may be transiently expressed (e.g., through use of an inducible promoter). AAV particles may be produced utilizing a triple transfection method using, e.g., the calcium phosphate method (Clontech) or Effectene reagent (Qiagen, Valencia, Calif.), according to manufacturer’s instructions. See also, Herzog et al., Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector, Nat Med.1999 Jan;5(l):56-63, incorporated herein by reference in its entirety, for a method employing the plasmid with the transgene, a helper plasmid containing AAV rep and cap, and a plasmid supplying adenovirus helper functions of E2A, E40rf6 and VA.

[0201] The rAAV virions can be produced by culturing a host cell containing an rAAV genome to be packaged into a rAAV virion, an AAV rep sequence and an AAV cap sequence under the control of regulatory sequences directing expression thereof. Suitable viral helper genes, e.g., adenovirus E2A, E40rf6, and VA, among other possible helper genes, may be provided to the culture in a variety of ways known to the art, preferably on a separate plasmid. Thereafter, the recombinant AAV virion, which directs expression of the transgene, is isolated from the cell or cell culture in the absence of contaminating helper virus or wildtype AAV.65180489702.1

[0202] rAAV purification processes can include one or more of the following phases: (i) harvest of the producer cells, occasionally with the supernatant, (ii) chemical (e.g., detergent and ion containing lysis buffers) and / or mechanical (e.g., freeze-thaw, microfluidization) cell lysis to liberate AAV particles, (iii) cellular and viral nucleic acid removal, e.g, by enzymatic digestion (Benzonase), (iv) one to three step particle separation by chromatography and optionally density gradients, and (v) concentration, formulation, and sterile filtration.

[0203] Purification methods for rAAV virions are known in the art and include density gradient ultracentrifugation, gradient sedimentation, nonionic iodixanol gradients followed by ion-exchange or heparin-affinity column chromatography, ion-exchange chromatography, mucin affinity chromatography columns, tangential flow filtration, etc.

[0204] Nucleic Acids

[0205] Also provided herein are nucleic acids encoding the modified AAV VP1, VP2, and / or VP3 capsid proteins disclosed herein, as well as expression constructs, vectors, and host cells.

[0206] The term “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0207] The nucleic acids encoding the modified AAV VP1, VP2, and / or VP3 capsid proteins disclosed herein may be, e.g, DNA, cDNA, RNA, synthetically produced DNA or RNA, or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides either alone or in combination. For example, provided is an expression vector comprising a polynucleotide sequence encoding a modified AAV VP1, VP2, and / or VP3 capsid proteins disclosed herein operably linked to expression control sequences suitable for expression in a eukaryotic and / or prokaryotic host cell.

[0208] The term “vector” refers to a vehicle capable of transporting another nucleic acid to which it has been linked. A “vector” includes, but is not limited to, a viral vector, a plasmid, an RNA vector or a linear or circular DNA or RNA molecule which may comprise a chromosomal, non-chromosomal, semi -synthetic, or synthetic nucleic acid. The vector can be a nucleic acid and or viral particle. In some embodiments, the employed vectors are those capable of autonomous replication (episomal vector) and / or expression of nucleic acids to which they are linked (expression vectors). A viral particle is a vector.66180489702.1

[0209] An “expression construct” is a nucleic acid that allows for the expression of a transgene.

[0210] Provided herein is a nucleic acid sequence encoding a modified AAV VP1 capsid protein, wherein the nucleic acid sequence is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:63. Provided herein is a nucleic acid sequence encoding a modified AAV VP1 capsid protein, wherein the nucleic acid sequence comprises SEQ ID NO:63.

[0211] ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTG AAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCG CAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACC TCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCG CGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACC CGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAG ATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGG TTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAA AGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAA AGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACG CAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGG TCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAA CGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCAC ATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCAC CTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGA CAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTC CACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGAT TCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCAC GCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGT GTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGA TGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCA CCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTA CTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTG AGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCAT GAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGT GGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATT67180489702.1CGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTAT CAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCA AGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAA GCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGG GAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAG ACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTG TATCTACCAACCTCCAGAGAGGCAACGCGGCCGCCACTGTGAATCTGGTTAAG GCGGCGGCGAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCC AGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAA GATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGA CTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATC CTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCAC GGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAAC GCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGA CTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGA TACCTGACTCGTAATCTGTAA (SEQ ID NO:63).

[0212] Provided herein is a nucleic acid sequence encoding a modified AAV VP1 capsid protein, wherein the nucleic acid sequence is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:64. Provided herein is a nucleic acid sequence encoding a modified AAV VP1 capsid protein, wherein the nucleic acid sequence comprises SEQ ID NO:64.

[0213] ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTG AAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCG CAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACC TCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCG CGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACC CGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAG ATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGG TTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAA AGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAA AGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACG CAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGG TCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAA68180489702.1CGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCAC ATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCAC CTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGA CAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTC CACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGAT TCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCAC GCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGT GTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGA TGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCA CCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTA CTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTG AGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCAT GAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGT GGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATT CGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTAT CAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCA AGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAA GCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGG GAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAG ACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTG TATCTACCAACCTCCAGAGAGGCAACGCGGCCGCCACTTCCGTTGGGACTATT AGGGCGGCGAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCC AGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAA GATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGA CTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATC CTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCAC GGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAAC GCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGA CTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGA TACCTGACTCGTAATCTGTAA (SEQ ID NO: 64).

[0214] Provided herein is a nucleic acid sequence encoding a modified AAV VP1 capsid protein, wherein the nucleic acid sequence is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:96. Provided herein is a nucleic acid sequence 69180489702.1encoding a modified AAV VP1 capsid protein, wherein the nucleic acid sequence comprises SEQ ID NO:96.

[0215] ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTG AAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCG CAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACC TCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCG CGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACC CGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAG ATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGG TTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAA AGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAA AGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACG CAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGG TCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAA CGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCAC ATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCAC CTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGA CAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTC CACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGAT TCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCAC GCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGT GTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGA TGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCA CCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTA CTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTG AGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCAT GAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGT GGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATT CGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTAT CAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCA AGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAA GCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGG GAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAG ACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTG70180489702.1TATCTACCAACCTCCAGAGAGGCAACGCGGCCGCCTTTAGCTCTGATCGGATT AAGGCGGCGAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCC AGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAA GATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGA CTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATC CTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCAC GGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAAC GCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGA CTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGA TACCTGACTCGTAATCTG (SEQ ID NO:96).

[0216] Provided herein is a nucleic acid sequence encoding a modified AAV VP1 capsid protein, wherein the nucleic acid sequence is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 129. Provided herein is a nucleic acid sequence encoding a modified AAV VP1 capsid protein, wherein the nucleic acid sequence comprises SEQ ID NO: 129.

