Bardet-biedl syndrome 10 (BBS10) protein gene therapy

Abstract

Provided herein are improved expression constructs for the expression of Bardet-Biedl syndrome 10 protein (BBS10 protein). Also provided are vectors and pharmaceutical compositions comprising such constructs and / or. Further provided are methods of treating disease, including, but not limited to, Bardet-Biedl syndrome (BBS).

Description

BARDET-BIEDL SYNDROME 10 (BBS10) PROTEIN GENE THERAPYREFERENCE TO A SEQUENCE LISTING

[0001] 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 April 30, 2025, is named SeqList-162027-54776.xml and is 27,464 bytes in size.FIELD

[0002] The present disclosure relates generally to the field of molecular biology and medicine. More particularly, the methods and compositions herein are useful for treating disease, such as Bardet-Biedl syndrome.BACKGROUND

[0003] Bardet-Biedl syndrome (BBS) is a group of inherited, autosomal recessive ciliopathies. In these disorders, mutations in genes important for maintaining the function of cilia cause cellular dysfunction in multiple cell types, leading to obesity, polydactyly, renal failure, and blindness.

[0004] Bardet-Biedl syndrome 10 (BBS 10), which is also referred to as Bardet-Biedl syndrome, BBSlO-related, is a serious disease that causes profound visual impairment from early years. The disease causes loss of vision with a rapid progression with no plateau or remission. Because symptoms are often observed shortly after birth and vision loss progresses rapidly, the treatment window of opportunity is during the patients’ childhood. Given the importance of vision in driving development, profoundly visually impaired children, such as those with BBS 10 ciliopathy, often have major delays in multiple areas of development (e.g., behavioral, social, and emotional development, as well as development of communication skills).

[0005] There are currently no treatments for BBS10 approved by the U.S. Food and Drug Administration. Presently, only supportive care is routinely offered. Where there is some residual sight, this is confined to correction of refractive error, use of low vision aids, and access to educational and work-related opportunities. This can lessen the debilitating consequences of vision loss somewhat but does not treat the condition itself. Further, none ofthe interventions currently available are able to prevent the continued deterioration in the subject’s sight which leads to eventual blindness.

[0006] Accordingly, improved treatment options for BBS10 are urgently needed.SUMMARY

[0007] The disclosure herein relates to expression constructs for the expression of a BBS 10 gene and compositions and methods for treating BBS10.

[0008] Provided is an expression construct comprising:(a) a rhodopsin kinase promoter;(b) a nucleic acid sequence encoding a Bardet-Biedl syndrome 10 protein (BBS 10 protein), wherein the nucleic acid sequence is operatively linked to the promoter; and(c) a polyadenylation signal.

[0009] In some embodiments, the rhodopsin kinase promoter 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. In one embodiment, the rhodopsin kinase promoter comprises SEQ ID NO:3.

[0010] In some embodiments, the sequence encoding the BBS10 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:4. In one embodiment, the sequence encoding the BBS10 protein comprises SEQ ID NO:4. In some embodiments, the BBS10 protein 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:9. In one embodiment, the BBS10 protein comprises SEQ ID NO:9.

[0011] In some embodiments, the polyadenylation signal comprises a sequence that is 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: 5. In one embodiment, the polyadenylation signal comprises SEQ ID NO: 5.

[0012] In one embodiment, the expression construct further comprises a post-transcriptional regulatory element. In some embodiments, the post-transcriptional regulatory element 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 a nucleic acid sequence selected from SEQ ID NOs: 10-13. In some embodiments,the post-transcriptional regulatory element comprises a nucleic acid sequence selected from SEQ IDNOs: 10-13.

[0013] Provided is a vector comprising an expression construct disclosed herein. In one embodiment, the vector is a viral vector. In one embodiment, the vector is an adeno-associated viral (AAV) vector. In one embodiment, the vector comprises a nucleic acid sequence comprising (i) an expression construct disclosed herein and (ii) one or more inverted terminal repeats (ITR). In one embodiment, the nucleic acid sequence comprises a 5' ITR and a 3' ITR. In one embodiment, the 5' ITR and the 3' ITR are derived from AAV serotype AAV2. In some embodiment, the 5' ITR comprises a sequence that is 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 (b) the 3' ITR 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:6. In one embodiment, (a) the 5' ITR comprises SEQ ID NO: 1 and (b) the 3' ITR comprises SEQ IDN0:6.

[0014] In some embodiments, the vector comprises a nucleic acid sequence that is 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:8. In one embodiment, the vector comprises a nucleic acid sequence comprising SEQ ID NO: 8.

[0015] Provided is a cell comprising an expression construct disclosed herein or a vector disclosed herein.

[0016] Provided is a pharmaceutical composition comprising (i) an expression construct disclosed herein or a vector disclosed herein and (ii) a pharmaceutically acceptable carrier.

[0017] Provided is a method of treating Bardet-Biedl syndrome 10 (BBS10) in a subject in need thereof, the method comprising administering to the subject a vector disclosed herein or a pharmaceutical composition disclosed herein. Provided is a method of reducing vision loss in a subject in need thereof, the method comprising administering to the subject a vector disclosed herein or a pharmaceutical composition disclosed herein, wherein the subject has a BBSlO-deficiency. Provided is a method of restoring photoreceptor function in a subject in need thereof, the method comprising administering to the subject a vector disclosed herein or a pharmaceutical composition disclosed herein, wherein the subject has a BBSlO-deficiency. Provided is a method of restoring vision in a subject in need thereof, the method comprising administering to the subject a vector disclosed herein or a pharmaceutical composition disclosed herein, wherein the subject has a BBSlO-deficiency. In one embodiment, the subjectis a human. In some embodiments, the vector or the pharmaceutical composition is administered via retinal injection, subretinal injection, intravitreal injection, or suprachoroidal injection. In one embodiment, the vector or the pharmaceutical composition is administered via subretinal injection.BRIEF DESCRIPTION OF THE FIGURES

[0018] FIG. 1. Schematic of electroretinography (ERG), which serves as a functional assessment of retinal function. Illustrative ERG traces are shown for rod- and cone-driven response in WT mice and a mutant (e.g., BBS 10 knockout) mouse.