[0217] ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTG AAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCG CAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACC TCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCG CGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACC CGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAG ATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGG TTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAA AGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAA AGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACG CAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGG TCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAA CGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCAC ATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCAC CTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGA CAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTC CACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGAT TCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCAC71180489702.1GCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGT GTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGA TGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCA CCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTA CTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTG AGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCAT GAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGT GGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATT CGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTAT CAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCA AGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAA GCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGG GAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAG ACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTG TATCTACCAACCTCCAGAGAGGCAACGCGGCCGCCAATGTCACTAATTTGCTC ACTGCGGCGAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCC AGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAA GATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGA CTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATC CTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCAC GGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAAC GCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGA CTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGA TACCTGACTCGTAATCTG (SEQ ID NO: 129).

[0218] Provided herein is a nucleic acid sequence encoding a modified AAV VP1 capsid protein, wherein the nucleic acid sequence is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 130. Provided herein is a nucleic acid sequence encoding a modified AAV VP1 capsid protein, wherein the nucleic acid sequence comprises SEQ ID NO: 130.

[0219] ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTG AAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCG CAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACC TCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCG72180489702.1CGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACC CGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAG ATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGG TTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAA AGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAA AGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACG CAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGG TCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAA CGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCAC ATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCAC CTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGA CAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTC CACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGAT TCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCAC GCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGT GTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGA TGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCA CCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTA CTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTG AGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCAT GAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGT GGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATT CGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTAT CAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCA AGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAA GCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGG GAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAG ACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTG TATCTACCAACCTCCAGAGAGGCAACGCGGCCGCCAATGTCACTAATGTGCTC ACTGCGGCGAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCC AGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAA GATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGA CTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATC CTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCAC73180489702.1GGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAAC GCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGA CTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGA TACCTGACTCGTAATCTG (SEQ ID NO: 130).

[0220] A person skilled in the art will appreciate that based on SEQ ID NOs:63, 64, 96, 129, or 130, nucleic acid sequences can be created that encode the VP2 portion or the VP3 portion of the VP1 protein encoded by SEQ ID NOs:63, 64, 96, 129, or 130, respectively.

[0221] Cells

[0222] Provided herein are cells comprising a nucleic acid or expression construct provided herein. In embodiments, the cell is a cell suitable for producing AAV capsid proteins and / or AAV viral particles. In some embodiments, the cell is a mammalian cell or an insect cell.

[0223] Pharmaceutical Compositions

[0224] Provided herein are pharmaceutical compositions comprising a composition, including, but not limited to a viral particle, disclosed herein and a pharmaceutically acceptable excipient.

[0225] The composition, including, but not limited to a viral particle, disclosed herein is preferably assessed for contamination by conventional methods and then formulated into a pharmaceutical composition suitable for storage and / or administration to a patient.

[0226] Formulations of the composition, including, but not limited to a viral particle, disclosed herein involve the use of a pharmaceutically and / or physiologically acceptable vehicle or carrier, particularly one suitable for injection or infusion, such as buffered saline or other buffers, e.g., HEPES, to maintain pH at appropriate physiological levels. The compositions, including, but not limited to viral particles disclosed herein can be formulated into pharmaceutical compositions. These compositions may comprise, in addition to the composition, a pharmaceutically and / or physiologically acceptable excipient, carrier, buffer, stabilizer, antioxidants, preservative, or other additives well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may be determined by the skilled person according to the route of administration. The pharmaceutical composition is typically in liquid form. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Additional carriers are provided in International Patent Publication No. W02000015822A1, entitled METHODS FOR 74180489702.1TREATMENT OF DEGENERATIVE RETINAL DISEASES, incorporated herein by reference in its entirety. Physiological saline solution, magnesium chloride, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. In some cases, a surfactant, such as pluronic acid (PF68) 0.001% may be used. In some cases, Ringer's Injection, Lactated Ringer's Injection, or Hartmann's solution is used. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included, as required.

[0227] Pharmaceutical compositions comprising a composition, including, but not limited to a viral particle, disclosed herein may be formulated with one or more pharmaceutically-acceptable excipients, which can be a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or stearic acid), solvent or encapsulating material, involved in carrying or transporting the therapeutic compound for administration to the subject, bulking agent, salt, surfactant and / or a preservative. Some examples of materials which can serve as pharmaceutically-acceptable excipients include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; gelatin; talc; waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as ethylene glycol and propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents; water; isotonic saline; pH buffered solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0228] A bulking agent is a compound which adds mass to a pharmaceutical formulation and contributes to the physical structure of the formulation in lyophilized form. Suitable bulking agents according to the present disclosure include mannitol, glycine, polyethylene glycol and sorbitol.

[0229] The use of a surfactant can reduce aggregation of the reconstituted particles and / or reduce the formation of particulates in the reconstituted formulation. The amount of surfactant added is such that it reduces aggregation of the reconstituted protein and minimizes the formation of particulates after reconstitution. Suitable surfactants according to the present disclosure include polysorbates (e.g. polysorbates 20 or 80); pol oxamers (e.g. pol oxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; 1 auroamidopropyl-, cocamidopropyl-, linoleamidopropyl- 75180489702.1, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol e.g. Pluronics, PF68, etc.).

[0230] Preservatives may be used in formulations of disclosure. Suitable preservatives for use in the formulation of the disclosure include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3 -pentanol, and m-cresol. Other suitable excipients can be found in standard pharmaceutical texts, e.g., in “Remington's Pharmaceutical Sciences,” The Science and Practice of Pharmacy, 19th Ed. Mack Publishing Company, Easton, Pa., (1995).

[0231] For delayed release, the composition, including, but not limited to a viral particle, disclosed herein may be included in a pharmaceutical composition which is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.

[0232] If a composition, including, but not limited to a viral particle, is to be stored longterm, it may be frozen in the presence of a cryoprotectant, such as glycerol.

[0233] Methods

[0234] Provided is a method of increasing expression of a transgene in a retinal cell as compared to expression in non-retinal cell, the method comprising contacting the retinal cell with an AAV particle comprising one or more modified VP capsid proteins provided herein.

[0235] Provided is a method of increasing expression of a transgene in a retinal cell type of interest as compared to expression other retinal cells or non-retinal cells, the method comprising contacting the retinal cell with an AAV particle comprising one or more modified VP capsid proteins provided herein.

[0236] A method of delivering a transgene to a retinal cell, the method comprising contacting the retinal cell with an AAV particle comprising one or more modified VP capsid proteins provided herein.76180489702.1

[0237] A method of delivering a transgene to a retinal cell type of interest, the method comprising contacting the retinal cell type of interest with an AAV particle comprising one or more modified VP capsid proteins provided herein.

[0238] The retinal cells may be rod cells, cone cells, bipolar cells, Mueller glia cells, horizontal cells, retinal astrocytes, retinal ganglion cells, RPE cells, or amacrine cells.