[0019] FIG. 2. Schematic of illustrative route of administration (ROA) and target cells.Abbreviations: AAV = adeno-associated virus; GCL = ganglion cell layer; ILM = internal limiting membrane; INL = inner nuclear layer; ONL = outer nuclear layer; RPE = retinal pigment epithelium.

[0020] FIG. 3. BBS10 knockout (KO) mouse model characterization. ERG responses of BBS10 mutant mice compared to WT and HET littermates showed diminished retinal sensitivity oiBBSlO KO mice over time. Abbreviations: WT = wild-type, HET = heterozygote, wk = week, N = number of eyes.

[0021] FIG. 4. BBS10 KO mouse model characterization. Analysis of whole retina thickness using optical coherence tomography (OCT) showed progressive thinning of the retina in BBS 10 mutant (ie., knockout) mice. A 2-way ANOVA was performed to determine significance between wild type and PBS-treated mutant mice. Bars for different treatment conditions (from left to right) are as shown in the figure legend (from top to bottom).

[0022] FIG. 5. Experimental setup for Examples 3-9. ERG = electroretinography; OCT = optical coherence tomography; KO = knockout, wk pi = week post injection.

[0023] FIG. 6. AAV vectors expressing human BBS10 and using an AAV5 capsid do not provide long-term rescue of retinal function in BBS10 O mice. Photopic, scotopic and flicker ERG amplitudes at 6-, 12- and 24 ± 1 weeks after vector administration in mutant mice. Treated eyes received high dose (2 x 1010VG / eye) of either AAV5-hRK-hBBS10 or AAV5-CAG-hBBSlO. Number of eyes for each group at each time point displayed in the Figure. Statistical analysis comparing vector-treated with PBS treated control group was assessed using 2-way ANOVA with Dunnett’s posthoc test. Abbreviations: WT = wild-type, HET = heterozygote, KO = knockout, wk = week, N = number of eyes. R&D grade vectors were used. hRK = human rhodopsin kinase promoter. CAG = CAG promoter.

[0024] FIG. 7. AAV8-RK-hBBS10 provides long-term rescue of retinal function in BBS10 KO mice. Photopic, scotopic and flicker ERG amplitudes at 6-, 12- and 24 ± 1 weeks after vector administration in mutant mice. Treated eyes received high dose (2 x IO10VG / eye = 5 x 1012VG / ml) of either AAV8-hRK-hBBS10 or AAV8-CAG-hBBS10. Number of eyes for each group at each time point displayed on the Figure. Statistical analysis against PBS treated control group assessed using 2-way ANOVA with Dunnett’s p.h. test. Abbreviations: KO = knockout; PBS = phosphate buffered saline; wk = week; N = number of eyes. AAV8-hRK-hBBSlO: top trace (by end point) for photopic B-wave, scotopic B-wave, and flicker; bottom trace (by endpoint) for scotopic A-wave. R&D grade vectors were used.

[0025] FIG. 8. Mid-dose of AAV8-RK-hBBS10 provides minimal rescue of retinal function in BBS10 Q mice. Photopic, scotopic and flicker ERG amplitudes at 6-, 12- and 24 ± 1 weeks after vector administration in mutant mice. Treated eyes received mid dose (4 x 109VG / eye = 1 x 1012VG / ml) of either AAV8-CAG-hBBS10 or AAV8-hRK-hBBS10 (R&D grade). Number of eyes for each group at each time point displayed on the Figure. Statistical analysis against PBS treated control group assessed using 2-way ANOVA with Dunnett’s p.h. test. Abbreviations: KO = knockout; PBS = phosphate buffered saline; wk = week; N = number of eyes.

[0026] FIG. 9. AAV8-RK-mBbslO (expressing murine BBS10) provides long-term rescue of retinal sensitivity in BBS10 KO mice in a dose-dependent manner. Photopic, scotopic and flicker ERG amplitudes at 6-, 12- and 24 ± 1 weeks after vector administration in mutant mice. Treated eyes received either mid dose (4 x 109VG / eye = 1 x 1012VG / ml) or high dose (2 x 1010VG / eye = 5 x 1012VG / ml) of AAV8-RK-mBBS10. Number of eyes for each group at each time point displayed on the Figure. Statistical analysis against PBS treated control group assessed using 2-way ANOVA with Dunnett’s p.h. test. Abbreviations: KO = knockout; N = number; pi = post injection; PBS = phosphate buffered saline; wk = week. Data for six-week timepoint: Photopic B-wave, scotopic B-wave, flicker - traces from top to bottom: AAV8-RK-mBBS10 high dose, AAV8-RK-mBBS10 mid dose, PBS. Scotopic A-wave -traces from bottom to top: AAV8-RK-mBBS10 high dose, AAV8-RK-mBBS10 mid dose, PBS.

[0027] FIG. 10. Structural preservation of the retina following treatment with AAV8-RK-hBBSlO and AAV8-RK-mBBS10. Whole retina thickness measurements focused on the quantification of three horizontal scans per eye including one central scan through optic nerve head (area outside of injection site) and two scans superior of optic nerve head (mid-superior retina and peri-superior retina; treated area). Statistical analysis against PBS treated controlgroup assessed using 2-way ANOVA with Dunnett’s p.h. test. Bars for different treatment conditions (from left to right) are as shown in the figure legend (from top to bottom).

[0028] FIG. 11. Immunohistochemistry demonstrates the partial correction of STX3 mis-localization following AAV8-RK-hBBS10 and AAV8-RK-mBBS10 treatment. In WT mice syntaxin 3 (STX3) is expressed in the inner photoreceptor segments (IS). In BBS10 KO mice, STX3 mis-localizes into the outer photoreceptor segments (OS), where it overlays with peripherin 2 (PRPH2). Following treatment STX3 mis-localization is partly corrected (retention of some STX3 in IS). Arrowheads point to the IS retina layer where STX3 should normally be expressed. WT, untreated BBS10 KO and treated BBS10 KO eyes were used to perform antibody staining for PRPH2 (mainly found in OS) and STX3 (mainly found in IS). DAPI staining was used to visualize cell nuclei (see particularly in ONL). White arrowheads indicate the IS area, which highlights the mis-localization of STX3 in untreated KO eyes (lack of fluorescence intensity in IS) and the partial correction of STX3 localization in treated BBS 10 KO eyes (restored fluorescence intensity in IS). Abbreviations: WT = wild-type; KO = knockout; INL = inner nuclear layer; ONL = outer nuclear layer; IS = inner segment; OS = outer segment.

[0029] FIG. 12. No detrimental effect in retinal function in a safety study in wild-type mice following 6 months of administration with AAV8-RK-hBBS10. Abbreviations: pi / p.i. = post injection; VG = viral genomes; w = week. Left bars: PBS. Right bars: AAV8-RK-hBBSlO.

[0030] FIG. 13A, FIG. 13B, FIG. 13C, and FIG. 13D. Histological assessment of mouse eyes after treatment with a mid dose of AAV8-RK-hBBS10 confirms a good safety profile. FIG. 13A. Loss of structure events. FIG. 13B. Rosette events. FIG. 13C. Bulge events. FIG.13D. All events (FIG. 13A, FIG. 13B, and FIG. 13C combined).

[0031] FIG. 14. AAVAnc80-CMV-mBBS10 (expressing murine BBS10), but not AAVAnc80-CMV-hBBS10 (expressing human BBS10), provides long-term rescue of retinal sensitivity following treatment of BBS10 KO mice. Photopic, scotopic and flicker ERG amplitudes at 6-, 12- and 24 ± 1 weeks after vector administration in mutant mice. Treated eyes received high dose (2 x 1010VG / eye) of AAVAnc80-CMV-mBBS10 or AAVAnc80-CMV-hBBSlO either by single or double subretinal injection. Number of eyes for each group at each time point displayed on the Figure. Statistical analysis against PBS treated control group assessed using 2-way ANOVA with Dunnett’s p.h test. Traces for 6 week data points (as determined by endpoint, from top to bottom): Photopic A- wave - PBS KO, Anc80-mouse, Anc80-human, uninjected KO; photopic B-wave - Anc80-mouse, Anc80-human, uninjectedKO, PBS KO; scotopic A- wave - PBS KO, Anc80-human, uninjected KO, Anc80-mouse; scotopic B-wave - Anc80-mouse, uninjected KO, Anc80-human, PBS KO; flicker - Anc80-mouse, uninjected KO, Anc80-human, PBS KO.

[0032] FIG. 15. Manufacturing Science and Technology (MSAT)-grade AAV8-RK-hBbslO provides long-term rescue of retinal sensitivity following treatment of BBS10 O mice. Photopic and scotopic ERG amplitudes at 6-, 12- and 24 ± 1 weeks after vector administration in mutant mice. Treated eyes received either mid (4 x 109VG / eye = 1 x 1012VG / ml) or high dose (2 x IO10VG / eye = 5 x 1012VG / ml) of AAV8-RK-hBBS10. Number of eyes for each group at each time point displayed on the Figure. Statistical analysis against PBS treated control group assessed using 2-way ANOVA with Dunnett’s p.h. test. Abbreviations: KO = knockout; N = number; pi = post injection; PBS = phosphate buffered saline; wk = week. Traces from top to bottom: AAV8-RK-hBBS10 5E12, AAV8-RK-hBBS10 1E12, PBS.

[0033] FIG. 16A and FIG. 16B. Treatment with MSAT-grade AAV8-RK-hBBS10 vector increases functional vision in BBS10 KO mice. FIG. 16A. Experimental setup for OMR experiments. FIG. 16B. HET represents the equivalent of wild-type mice (positive control). KO PBS and KO uninjected are untreated BBS10 KO mice (negative controls). Traces from top to bottom for spatial frequency of 0.25 eye / 0: HET, AAV8-RK-hBBS105E12, AAV8-RK-hBBSlO 1E12, KO PBS, KO uninjected.DETAILED DESCRIPTION

[0034] Provided herein are nucleic acids, expression constructs, vectors, and other compositions as well as methods for the expression of the BBS10 gene and for treating disease.

[0035] BBS, the BBSome and BBS10 protein

[0036] BBS is an inherited ciliopathy. In this disorder, mutations in genes important for maintaining the function of cilia cause cellular dysfunction in multiple cell types, leading to obesity, polydactyly, renal failure, and blindness. Many of these mutations disable the function of a protein complex called the BBSome. This protein complex regulates the movement of cargo proteins in and out of cilia. For example, the BBSome acts as a cargo adaptor to the intraflagellar transport (IFT) complex, expands the cargo range of IFT in ciliary trafficking, and regulates the movement of cargo proteins in and out of cilia. BBS proteins are not acting individually, but are dependent on their associations with other BBSome counterparts. BBS 10, BBS6 and BBS 12 are chaperon-like BBS proteins that interact to form the BBS chaperon complex (BBScc) and facilitate the assembly of the BBSome.

[0037] More than 20 causative genes are known with many mutations disabling the function of the BBSome. Mutations in the BBS10 gene are the second most common cause of BBS and account for more than 20% of all BBS cases. Although not a part of the BBSome complex, BBS10 is indispensable for the assembly of the BBSome complex. Therefore, intact ciliary transport requires a functional BBS 10 gene. BBS10 belongs to the group II chaperonins and possesses a conserved ATP -binding site for adenosine 5'-triphosphate (ATP) hydrolysis, suggesting that it may be a catalytically active enzyme. BBS10 is also known as Bardet-Biedl syndrome 10 protein or BBSome complex assembly protein BBS10.

[0038] Total BBS prevalence varies from 1:160,000 in northern European populations and 1:13,500 in some Arab populations. As such, BBS10 prevalence can range between 1:67,500 and 1: 800,000.

[0039] In the retina, the primary cilium of the photoreceptor have evolved into a structure specializing in phototransduction called the outer segment (OS), which occupies up to 40% of the photoreceptor cell volume. These enlarged cilia of photoreceptors require more active protein trafficking for their maintenance compared with the primary cilia of other cell types, and they in turn render the photoreceptors particularly vulnerable to mutations that disrupt ciliary transport or function. The disruption of the BBSome function leads to the inundation of photoreceptor OS by more than 100 different proteins normally found in other parts of the cell, including syntaxin-3 (STX3). Cellular stress caused by protein mis-localization contributes to photoreceptor cell death in BBS. In addition, disrupting the BBSome causes malformation of the photoreceptor OS prior to degeneration, increased cellular stress, and eventually photoreceptor cell death, leading to blindness.