[0239] Provided are methods of treating or preventing ocular disorders using the compositions disclosed herein in a subject in need thereof. Both acquired and congenital diseases are amenable to the gene therapy methods disclosed herein.

[0240] The terms “treat,” “treated,” “treating,” or “treatment” as used herein refer to therapeutic treatment, wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (z.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0241] The terms “prevent”, “prevention”, and the like refer to acting prior to overt disease or disorder onset, to prevent the disease or disorder from developing or to minimize the extent of the disease or disorder or slow its course of development.

[0242] An “effective amount” or “therapeutically effective amount” refers to an amount of the compound or agent that is capable of producing a medically desirable result in a treated subject. The treatment method can be performed in vivo or ex vivo, alone or in conjunction with other drugs or therapy. A therapeutically effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

[0243] As used herein, the terms “subject” and “patient” are used interchangeably irrespective of whether the subject has undergone treatment in the past or is currently undergoing any form of treatment. As used herein, the terms “subject” and “subjects” may refer to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, mouse, non-human primate (for example,77180489702.1a monkey, such as a cynomolgus monkey, chimpanzee, etc.) or a human). The subject may be a human or a non-human. In some embodiments, the subject is a human.

[0244] Examples of disorders that can be treated using the compositions and methods disclosed herein include retinal dystrophies. Retinal dystrophies cause blindness due to destruction of the photoreceptors in the outer retina ( / .< ., the rods and cones). These conditions may be a result of direct damage to the photoreceptors, or photoreceptors being indirectly destroyed as a result of pathology in the retinal pigment epithelium and / or choroid. Severe visual impairment is common in advanced stages of the degeneration. Retinal dystrophies can be divided into rod-cone dystrophies (also called retinitis pigmentosa), cone-rod dystrophies and macular dystrophies. In rod-cone dystrophies the rod photoreceptors degenerate resulting in a loss of peripheral vision and night vision, and frequently this is followed by cone destruction leading to a loss of central and color vision. Conversely, in the cone-rod dystrophies there is initially a loss of cone photoreceptors leading to a loss of detailed and color vision and this is then followed by rod degeneration resulting in a loss of peripheral vision and night blindness. Both forms can result in blindness with extensive or complete loss of visual field. Another type of retinal dystrophy called macular dystrophy results in a loss of central vision, but peripheral vision is preserved.

[0245] Diseases and disorders that can be treated using the compositions and methods disclosed herein include, but are not limited to, cone dysfunction, rod dysfunction, age related macular degeneration (AMD) (including dry AMD, geographic atrophy (GA), and wet AMD), ocular angiogenesis, Vitelliform macular dystrophy, or North Carolina macular dystrophy, Leber’s congenital amaurosis (LCA), choroideremia (CHM), achromatopsia (ACHM), usher syndrome (USH), X-linked retinoschisis (often also referred to as X-linked juvenile retinoschisis (XLRS)), Stargardt disease (including Autosomal recessive Stargardt disease (STGD1)), Retinitis pigmentosa (RP) (including X-linked RP (XLRP), MERTK-associated RP, PDE6B-associated RP, RLBP1 -associated RP, and non-syndromic RP), diabetic macular edema, diabetic retinopathy, Leber hereditary optic neuropathy (LHON), Congenital stational nightblindness, cone-dystrophy with supernormal rod response (CDSSR), glaucoma, acquired renal cystic disease (arCD), Best disease (vitelliform macular dystrophy (BVMD)), and autosomal dominant cone-rod dystrophy (adCRD). In one embodiment, the LCA is Leber congenital amaurosis type 1 (arLCA orLCAl).

[0246] LCA is a progressive visual disorder with a prevalence of 1 : 50,000-100,000. It is an inherited retinal dystrophy owing to a defect in the retinal pigment epithelium (RPE) that often results in profound vision loss and nystagmus in childhood. Biallelic mutations in the gene for 78180489702.1RPE65, an essential isomerase of the retinoid visual cycle responsible for the conversion of all-trans-retinyl ester to 11 -cis-retinol, account for 3-16% of LCA cases. The absence of RPE65 results in the disruption of the visual cycle leading to absent rod function and, consequently, to photoreceptor degeneration. Provided is a method of treating LCA in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an AAV vector comprising one or more modified VP capsid proteins provided herein and a nucleic acid comprising an RPE65 gene. The compositions and methods for treating LCA disclosed herein may be used in conjunction with any expression constructs disclosed in International Patent Publication No. WO2016128722A1, entitled OPTIMIZED RPE65 PROMOTER and coding sequences, which is incorporated herein by reference in its entirety. Provided is a method of treating LCA in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an AAV vector comprising one or more modified VP capsid proteins provided herein and a nucleic acid comprising an RDH12 gene. The compositions and methods for treating LCA disclosed herein may be used in conjunction with any expression constructs disclosed in International Patent Publication No. WO2019099696A1, entitled VIRAL VECTORS COMPRISING RDH12 CODING REGIONS AND METHODS OF TREATING RETINAL DYSTROPHIES, which is incorporated herein by reference in its entirety.

[0247] Retinal membrane guanylyl cyclase (RetGC) is located in disc membranes of photoreceptor outer segments and is one of the key enzymes in photoreceptor physiology, producing a second messenger of phototransduction, cyclic guanosine monophosphate (cGMP), in mammalian rods and cones. During photoreceptor excitation and recovery, two RetGC isozymes, RetGC 1 and RetGC2 (also known as GC-E and GC-F or ROSGC1 and ROSGC2, respectively), are tightly regulated by calcium feedback mediated by guanylyl cyclase-activating proteins (GCAPs). Over 100 mutations in GUCY2D, the gene that encodes RetGC are known to cause two major diseases: autosomal recessive Leber congenital amaurosis type 1 (arLCA or LCA1) or autosomal dominant cone-rod dystrophy (adCRD). In CRD, degeneration starts in the cones and leads to loss of the central visual field due to the high presence of cones in the macula of a non-affected retina. CRD can lead to complete blindness when degeneration of rods follows those of cones. The LCA1 phenotype appears even more severe, with photoreceptor function loss and blindness emerging very early in life. Provided is a method of treating a retinal disease that is associated with one or more mutations in the GUCY2D gene in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an AAV vector comprising one or more 79180489702.1modified VP capsid proteins provided herein and a nucleic acid comprising an RetGCl or RetGC2 gene. In some embodiments, the retinal disease is CRD or LCA1. The compositions and methods for treating a retinal disease that is associated with one or more mutations in the GUCY2D gene disclosed herein may be used in conjunction with any expression constructs disclosed in International Patent Publication No. WO2023285987A1, entitled RETGC GENE THERAPY, which is incorporated herein by reference in its entirety.