[0040] Nucleic acids and expression constructs

[0041] Provided herein are nucleic acids encoding BBS10 and nucleic acids comprising expression constructs for the expression ofBBSlO.

[0042] 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. The nucleic acids disclosed herein may be recombinantly produced chimeric nucleic acid molecules comprising any of those polynucleotides either alone or in combination.

[0043] As used herein, an “expression construct” is a nucleic acid that allows for the expression of a transgene (such as BBS 10). Expression of a transgene may be measured in waysknown 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.

[0044] Provided herein is an expression construct comprising a nucleic acid sequence encoding BBS 10, wherein the nucleic acid sequence is operatively linked to one or more expression control sequences.

[0045] As used herein, “operably 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, a promoter is operably linked to a transcribable polynucleotide molecule if the promoter modulates transcription of the transcribable polynucleotide molecule of interest in a cell. Additionally, two portions of a transcription regulatory element are operably 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 transcription regulatory elements may be operably linked to one another by way of a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operably linked to one another with no intervening nucleotides present.

[0046] Expression control sequences (or transcriptional control elements) 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 (z.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein processing and / or secretion. A transcriptional control element may be functional in a eukaryotic cell, e.g., a mammalian cell; or a prokaryotic cell (e.g., bacterial or archaeal cell). In some embodiments, a nucleotide sequence encoding a polypeptide disclosed herein is operably linked to multiple control elements that allow expression of the nucleotide sequence encoding the polypeptide disclosed herein in both prokaryotic and eukaryotic cells. A great number of expression control sequences, e.g, native, constitutive, inducible and / or tissuespecific, 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 sequencewhich may include splice donor and acceptor sites. The polyadenylation sequence generally is inserted following the transgene.

[0047] In some embodiments, the specific genetic elements are linked to one another by way of a linker nucleic acid (e.g., an intervening non-coding nucleic acid). In some embodiments, the specific genetic elements are linked to one another with no intervening nucleotides present. In some embodiments, some of the specific genetic elements are linked to one another by way of a linker nucleic acid, while others are linked to one another with no intervening nucleotides present.

[0048] In one embodiment, the expression cassette comprises a promoter. As used herein, the term “promoter” usually 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 transcription initiation site of the coding sequence. A promoter may be 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.

[0049] In one embodiment, the expression cassette comprises an enhancer. “Enhancers” are usually defined as DNA regions that amplify transcription initiation by directly interplaying with their target promoters. Enhancer sequences, distal from their target promoters, can contain DNA motifs that act as binding sites for TFs and cofactors.

[0050] At times, the term “promoter” may be used as a shorthand to refer to a nucleic acid sequence that comprises multiple regulatory elements. The control promoter CAG, for example, contains a CMV enhancer, P actin promoter region and intron, but may be referred to as a promoter. Specifically, the CAG promoter consists of (1) the cytomegalovirus (CMV) early enhancer element, (2) the promoter, the first exon and the first intron of chicken betaactin gene, and (3) the splice acceptor of the rabbit beta-globin gene.

[0051] In one aspect, provided is an expression construct comprising:(a) a rhodopsin kinase (RK) promoter;(b) a nucleic acid sequence encoding BBS 10, wherein the nucleic acid sequence is operatively linked to the promoter; and(c) a polyadenylation signal.

[0052] In one aspect, provided is an expression construct comprising from 5’ to 3’:(a) a rhodopsin kinase promoter;(b) a nucleic acid sequence encoding BBS 10, wherein the nucleic acid sequence is operatively linked to the promoter; and(c) a polyadenylation signal.

[0053] As used herein, the term “from 5' to 3'” refers to the order of the specific genetic elements in a nucleic sequence.

[0054] In one embodiment, the RK promoter is a human RK promoter. In some embodiments, the RK promoter 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. In one embodiment, the RK promoter comprises SEQ ID NO:3.

[0055] In one embodiment, the expression construct comprises a nucleic acid sequence encoding BBS 10, wherein the BBSlO-encoding sequence is codon-optimized.

[0056] In one embodiment, the expression construct comprises a nucleic acid sequence encoding human BBS10 or a derivative thereof. In some embodiments, the BBS10 derivative comprises one or more conservative mutations as compared to its parental (e.g., wild-type) counterpart. In some embodiments, the BBS10 derivative comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative mutations as compared to its parental counterpart.

[0057] As used herein, the terms “conservative amino acid substitutions”, “conservative mutations” and “conservative modifications” refer to amino acid substitutions or modifications (or changes in coding sequence that results in a conservative substitution of and amino acid) 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.

[0058] 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, phenylalanine, 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.

[0059] In some embodiments, the expression construct comprises a nucleic acid sequence encoding BBS 10, wherein the BBSlO-encoding sequence 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:4. In one embodiment, the BBSlO-encoding sequence comprises SEQ ID NO:4. In some embodiments, the expression construct comprises a nucleic acid sequence encoding BBS 10, wherein the BBS10 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: 9. In one embodiment, the expression construct comprises a nucleic acid sequence encoding BBS10, wherein the BBS10 comprises SEQ ID NO:9. The sequence encoding BBS 10 may be in frame with one or more stop codons located at the 3’ end of the BBSlO-encoding sequence.

[0060] In some embodiments, the expression construct comprises a poly adenylation signal comprising 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: 5. In one embodiment, the expression construct comprises a polyadenylation signal comprising SEQ ID NO: 5.

[0061] In one embodiment, the expression construct comprises:(a) an RK promoter comprising 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;(b) a nucleic acid sequence encoding BBS 10, wherein the nucleic acid sequence is operatively linked to the promoter, comprising 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 NON; and / or(c) a polyadenylation signal comprising a sequence that is 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: 5.

[0062] In one embodiment, the expression construct comprises:(a) an RK promoter comprising SEQ ID NON;(b) a nucleic acid sequence encoding BBS 10, wherein the nucleic acid sequence is operatively linked to the promoter, comprising SEQ ID NO:4; and / or(c) a polyadenylation signal comprising SEQ ID NO: 5.