[0248] Potassium voltage-gated channel subfamily V member 2 (Kv8.2) is a voltage gated potassium channel subunit encoded by the KCNV2 gene. Variants / mutations of the KCNV2 gene cause a severe inherited photoreceptor dystrophy known as “cone-dystrophy with supernormal rod response” (CDSSR). Symptoms of CDSSR include reduced visual acuity, color vision defects, and altered electroretinogram responses, including elevated b-wave amplitudes. Provided is a method of treating CDSSR in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an AAV vector comprising one or more modified VP capsid proteins provided herein and a nucleic acid comprising an KCNV2 gene. The compositions and methods for treating LCA disclosed herein may be used in conjunction with any expression constructs disclosed in International Patent Publication No. WO2023285986A1, entitled KCNV2 GENE THERAPY, which is incorporated herein by reference in its entirety.

[0249] ACHM is a congenital autosomal recessive cone dysfunction disorder. It presents with early-onset pendular nystagmus, decreased visual acuity, photosensitivity, and impaired color perception. Six phototransduction genes (CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, and ATF6) are known to be implicated in ACHM. Out of these, loss-of-function mutations in CNGA3 and CNGB3 account for over 70% of ACHM cases. CNGA3 and CNGB3 encode the alpha- and beta-subunits of the cyclic nucleotide-gated (CNG) cation channel in cones. The deficiency in, for example, the CNGA3 or CNGB3 protein in the cone photoreceptor cells leads to an inability of the cells to hyperpolarize in response to light. As a result, the cones initially survive, but do not function. Patients presumably maintain a normal rod function. Provided is a method of treating ACHM in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an AAV vector comprising one or more modified VP capsid proteins provided herein and a nucleic acid comprising a CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, and / or ATF6 gene. The compositions and methods for treating ACHM disclosed herein may be used in conjunction with any expression constructs disclosed in International Patent Publication No. WO2019138250A1, entitled80180489702.1COMPOSITIONS AND METHODS FOR TREATING RETINAL DISORDER, which is incorporated herein by reference in its entirety.

[0250] STGD1 is caused by mutations in the ABCA4 gene, a photoreceptor ATP-binding cassette (ABC) transporter. This disease is typically diagnosed in adolescence. ABCA4-associated retinopathy causes progressive central or pericentral visual field loss. Provided is a method of treating Stargardt disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an AAV vector comprising one or more modified VP capsid proteins provided herein and a nucleic acid comprising an ABCA4 gene. Provided is a method of treating an ABCA4-associated retinopathy in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an AAV vector comprising one or more modified VP capsid proteins provided herein and a nucleic acid comprising an ABCA4 gene.

[0251] X-linked juvenile retinoschisis (XLRS) is hereditary retinal degeneration that affects 1:5,000 to 1:20,000 young males. The disease is caused by mutations in the gene for RSI, which is a secretory protein essential for retinal organization and intracellular adhesion. Patients typically present with a slowly progressive loss of central vision in early childhood. Complications include retinal detachment and vitreous hemorrhage. Provided is a method of treating XLRS in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an AAV vector comprising one or more modified VP capsid proteins provided herein and a nucleic acid comprising an RSI gene.

[0252] RP is a leading form of inherited blindness in humans. Of the three general modes of inheritance (autosomal dominant, autosomal recessive, and X-linked), XLRP is associated with a severe form of disease involving both rod and cone photoreceptors as primary targets. Over 70% of XLRP and 10% - 20% of all RP cases are caused by mutations in the gene encoding RPGR. Given that mutations in well over 100 genes are currently known to cause RP and the greater severity of X-linked disease, RPGR is one of the most important RP disease genes. RPGR is involved in ciliary transport and critical in maintaining photoreceptor integrity. Male patients typically present in early childhood with progressive nyctalopia and narrowing of peripheral visual field that result in legal blindness. Provided is a method of treating RP in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an AAV vector comprising one or more modified VP capsid proteins provided herein and a nucleic acid comprising an RPGR gene. Provided is a method of treating XLRP in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an AAV vector comprising one or more modified VP 81180489702.1capsid proteins provided herein and a nucleic acid comprising an RPGR gene. The compositions and methods for treating RP / XLRP disclosed herein may be used in conjunction with any expression constructs disclosed in International Patent Publication No. WO2016014353A1, entitled RPGR GENE THERAPY FOR RETINITIS PIGMENTOSA, which is incorporated herein by reference in its entirety.

[0253] MERTK-associated RP, similar to RPE65-associated LCA, involves dysfunction of the RPE. The disease is caused by mutations in MERTK implicated in RPE phagocytosis of photoreceptor segments. Provided is a method of treating MERTK-associated RP in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an AAV vector comprising one or more modified VP capsid proteins provided herein and a nucleic acid comprising a MERTK gene. Provided is a method of treating PDE6B-associated RP in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an AAV vector comprising one or more modified VP capsid proteins provided herein and a nucleic acid comprising a PDE6B gene. Provided is a method of treating RLBP1 -associated RP in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an AAV vector comprising one or more modified VP capsid proteins provided herein and a nucleic acid comprising an RLBPlgene.

[0254] LHON is a mitochondrial DNA disorder that typically presents in young males as concurrent or consecutive loss of central vision bilaterally. The majority of LHON cases are caused by mitochondrial DNA (mtDNA) point mutations in ND4, a critical component of the respiratory chain. The resulting increased generation of reactive oxygen species caused by a dysfunctional respiratory chain leads to retinal ganglion cell damage and subsequent optic atrophy. Provided is a method of treating LHON in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an AAV vector comprising one or more modified VP capsid proteins provided herein and a nucleic acid comprising a ND4 gene.

[0255] Also provided are methods of prolonging the survival of cones that otherwise degenerate due to a bystander effect in a subject in need thereof using the compositions disclosed herein. The bystander effect refers to a phenomenon where disease spreads from dying rods to healthy cones. Provided is a method of reducing or eliminating a bystander effect in the retina of a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an AAV vector comprising one or more modified VP capsid proteins provided herein and a nucleic acid comprising a transgene disclosed herein.82180489702.1