[0063] In one embodiment, the expression construct comprises:(a) an RK promoter comprising 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;(b) a nucleic acid sequence encoding BBS 10, wherein the nucleic acid sequence is operatively linked to the promoter, wherein the BBS 10 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: 9; and / or(c) a polyadenylation signal comprising a sequence that is 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: 5.

[0064] In one embodiment, the expression construct comprises:(a) an RK promoter comprising SEQ ID NO: 3;(b) a nucleic acid sequence encoding BBS 10, wherein the nucleic acid sequence is operatively linked to the promoter, wherein the BBS 10 comprises SEQ ID NO:9; and / or (c) a polyadenylation signal comprising SEQ ID NO: 5.

[0065] In some embodiments, the expression construct comprises a Kozak sequence for initiating protein translation. In some embodiments, the expression construct comprises a Translation Initiator of Short 5' UTR (TISU) sequence for initiating protein translation. See, e.g., Elfakess et al., Unique translation initiation of mRNAs-containing TISU element, Nucleic Acids Res. 2011 Sep l;39(17):7598-609.

[0066] In some embodiments, the expression construct comprises a post-transcriptional regulatory element. In some embodiments, the expression construct comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0067] In some embodiments, the post-transcriptional regulatory element 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 a nucleic acid sequence selected from SEQ ID NOs: 10-13. In some embodiments, the post-transcriptional regulatory element comprises a nucleic acid sequence selected from SEQ ID NOs: 10-13.

[0068] In some embodiments, the expression construct comprises one or more of the genetic elements provided in Table 2 (or variants thereof) or encodes for a sequence shown in Table 3 (or variants thereof).

[0069] Vectors

[0070] In one aspect, provided are recombinant vectors and their use for the introduction of a transgene or an expression construct into a cell. In some embodiments, the recombinant vectors comprise recombinant DNA constructs that include additional DNA elements, including DNA segments that provide for the replication of the DNA in a host cell and expression of the target gene in target cells at appropriate levels. The ordinarily skilled artisan appreciates that expression control sequences (promoters, enhancers, and the like) are selected based on their ability to promote expression of the target gene in the target cell.

[0071] “Vector,” as used herein, means a vehicle that comprises a polynucleotide to be delivered into a host cell, either in vitro or in vivo. Non-limiting examples of vectors include a recombinant plasmid, yeast artificial chromosome (YAC), mini chromosome, DNA minicircle, or a virus (including virus-derived sequences). 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 consist of chromosomal, non-chromosomal, semi- synthetic or synthetic nucleic acids. 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). Large numbers of suitable vectors are known to those of skill in the art and commercially available. A vector may also refer to a virion comprising a nucleic acid to be delivered into a host cell, either in vitro or in vivo. In some embodiments, a vector refers to a virion comprising a recombinant viral genome, wherein the viral genome comprises one or more ITRs and an BBS 10 transgene.

[0072] In one embodiment, the recombinant vector is a viral vector or a combination of multiple viral vectors. In one aspect, provided is a vector comprising any of the expression constructs disclosed herein. Viral vectors for the expression of a target gene in a target cell, tissue, or organism are known in the art and include, for example, an AAV vector, adenovirus vector, lentivirus vector, retrovirus vector, poxvirus vector, baculovirus vector, herpes simplex virus vector, vaccinia virus vector, or a synthetic virus vector (e.g., a chimeric virus, mosaic virus, or pseudotyped virus, and / or a virus that contains a foreign protein, synthetic polymer, nanoparticle, or small molecule).

[0073] Provided herein is a vector that is derived from a recombinant, preferably replication incompetent, adeno-associated virus (AAV) viral vector platform.

[0074] Adeno-associated viruses (AAV) are small, single-stranded DNA viruses which require 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, respectively. The Rep reading frame encodes four proteins of molecular weight 78 kD, 68 kD, 52 kD, and 40 kD. These proteins function mainly in regulating AAV replication and rescue and integration of the AAV into a host cell's chromosomes. The Cap reading frame encodes three structural proteins of molecular weight (VP1-VP3), which form the virion capsid. Capsid proteins VP1, VP2, and VP3 are 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 57aa 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. More than 80% of total proteins in AAV virion comprise VP3.

[0075] Flanking the rep and cap open reading frames at the 5' and 3' ends are about 145 bp long inverted terminal repeats (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.

[0076] Provided herein is an AAV genome comprising (a) an BBS 10 gene and (b) one or more ITRs. Provided herein is an AAV genome comprising (a) an expression construct and (b) one or more ITRs. Provided is an AAV genome comprising a nucleic acid comprising an AAV 5' ITR and 3' ITR located 5' and 3' of the BBS 10 gene, respectively. However, in certain embodiments, it may be desirable for the nucleic acid to contain the 5' ITR and 3' ITR sequences arranged in tandem, e.g., 5' to 3' or a head-to-tail, or in another alternative configuration. In still other embodiments, it may be desirable for the nucleic acid to contain multiple copies of the ITRs or to have 5' ITRs (or conversely, 3' ITRs) located both 5' and 3'of the BBS 10 gene. The ITRs sequences may be located immediately upstream and / or downstream of the BBS 10 gene, or there may be intervening sequences.

[0077] 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 wildtype 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.

[0078] The AAV genome may have one or all 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.

[0079] 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, 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.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-HBK0, 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.

[0080] In one embodiment, the ITR sequence(s) are derived from AAV2.

[0081] In some embodiments, provided is a vector comprising a viral genome comprising (i) an expression construct disclosed herein and (ii) one or more inverted terminal repeats(ITR). In one embodiment, the nucleic acid sequence comprises a 5' ITR and a 3' ITR. In one embodiment, the 5' ITR and the 3' ITR are derived from AAV serotype AAV2.

[0082] In some embodiments, the 5' ITR sequence 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. In one embodiment, the 5' ITR sequence comprises SEQ ID NO:1.

[0083] In some embodiments, the 3' ITR sequence 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: 6. In one embodiment, the 3' ITR sequence comprises SEQ ID NO:6.

[0084] In some embodiments, the vector comprises a nucleic acid 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:8. In one embodiment, the vector comprises a nucleic acid sequence comprising SEQ ID NO: 8.

[0085] 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.

[0086] Recombinant adeno-associated virus “rAAV” vectors include any vector derived from any adeno-associated virus serotype. rAAV vectors can have one or more of the AAV wild-type genes deleted in whole or in part, preferably the Rep and / or Cap genes, but retain functional flanking ITR sequences.

[0087] In some embodiments, the viral vector is an rAAV virion. 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 and (ii) at least one AAV ITR sequence, wherein the AAV genome is encapsidated by capsid proteins.

[0088] In some embodiments, the AAV vector comprises capsids 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.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, JBL4JBL5, JBB1, JBB2, JBB3, JBB4, JBB6, JBB7, JBB8, JBB9, JBB11, JBB12, JBB13, CONB23, CONB36, CONB37, CONB39, C0NS3, C0NS6, 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, 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.

[0089] In some embodiments, the viral vector is an AAV vector, such as an AAV1 (z.e., an AAV containing AAV1 ITRs and AAV1 capsid proteins), AAV2 (z.e., an AAV containing AAV2 ITRs and AAV2 capsid proteins), AAV3 (i.e., an AAV containing AAV3 ITRs and AAV3 capsid proteins), AAV4 (z.e., an AAV containing AAV4 ITRs and AAV4 capsid proteins), AAV5 (z.e., an AAV containing AAV5 ITRs and AAV5 capsid proteins), AAV6 (z.e., an AAV containing AAV6 ITRs and AAV6 capsid proteins), AAV7 (z.e., an AAV containing AAV7 ITRs and AAV7 capsid proteins), AAV8 (z.e., an AAV containing AAV8 ITRs and AAV8 capsid proteins), AAV9 (z.e., an AAV containing AAV9 ITRs and AAV9 capsid proteins), AAVrh74 (z.e., an AAV containing AAVrh74 ITRs and AAVrh74 capsid proteins), AAVrh.8 (z.e., an AAV containing AAVrh.8 ITRs and AAVrh.8 capsid proteins), or AAVrh.10 (z.e., an AAV containing AAVrh.10 ITRs and AAVrh.10 capsid proteins).

[0090] In some embodiments, the viral vector is a pseudotyped AAV vector, containing ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped AAV is AAV2 / 9 (z.e., an AAV containing AAV2 ITRs and AAV9 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 10 (z.e., an AAV containing AAV2 ITRs and AAV10 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 7m8 (z.e., an AAV containing AAV2 ITRs and AAV7m8 capsid proteins).

[0091] In some embodiments, the AAV vector contains a recombinant capsid protein, such as a capsid protein containing a chimera of one or more of capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVrh.8, or AAVrh.10 or any of the other AAV serotypes discloses herein.

[0092] In some embodiments, the AAV vector contains two or more capsid proteins selected from different serotypes. In some embodiments, the AAV vector contains an rAAV2-retro and an AAVrh.10 capsid protein. In some embodiments, the AAV vector contains rAAV2-retro and AAVrh.10 capsid proteins respectively, in a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some embodiments, the AAV vector contains AAVrh.10 and rAAV2-retro capsid proteins, respectively, in a ratioof 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

[0093] Provided herein is an AAV vector that comprises an AAV serotype 8 capsid. As with other rAAV vectors, the AAV8-BBS10 vector may have a compact macromolecular structure and may form stable viral particles approximately 25 nm in diameter.

[0094] 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 a 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. Further, methods suitable for producing a rAAV cassette in an adenoviral capsid have been described in U.S. Pat. No. 5,856,152 (entitled Hybrid adenovirus-AAV vector and methods of use therefor) and U.S. Pat. No. 5,871,982 (entitled Hybrid adenovirus- AAV virus and methods of use thereof), both of which are incorporated herein in their entireties. See also Asaad W et al., AAV genome modification for efficient AAV production, Heliyon. 2023 Apr l;9(4):el5071.

[0095] 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.

[0096] 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 fragmentsthereof as well as helper genes essential for AAV production. The AAV rep and cap sequences are obtained from an AAV source as identified herein. 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, infection, calcium phosphate precipitation, 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 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. Alternatively, infection of proviral cell lines with adenovirus or herpes simplex virus vector carrying a Rep and Cap expression cassette can be used. Further, baculovirus expression vector systems for rAAV vector production in insect SF9 cells have been developed.

[0097] 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 into a cell.

[0098] Although the molecule providing rep and cap may be transiently transfected 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).

[0099] The methods employed for constructing embodiments of this disclosure are conventional genetic engineering or recombinant engineering techniques such as those described in the references above. For example, the rAAV may be produced utilizing a tripletransfection method using either 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, 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. While this specification provides illustrative examples of specific constructs, using the information provided herein, one of skill in the art may select and design other suitable constructs, using a choice of spacers, promoters, and other elements, including at least one translational start and stop signal, and the optional addition of polyadenylation sites.

[0100] The rAAV virions can be produced by culturing a host cell containing a rAAV virus as described herein which contains a 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.

[0101] In some embodiment, the nucleic acids or vectors disclosed herein are purified. The terms “purified” or “isolated” nucleic acid or vector refer to a nucleic acid or vector that has been separated from other proteins, lipids, and / or nucleic acids with which it may be naturally associated. The nucleic acid or vector can constitute at least 10% (z.e., any percentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%, 60%, 70 %, 80%, 85%, 90%, 95%, and 99%) by dry weight of the purified preparation.

[0102] Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0103] 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.

[0104] Purification methods for rAAV virions are known in the art and include density gradient ultracentrifugation, gradient sedimentation, nonionic iodixanol gradients followed byion-exchange or heparin-affinity column chromatography, ion-exchange chromatography, mucin columns, tangential flow filtration, etc.

[0105] Pharmaceutical compositions

[0106] Provided herein are pharmaceutical compositions comprising any of the vectors disclosed herein and a pharmaceutically acceptable excipient or carrier.

[0107] In some embodiments, the rAAV comprising the gene encoding BBS 10 is assessed for contamination by conventional methods and formulated into a pharmaceutical composition suitable for storage and / or administration to a patient.