[0256] Also provided herein are methods of making rods more cone-like as disclosed in International Patent Publication No. WO2016 / 135457, entitled GENE THERAPY TO IMPROVE VISION, which is incorporated herein in its entirety, using the compositions disclosed herein. In many mammalian species including mice and humans, the number of rod photoreceptors that mediate vision under dim light outnumbers greatly that of cone photoreceptors. However, in an industrialized world where illumination allows cones to operate throughout the day, rod-mediated vision can be less important. Many patients with absent rod function from birth are identified only incidentally and, in fact, cannot recognize their abnormal vision. On the contrary, when cone dysfunction is present, patients are always symptomatic and often suffer visual handicap dependent on the degree of their cone dysfunction. In some conditions, however, only (or mostly) the cones are lost or dysfunctional and rods remain relatively preserved. For example, achromatopsia is a severe hereditary retinal dystrophy with a complete absence of cone function from birth but, presumably, with a normal rod function. Mutations in multiple genes including CNGA3 and PDE6C have been associated with the disease. Each of the disease-causing genes encodes a component of the cone phototransduction cascade that translates light into an electric signal by causing hyperpolarization of the photoreceptor cell. In AMD, visual impairment is caused primarily by degeneration of the cone-rich fovea in the central macula. Thus, patients lose central vision and acuity, but often have relatively well-preserved peripheral macula and thus have some useful residual vision that is limited by the paucity of cones outside the fovea. Accordingly, provided is a method of improving vision in a subject with cone photoreceptor dysfunction and / or degeneration, wherein the patient has healthy rod photoreceptors in the subject’s retina, the method comprising administering to the subject an AAV vector comprising one or more modified VP capsid proteins provided herein and a nucleic acid comprising a transgene encoding a gene product that is light-sensitive and / or that modulates endogenous light-sensitive signaling in a photoreceptor cell, whereby administration of the vector results in introducing said transgene into the healthy rod photoreceptors in the subject’s retina and expression of said gene product therein and thereby increasing the intensity of the light to which the rod photoreceptor responds and / or increasing the speed at which the rod photoreceptor responds to light as compared to a healthy rod photoreceptor that does not express said gene product. In embodiments, the gene product is ArchT, R9AP, Jaws, iClC2, Rhodopsin, GNAT1, CNGB1, APOE, HTRA1, or C3.

[0257] The compositions disclosed herein may be administered in order to prevent or delay the onset of one or more symptoms of an ocular disorder. The subject may be asymptomatic or83180489702.1may have a predisposition to the disease. The method or use may comprise a step of identifying a subject that has, or is at risk of developing, an ocular disorder.

[0258] The dose of a vector of the disclosure may be determined according to various parameters, especially according to the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. A typical single dose is about 108, about 109, about IO10, about 1011, about 1012, about 1013, or about 1014, genome particles, depending on the amount of retinal tissue that requires transduction. A genome particle is defined herein as an AAV capsid that contains a viral genome (VG) that can be quantified with a sequence specific method (such as real-time PCR). That dose may be provided as a single dose, but may be repeated for the fellow eye or in cases where vector may not have targeted the correct region of retina for whatever reason (such as surgical complication). The treatment is preferably a single permanent treatment for each eye, but repeat injections, for example in future years and / or with different AAV serotypes may be considered.

[0259] In some embodiments, AAV particles disclosed herein are provided in a volume of between about 5 pL to about 5 mL. In some embodiments, AAV particles are provided in a volume of between about 20 pL to about 3 mL. In some embodiments, AAV particles are provided in a volume of about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000 pL.

[0260] The AAV particles disclosed herein (or pharmaceutical compositions comprising AAV particles disclosed herein) may be administered using any method compatible with transduction of cells in the eye. In some embodiments, the AAV particles disclosed herein (or pharmaceutical compositions disclosing AAV particles disclosed herein) are administered via direct retinal injection, subretinal injection, intravitreal injection, suprachoroidal injection, topical instillation, or intravenous injection.

[0261] In embodiments, the expression construct, vector, or pharmaceutical composition disclosed herein is administered with a device described in any one of International Patent Publication No. WO2018 / 112305 (entitled SYSTEM AND METHOD FOR RESISTANCEDEPENDENT, SELF-REGULATED MEDICAL PENETRATION), W02021 / 055906 (entitled INJECTION SYSTEMS AND METHODS OF THEIR USE), and WO2022 / 036256 (entitled MOTORIZED INJECTION SYSTEM AND METHODS OF USE), which are incorporated by reference herein in their entireties.

[0262] Kits84180489702.1

[0263] The compositions, including, but not limited to viral particles, disclosed herein can be packaged into a kit. Provided are kits for the manufacturing, preparation, and development of the compositions described above, comprising at least one or more containers, each with a different reagent for the manufacturing of the compositions disclosed herein. Kits may include a set of instructions in the use of the reagents, essential information on how performing the procedures for the manufacturing.

[0264] In certain embodiments, kits are provided for the preparation of a pharmaceutical composition comprising a therapeutically effective amount of a composition disclosed herein and a pharmaceutically acceptable carrier. The kit may comprise at least one, or more containers, each with a different reagent. Kits may include instruction for the preparation, for the therapeutic regimen to be used, and periods of administration.

[0265] The compositions or the pharmaceutical compositions described herein can be provided in a kit. In one embodiment, the kit includes (a) a container that contains the composition and optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and / or the use of the agents for therapeutic benefit. For example, kits may include instruction for the preparation and / or administration of the compositions described herein. In an embodiment, the kit includes also includes an additional therapeutic agent. The kit may comprise one or more containers, each with a different reagent.

[0266] The containers can include a unit dosage of the pharmaceutical composition. In addition to the composition, the kit can include other ingredients, such as a solvent or buffer, an adjuvant, a stabilizer, or a preservative.

[0267] The kit optionally includes a device suitable for administration of the composition, e.g., a syringe or other suitable delivery device. The device can be provided pre-loaded with one or both of the agents or can be empty, but suitable for loading.

[0268] It is to be understood that this invention is not limited to the particular molecules, compositions, methodologies, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention. It is further to be understood that the disclosure of the invention in this specification includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the invention, or a particular claim, that feature can also be used, to the extent85180489702.1possible, in combination with and / or in the context of other particular aspects and embodiments of the invention, and in the invention generally.

[0269] Where reference is made herein to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes those possibilities).

[0270] All referenced patents and applications, scientific papers, book chapters, etc., are incorporated herein by reference in their entireties. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0271] To facilitate a better understanding of the present invention, the following Examples of specific embodiments are given. The following examples should not be read to limit or define the entire scope of the invention.EXAMPLES

[0272] Example 1: Isolation of Modified AAV Capsid Proteins Particularly Suitable for Transducing Cells in the Eye

[0273] In Vivo Directed Evolution Capsid Screen

[0274] An in vivo directed evolution capsid screen was performed in non-human primates to identify novel capsids that target the back of the eye. This process involved administering a library of modified AAV capsids intravitreally into the eyes of non-human primates (NHPs). By deselecting the fovea, which most capsids readily transduce, this approach aimed to identify capsids with a broader transduction profile, extending to the perimacular region and beyond.

[0275] Library Construction

[0276] The library construction involved generating a diverse array of modified AAV VP1 capsid through inserting random peptides into a specific location in the capsid. Specifically, a 7mer peptide display library based on capsid protein VP1 from AAV2 was created, containing a 7mer peptide insertion before amino acid 588, surrounded by an N-terminal AAA linker and a C-terminal AA linker. In other words, 12 amino acids inserted between amino acid residues86180489702.1587 and 588 of the AAV2 VP1 capsid protein. The construction process included synthesizing a large number of peptide sequences and incorporating them into the AAV2 VP1 capsid protein, creating a vast pool of potential candidates for screening. As discussed herein, the 587 / 588 VP1 insertion site is located in the VP portion that is common to VP1, VP2, and VP3. As such, the capsid in the library included modified VP2 and VP3 proteins as well.