[0108] Pharmaceutically-acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. The pharmaceutical compositions may generally be formulated in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0109] The terms “pharmaceutically acceptable,” “physiologically tolerable,” as referred to compositions, carriers, diluents, and reagents, are used interchangeably and include materials are capable of administration to or upon a subject without the production of undesirable physiological effects to the degree that would prohibit administration of the composition. For example, “pharmaceutically-acceptable excipient” includes an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use.

[0110] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer’s solutions, dextrose solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the compositions disclosed herein, use of the media or compound in the compositions disclosed herein is contemplated. In some embodiments, a second therapeutic agent, such as an anti-cancer or anti-tumor agent, can also be incorporated into pharmaceutical compositions.[OHl] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate-buffered saline (PBS). The composition may be sterile and fluid to the extent that easy syringeability exists. In embodiments, the compositions disclosed hereinare stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0112] In some embodiments, the pharmaceutical composition further includes a cryoprotectant e.g., glycerol, DMSO, PEG).

[0113] In some embodiments, the pharmaceutical composition will be suitable for administration to a subject, e.g, will be sterile. For example, in some embodiments, a subject pharmaceutical composition will be suitable for administration to a human subject, e.g, where the composition is sterile and is free of detectable pyrogens and / or other toxins and / or such detectable pyrogens and / or other toxins are below permissible limits.

[0114] Where a composition disclosed herein is administered as an injectable (e.g., subcutaneously, intraperitoneally, intramuscularly, and / or intravenously) directly into a tissue, a formulation can be provided as a ready-to-use dosage form, a non-aqueous form (e.g., a reconstitutable storage-stable powder) or an aqueous form, such as liquid composed of pharmaceutically acceptable carriers and excipients. The formulations comprising an antigenbinding protein or antigen-binding fragment thereof disclosed herein may also be provided so as to enhance serum half-life of the subject protein following administration. For example, the antigen-binding protein or antigen-binding fragment thereof may be provided in a liposome formulation, prepared as a colloid, or other conventional techniques for extending serum halflife. A variety of methods are available for preparing liposomes are known in the art. The preparations may also be provided in controlled release or slow-release forms.

[0115] Other examples of formulations suitable for parenteral administration include isotonic sterile injection solutions, anti-oxidants, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. For example, a pharmaceutical composition can be present in a container, e.g., a sterile container, such as a syringe. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0116] The concentration of a vector disclosed herein in a formulation can vary widely (e.g. , from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight) and will usually be selected primarily based on fluid volumes, viscosities, and patient-based factors in accordance with the particular mode of administration selected and the patient’s needs.

[0117] In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, and other species. Excipients such as waxes, butters, coloring agents, coating agents, flavorings, and perfuming agents may also be included. Pharmaceutically acceptable excipients are well known in the art (see, e.g., Remington's The Science and Practice of Pharmacy, 21stEdition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006).

[0118] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and / or combinations thereof. Granulating and dispersing agents may be selected from the non-limiting list consisting of potato starch, com starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0119] Surface active agents and / or emulsifiers may include, but are not limited to, natural emulsifiers e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC®F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof.

[0120] A binding agent may be starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; and combinations thereof, or any other suitable binding agent.

[0121] Preservatives include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Antimicrobial preservatives include, but are notlimited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, benzyl alcohol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®II, NEOLONE™, KATHON™, and / or EUXYL®.

[0122] Examples of buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffers (e.g. HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof. Lubricating agents may be selected from the non-limiting group consisting of magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0123] Examples of oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils as well as butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, simethicone, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and / or combinations thereof.

[0124] If a vector disclosed herein is to be stored long-term, it may be frozen in the presence of a cryoprotective agent such as glycerol.

[0125] Methods, including methods of treatment

[0126] Provided herein are methods of treating a disease in a subject in need thereof using expression constructs, vectors, and pharmaceutical compositions disclosed herein.

[0127] Effective intervention with respect to BBS 10 benefits from the introduction of a normal functional copy of the BBS 10 gene into rod and cone photoreceptors early in a patient’s life while some retinal structure remains, in order to activate function and survival of the photoreceptors that are still present.

[0128] In some embodiments, the subject is a mammal. The term “mammal” as used herein is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and farm animals. Mammals, include, but are not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline, etc. Individuals and patients are also subjects herein.

[0129] 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 (i.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 one or more symptoms of the condition, disorder or disease state; and remission (whether partial ortotal), or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects.

[0130] 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.

[0131] The medical uses contemplated by the specification may include formulation of an expression construct or vector disclosed herein for use as a medicament for prevention or treatment of the disease(s) and / or disorder(s) defined herein (or for the prevention of treatment of symptoms associated with the disease(s) and / or disorder(s) defined herein), but could equally be formulated for use as (i) a medicament for a method of prevention or treatment of the disease(s) and / or disorder(s) defined herein (or prevention of treatment of symptoms associated with the disease(s) and / or disorder(s) defined herein), as (ii) a vector for use in the preparation of a medicament for the prevention or treatment of the disease(s) and / or disorder(s) defined herein (or for the prevention of treatment of symptoms associated with the disease(s) and / or disorder(s) defined herein), or as (iii) use of a vector according for the prevention or treatment of the disease(s) and / or disorder(s) defined herein (or for the prevention of treatment of symptoms associated with the disease(s) and / or disorder(s) defined herein). Such medical uses are all envisaged by the present disclosure.

[0132] Provided herein is a method of treating BBS 10 in a subject in need thereof, the method comprising administering to the subject a vector or a pharmaceutical composition disclosed herein. Provided is a vector or a pharmaceutical composition disclosed herein for use in treating BBS10 in a subject in need thereof. Provided is the use of a vector disclosed herein in the manufacture of a medicament for treating BBS 10 in a subject in need thereof.

[0133] Provided herein is a method of treating or reducing vision loss in a subject in need thereof, the method comprising administering to the subject a vector or a pharmaceutical composition disclosed herein, wherein the subject has a BBSlO-deficiency.