[0277] In Vivo Selection

[0278] To identify modified capsids targeting the back of the eye, AAV particles comprising the modified VP capsid proteins from the library were administered intravitreally into NHP eyes. Following administration, retinal cells were harvested. Viral genomes were recovered.

[0279] Deep Sequencing

[0280] High-throughput sequencing was employed to analyze the recovered viral genomes. This allowed for the identification of modified AAV capsid proteins that were enriched in the retinal cells, indicating superior transduction efficiency.

[0281] Validation and Characterization

[0282] Out of a library of about 10A7 modified capsid proteins, variants that performed particularly well, including DE12 and DE25, were selected and further validated, as discussed in Examples 2-5.

[0283] Example 2: Validation of Isolated Modified AAV Capsid Proteins in the Mouse Retina

[0284] The ability of AAV particles comprising modified AAV capsid proteins DE25 (see SEQ ID NOs:33-35) and DE12 (see SEQ ID NOs:30-32) to transduce cells in the mouse retina following intravitreal delivery was assessed. AAV particles comprising AAV2.7m8 VP capsid proteins served as controls. AAV2.7m8 is currently among the best capsids for transducing the retina following intravitreal injection and served as a benchmark. See Khabou et al (2016).

[0285] All vectors were titre-matched, injected intravitreally (lA10VG / eye) and assessed five weeks after delivery by immunohistochemistry and three weeks after delivery by absolute quantification.

[0286] AAV particles comprising AAV 7m8 and DE25 capsid proteins, respectively, showed a similar transduction profile, targeting the inner retina more prominently, followed by outer retina (Fig. 1). In contrast, AAV particles comprising DE12 capsid proteins also showed (in addition to inner retina transduction) a higher degree of Muller glia transduction (Fig. 1).Absolute quantification of total retinal expression in injected retinas showed a trend for higher 87180489702.1overall expression levels provided by AAV particles comprising DE12 or DE25 capsid proteins, respectively, as compared to AAV particles comprising 7m8 capsid proteins (Fig.2).

[0287] In sum, this data shows that AAV particles comprising modified AAV VP capsid proteins can transduce retinal cells with high efficiency and specificity following in vivo delivery in a mammalian model.

[0288] Example 3: Validation of Modified AAV VP Capsid Proteins in Mature Human Retinal Organoids

[0289] Next, the expression profiles of AAV particles comprising modified capsid proteins DE12 and DE25, respectively, as compared to AAV particles comprising AAV2.7m8 capsid proteins or the parental AAV2 capsid proteins, respectively, were assessed in human retinal organoids. Additional experiments were performed with modified capsid protein DE39 (see SEQ ID NOs:93-95).

[0290] Macroscopic evaluation of transduced human retinal organoids showed robust transgene expression when transduced with AAV virions comprising the DE12, DE25, and DE39 capsid proteins, respectively, as compared to AAV virions comprising parental AAV2 VP capsid proteins (Fig. 3A and Fig. 3B). Flow cytometric analysis of human photoreceptor expression levels showed, similarly to the mouse retinal data in Fig.2, a trend for higher overall expression levels for AAV virions comprising capsid proteins DE12, DE25, and DE39 as compared to AAV virions comprising AAV.7m8 capsid proteins (Fig. 4A and Fig. 4B - right panel). Expression levels were quantified using median GFP intensity measures in transduced human photoreceptors. The percentage of transduced photoreceptors was similar for AAV virions comprising DE12, DE25, or 7m8 capsid proteins, respectively (Fig. 4A and Fig. 4B -left panel). AAV virions comprising AAV2 capsid proteins significantly underperformed in both readouts (percentage transduction and median GFP intensity).

[0291] Immunohistochemistry (IHC) analysis of human retinal organoids transduced with AAV virions comprising DE12 and DE25 capsid proteins showed robust outer nuclear layer (ONL) expression (Fig. 5). Qualitative assessment of human retinal organoids transduced with AAV virions comprising DE12 capsid proteins also indicated that these virions exhibited similar transduction efficiency and resulting transgene expression levels in photoreceptor cells compared to AAV virions comprising 7m8 capsid proteins. In contrast, human retinal organoids transduced with AAV virions comprising DE25 capsid proteins showed a marked increase in ONL expression levels compared to both expression levels promoted by AAV virions comprising DE12 or 7m8 capsid proteins, respectively. Control AAV virions 88180489702.1comprising AAV2 capsid proteins showed markedly weaker overall expression levels when compared to AAV virions comprising either 7m8, DE12, or DE25 capsid proteins, respectively.

[0292] Example 4: Modified AAV VP Capsid Proteins Show Differential Expression in Different Retinal Cell Populations

[0293] Single-cell RNA sequencing was employed to acquire a deep transcriptional profile and quantify transduction levels in the whole organoid as well as in specific retinal cell subtypes. This high-resolution technique allowed dissecting the transduction efficiency at a cellular level.

[0294] AAV virions comprising DE25 capsid proteins achieved over 2-fold higher transduction efficiency compared to AAV virions comprising AAV2.7m8 capsid proteins, both when assessing overall transduction levels (Fig.6A) and in specific retinal cell types (Fig. 6B).This finding underscores the superior performance of AAV virions comprising DE25 capsid proteins in targeting and transducing retinal cells, highlighting its value for clinical applications. AAV virions comprising DE12 capsid proteins showed similar levels of overall transduction efficiency compared to AAV virions comprising AAV2.7m8 capsid proteins (Fig.6A). However, on the cellular level, a marked increase in Muller glia transduction was observed over AAV virions comprising AAV2.7m8 capsid proteins (Fig. 6B).

[0295] Example 5: RPE Screening Identifies Capsids with Enhanced Transduction Efficiency in IPSC-Derived RPE Cells

[0296] IPSC-RPE were differentiated from wild-type Induced pluripotent stem cells (IPSC) using STEMdiff™- animal component-free (ACF) differentiation kit and cultured in STEMdiff™-XF RPE maturation medium (MM) from Stem Cell Technologies (Cat no. 1 GO-1367).

[0297] RPE cell identity was confirmed by PMEL17 staining and flow cytometry (90 % PMEL17 positive at day of passaging). PMEL17 is a transmembrane glycoprotein used for identifying and confirming RPE cells. IPSC-RPE cells were cultured in 48 well plates and matured for 8-12 weeks prior to transduction. Cells were counted and transduced at the indicated MOIs calculated based on vector genome titration (VG) in 200 pl MM. Three weeks post transduction, RPE cell were imaged (EVOS M5000 - Thermo Fisher) before dissociation into single cells (TrypLE - Thermo Fisher Cat no.12604013). GFP expression was assessed in live cells by flow cytometry (percent GFP positive single cells, median GFP intensity in GFP89180489702.1population) on a BD FACS lyric flow cytometer with non-transduced RPE used to set the negative gate.

[0298] As shown in Figs. 7A, 7B, 7C, 7D and 7E, capsid protein variants DEI 8 (SEQ ID NOs: 123-125) andDE61 (SEQ ID NOs: 126-128) are particularly suitable for transducing RPE cells.

[0299] In sum, using a differentiated NHP screening approach, modified AAV VP capsid proteins were identified with significantly more potent transduction power compared to the best performing intravitreal capsids currently available, such as AAV2.7m8. The validation of the capsid variants disclosed herein in human retinal organoids has significant implications for their use in therapeutic applications. The enhanced transduction efficiency and specificity of virus particles comprising these capsids make them ideal candidates for gene therapies targeting retinal cells. The ability to achieve high levels of gene expression in specific retinal cell types opens up new possibilities for treating a wide range of retinal diseases, including inherited retinal disorders and age-related macular degeneration. The superior performance of AAV virions comprising capsid proteins disclosed herein, indicated that these capsids can improve the efficacy and safety of gene therapies, providing better outcomes for patients.90180489702.1

Claims

CLAIMS1. A modified adeno-associated virus (AAV) viral protein (VP), wherein:(a) the modified AAV VP comprises a framework protein and a peptide that is inserted into the framework protein;(b) (i) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1 and the peptide is inserted into the framework protein at a position between residues 587 and 588, numbered relative to SEQ ID NO: 1,(ii) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2 and the peptide is inserted into the framework protein at a position between residues 450 and 451, numbered relative to SEQ ID NO:2; or(iii) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3 and the peptide is inserted into the framework protein at a position between residues 385 and 386, numbered relative to SEQ IDN0:3; and(c) the peptide comprises (i) a core sequence comprising a sequence that has 0, 1, 2, or 3 amino acid substitutions as compared to SEQ ID NO:4, SEQ ID NO: 17, SEQ ID NO:80, SEQ ID NO:97, SEQ ID NO: 110, SEQ ID NO: 131, or SEQ ID NO: 132, (ii) optionally a first linker sequence located at the N-terminus of the core sequence, and (iii) optionally a second linker sequence located at the C-terminus of the core sequence.

2. The modified AAV VP of claim 1, wherein:(a) the modified AAV VP comprises a framework protein and a peptide that is inserted into the framework protein;(b) (i) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1 and the peptide is inserted into the framework protein at a position between residues 587 and 588, numbered relative to SEQ ID NO: 1,180489702.1(ii) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2 and the peptide is inserted into the framework protein at a position between residues 450 and 451, numbered relative to SEQ ID NO:2; or(iii) the framework protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3 and the peptide is inserted into the framework protein at a position between residues 385 and 386, numbered relative to SEQ IDN0:3; and(c) the peptide comprises (i) a core sequence comprising a sequence that has 0, 1, 2, or 3 amino acid substitutions as compared to SEQ ID NO:4, SEQ ID NO: 17, SEQ ID NO:80, SEQ ID NO:97, SEQ ID NO: 110, SEQ ID NO: 131, or SEQ ID NO: 132, (ii) a first linker sequence located at the N-terminus of the core sequence, and (iii) a second linker sequence located at the C-terminus of the core sequence.

3. The modified AAV VP of claim 1 or 2, wherein the peptide comprises a core sequence comprising SEQ IDNO:4, SEQ ID NO: 17, SEQ IDNO:80, SEQ IDNO:97, SEQ ID NO: 110, SEQ ID NO: 131, or SEQ ID NO: 132.

4. The modified AAV VP of any one of claims 1-3, wherein the first and / or the second linker predominantly consists of alanines, glycines, or serines.

5. The modified AAV VP of claim 4, wherein the first and / or the second linker predominantly consists of alanines.

6. The modified AAV VP of claim 5, wherein the first linker comprises the sequence AAA and the second linker comprises the sequence AA.

7. The modified AAV VP of any one of claims 1-3, wherein the peptide comprises a sequence that has 0, 1, 2, 3, 4, or 5 amino acid substitutions as compared to any one of SEQ ID NOs:4-29, SEQ ID NOs:80-92, or SEQ ID NOs:97-122.92180489702.

18. The modified AAV VP of claim 7, wherein the peptide comprises any one of SEQ ID NOs:4-29, SEQ ID NOs:80-92, or SEQ ID NOs:97-122.

9. The modified AAV VP of claim 8, wherein the peptide comprises SEQ ID NO: 12, SEQ IDNO:25, or SEQ IDNO:88, SEQ ID NO: 105, or SEQ ID NO: 118.

10. The modified AAV VP of any one of the preceding claims, wherein:(a) the framework protein comprises SEQ ID NO:1 and the peptide is inserted into the framework protein at a position between residues 587 and 588, numbered relative to SEQ ID NO:1;(b) the framework protein comprises SEQ ID NO:2 and the peptide is inserted into the framework protein at a position between residues 450 and 451, numbered relative to SEQ ID NO:2; or(c) the framework protein comprises SEQ ID NO:3 and the peptide is inserted into the framework protein at a position between residues 385 and 386, numbered relative to SEQ ID NO:3.

11. The modified AAV VP of claim 1, wherein the AAV VP comprises a sequence that is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:30-32.

12. The modified AAV VP of claim 11, wherein the modified AAV VP comprises any one of SEQ ID NOs:30-32.

13. The modified AAV VP of claim 1, wherein the AAV VP comprises a sequence that is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:33-35.

14. The modified AAV VP of claim 13, wherein the AAV VP comprises any one of SEQ IDNOs:33-35.93180489702.

115. The modified AAV VP of claim 1, wherein the AAV VP comprises a sequence that is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:93-95.

16. The modified AAV VP of claim 15, wherein the modified AAV VP comprises any one of SEQ ID NOs: 93 -95.

17. The modified AAV VP of claim 1, wherein the AAV VP comprises a sequence that is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 123-125.

18. The modified AAV VP of claim 17, wherein the modified AAV VP comprises any one of SEQ ID NOs: 123-125.

19. The modified AAV VP of claim 1, wherein the AAV VP comprises a sequence that is at least 80 %, at least 85 %, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 126- 128.

20. The modified AAV VP of claim 19, wherein the modified AAV VP comprises any one of SEQ ID NOs: 126-128.

21. An AAV particle comprising one or more of the modified AAV VP of any of the preceding claims.

22. The AAV particle of claim 21, wherein the viral particle comprises an AAV genome.

23. The AAV particle of claim 22, wherein the AAV genome comprises:(a) one or more inverted terminal repeats (ITRs);(b) a promoter; and(c) a transgene encoding a gene product, wherein the transgene is operatively linked to the promoter.94180489702.

124. The AAV particle of claim 23, wherein the one or more ITRs are derived from AAV serotype AAV2.

25. The AAV particle of claim 23 or 24, wherein the promoter is selected from the group consisting of metabotropic glutamate receptor 6 (GRM6) promoter, cytomegalovirus (CMV) promoter, cytomegalovirus early enhancer / chicken b-actin (CAG) promoter, elongation factor 1 -alpha 1 (EFla) promoter, SV40 promoter, CBA (chicken beta-actin (CBA)) promoter, rhodopsin (rho) promoter, neural retina-specific leucine zipper protein (NRL) promoter, phosphodiesterase 6B (PDE6B) promoter, human rhodopsin kinase (hRK) promoter, human L-opsin promoter or a promoter derived therefrom, human M-opsin promoter or a promoter derived therefrom, human S-opsin promoter or a promoter derived therefrom, retinoid isomerohydrolase (retinal pigment epithelium-specific 65 kDa protein, RPE65) promoter, bestrophin-1 (BEST1) promoter, membrane protein MLC1 (Mlcl) promoter, interphotoreceptor retinoid-binding protein (IRBP) promoter, and neurofilament heavy (NEFH) promoter.

26. The AAV particle of claim 23 or 24, wherein the promoter is selected from the group consisting of CAG promoter, CMV promoter, RK promoter, L-opsin derived promoter, and M-opsin derived promoter.

27. The AAV particle of any one of claims 23-26, wherein the transgene encodes a polypeptide or an RNA.

28. The AAV particle of claim 27, wherein the RNA is selected from the group consisting of inhibitory antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA), a Piwi-interacting RNAs (piRNAs), a short-hairpin RNA (shRNA), small noncoding RNA (sncRNA), and a long non-coding RNA (IncRNA).

29. The AAV particle of claim 27, wherein the polypeptide is selected from the group consisting of channel rhodopsin, halorhodopsin (halo), melanopsin (Opn4), rhodopsin (RHO), blue opsin, red opsin, halorhodopsin (NpHR), enhanced halorhodopsin (eNpHR), archaerhodopsin-3 (Arch), ArchT, leptosphaeria maculans (Mac), retinoid isomerohydrolase (retinal pigment epithelium-specific 65 kDa protein, RPE65), cyclic nucleotide-gated channel 95180489702.1alpha-3 (CNGA3), cyclic nucleotide-gated channel beta-3 (CNGB3), cone cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha (PDE6C), Jaws (cruxhalorhodopsin), iClC2, retinal cone rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma (PDE6H), G Protein Subunit Alpha Transducin 1 (GNAT1), GNAT2, KCNV2, voltagedependent calcium channel subunit alpha-2 / delta-4 (CACNA2D4), R9AP (Rgs9-anchor protein or RGS9BP), iClC2, apolipoprotein E (APOE), HtrA serine peptidase 1 (HTRA1), complement 3 (C3), C3 modulator, C5, C5 modulator, rab escort protein-1 (REP1), retinal-specific phospholipid-transporting ATPase (ABCA4), cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6), retinoschisin 1 (RSI), nicotinamide adenine dinucleotide dehydrogenase subunit 4 (ND4), tyrosine-protein kinase Mer (MERTK), rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit beta (PDE6B), retinaldehyde-binding protein (RLBP1), retinal membrane guanylyl cyclase 1 (RetGCl / GUCY2D), inosine-5'-monophosphate dehydrogenase 1 (IMPDH1), peripherin-2 (Prph2 / RDS), complement factor H (CFH) modulator, complement factor I (CFI) modulator, and Retinol Dehydrogenase 12 (RDH12).

30. The AAV particle of claim 29, wherein the polypeptide is selected from the group consisting of C3 modulator, C5 modulator, CFH modulator, CFI modulator, ABCA4, channel rhodopsin, halo, and Opn4.

31. The AAV particle of claim 29 or 30, wherein the channel rhodopsin is selected from the group consisting of channel rhodopsin-1 (ChRl), channel rhodopsin-2 (ChR2), ChETA, (C123T / E134R), ChR2 C128A, ChR2 C128S, ChR2 C128T, and ChRl-ChR2 hybrids / chimera.

32. A method of increasing expression of a transgene in a retinal cell as compared to expression of the transgene in a non-retinal cell, the method comprising contacting the retinal cell with the AAV particle of any one of claims 23-31.

33. A method of delivering a transgene to a retinal cell, the method comprising contacting the retinal cell with the AAV particle of any one of claims 23-31.

34. The method of claim 32 or 33, wherein the retinal cell is selected from the group consisting of rod cell, cone cell, bipolar cell, Mueller glia cell, horizontal cell, retinal astrocyte, retinal ganglion cell, retinal pigment epithelium (RPE) cell, or amacrine cell.96180489702.

135. A pharmaceutical composition comprising (a) the AAV particle of any one of claims 23-31 and (b) a pharmaceutical acceptable excipient.

36. A method of treating a retinal disease in a subject in needed thereof, the method comprising administering to the subject the AAV particle of any one of claims 23-31 or the pharmaceutical composition of claim 35.

37. The method of claim 36, wherein the retinal disease is a cone dysfunction or a rod dysfunction.

38. The method of claim 36, wherein the retinal disease is selected from the group consisting of age related macular degeneration (AMD), Vitelliform macular dystrophy, or North Carolina macular dystrophy, Leber’s congenital amaurosis (LCA), choroideremia (CHM), achromatopsia (ACHM), usher syndrome (USH), X-linked retinoschisis, Stargardt disease, Retinitis pigmentosa (RP), diabetic macular edema, diabetic retinopathy, Leber hereditary optic neuropathy (LHON), Congenital stational night blindness, cone-dystrophy with supernormal rod response (CDSSR), glaucoma, acquired renal cystic disease (arCD), Best Disease (vitelliform macular dystrophy (BVMD)), ocular angiogenesis, and autosomal dominant cone-rod dystrophy (adCRD).

39. The method of claim 38, wherein the retinal disease is selected from the group consisting of AMD, Stargardt disease, glaucoma, and RP.

40. The method of claim 38 or 39, wherein the AMD is selected from the group consisting of dry AMD, geographic atrophy (GA), and wet AMD.

41. The method of claim 38 or 39, wherein the RP is selected from the group consisting of X-linked RP (XLRP), MERTK-associated RP, PDE6B-associated RP, RLBP1 -associated RP, and non-syndromic RP.

42. The method of any one of claims 36-41, wherein the subject is a human.97180489702.

143. The method of any one of claims 36-42, wherein the AAV particle or the pharmaceutical composition is administered via direct retinal injection, subretinal injection, intravitreal injection, suprachoroidal injection, topical instillation, or intravenous injection.98180489702.1

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