[0134] In embodiments, the BBSlO-deficiency is due to one or more deletions, insertions, frameshifts, or other mutations in the BBS10 gene. In some embodiments, the mutation(s) lead to the production of a truncated BBS 10 protein. In some embodiments, the mutation(s) lead to the production of a protein comprising BBS 10 protein and additional amino acid sequence. In some embodiments, the mutation(s) lead to the production of a BBS10 protein with one or more amino acid substitutions. In one embodiment, the patient has one or more genomic variations that lead to the production of no or only very little BBS 10 protein.

[0135] Provided herein is a method of restoring vision in a subject in need thereof, the method comprising administering to the subject a vector or a pharmaceutical composition disclosed herein, wherein the subject has a BBSlO-deficiency.

[0136] Provided herein is a method of restoring photoreceptor function in a subject in need thereof, the method comprising administering to the subject a vector or a pharmaceutical composition disclosed herein, wherein the subject has a BBSlO-deficiency.

[0137] Methods of measuring vison loss, restoration of vision, and / or photoreceptor function are known to a person skilled in the art and include any of the methods used herein, including electroretinography, optical coherence tomography, or Spatio-Temporal Optical Coherence Tomography (STOC-T). See, e.g., Caltrider D, Gupta A, Tripathy K. Evaluation of Visual Acuity. [Updated 2024 May 1], In: StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2024 Jan-. See also Levenson JH, Kozarsky A. Visual Acuity. In: Walker HK, Hall WD, Hurst JW, editors. Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition. Boston: Butterworths; 1990. Chapter 115.

[0138] In some embodiments, the vector or pharmaceutical composition described herein is administered to a subject in combination with one or more additional therapies to treat BBS10 or the symptoms of BBS10, including obesity and kidney disease.

[0139] The eye has a combination of features that make it well- suited as a target organ for gene therapy. To start, the eye’s compartmentalized anatomy facilitates accurate delivery of vector suspensions at target sites to specific tissues under direct visualization using microsurgical techniques while minimizing systemic dissemination and the possibility of unwanted systemic effects. Second, intraocular tissues comprise small but stable populations of cells and may be transduced efficiently and stably by small volumes of vector suspension. Third, immune responses following intraocular administration of vectors are typically attenuated compared to those following systemic administration; anatomical barriers and a unique immune environment maintain a degree of protection from immune responses directed against vector antigens that might otherwise cause inflammation and limit transgene expression. Fourth, the eye’s unique optical transparency enables non-invasive imaging of ocular tissues in vivo. Finally, visual function is readily quantifiable in terms of both psychophysical and electrophysiological parameters, and the contralateral ‘fellow’ eye offers an invaluable experimental control.

[0140] In embodiments, the vector or pharmaceutical composition described herein is administered to the eye. Methods and devices for the administration of pharmaceutical compositions to the eye are known in the art and include, but are not limited, to the methodsand devices described in International Patent Publication No. WO2021 / 055906 (entitled Injection systems and methods of their use), International Patent Publication No. WO2022 / 036256 (entitled Motorized injection system and methods of use), and International Patent Publication No. W02018112305 (entitled System and method for resistance-dependent, self-regulated medical penetration).

[0141] In embodiments, the vector or pharmaceutical composition described herein is administered to the subretinal space. This can be achieved by intraocular subretinal injection using a fine cannula introduced through one or more small retinotomies (sites of injection in the retina).

[0142] In embodiments, the vector or pharmaceutical composition described herein is administered via retinal injection, subretinal injection, intravitreal injection, suprachoroidal injection, topical instillation, or intravenous injection.

[0143] The amount of the vector or pharmaceutical composition described herein that is effective for treating disease can be determined using standard clinical techniques known to those with skill in the art. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed can also depend on the route of administration, the condition, the seriousness of the condition being treated, as well as various physical factors related to the individual being treated, and can be decided according to the judgment of a health-care practitioner.

[0144] An effective amount of an rAAV carrying a nucleic acid sequence encoding BBS 10 under the control of the promoter may, for example, range between about P IO10to 5 x 1011viral genomes or genome particles per eye. A “genome particle” is defined herein as an AAV capsid that contains a single stranded DNA molecule that can be quantified with a sequence specific method (such as real-time PCR). Still other dosages in these ranges may be selected by the attending physician. In some embodiments, a dose of about 1-10 *109viral genomes is administered per eye. In some embodiments, a dose of about 1-5 *1010viral genomes is administered per eye. In some embodiments, a dose of about 1-5 MO11viral genomes is administered per eye. In some embodiments, a dose of about 4 *109viral genomes is administered per eye. In some embodiments, a dose of about 2 xlO10viral genomes is administered per eye.

[0145] Methods of translating AAV doses from preclinical models to does for humans are known in the art. See, e.g., Zhang & Zou, Interspecies Scaling of Transgene Products for Viral Vector Gene Therapies: Method Assessment Using Data from Eleven Viral Vectors, AAPS J.2023 Oct 27;25(6):101.

[0146] It is to be understood that for any particular subject, specific dosage regimens can be adjusted according to the individual need and the professional judgment of the person administering or supervising the administration of the expression construct of vector and that dosage ranges set forth herein are illustrative only and are not intended to limit the scope or practice of the claimed disclosure.In some embodiments, it may be desirable to administer multiple “booster” dosages of a pharmaceutical compositions disclosed herein. For example, depending upon the duration of the transgene within the target cell, one may deliver booster dosages at 6-month intervals, yearly, or longer following the first administration.

[0147] Articles of manufacture and kits

[0148] Also provided are kits or articles of manufacture for use in the methods described herein. In aspects, the kits comprise the compositions described herein (c.g, compositions for delivery of a BBS 10 encoding transgene) in suitable packaging. Suitable packaging for compositions (such as ocular compositions for injection) described herein are known in the art, and include, for example, vials (such as sealed vials), vessels, ampules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may further be sterilized and / or sealed.

[0149] Also provided are kits comprising the compositions described herein. These kits may further comprise instruction(s) on methods of using the composition, such as uses described herein. The kits described herein may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing the administration of the composition or performing any methods described herein. For example, in some embodiments, the kit comprises an rAAV for the expression of a BBS10 encoding transgene in target cells, a pharmaceutically acceptable carrier suitable for injection, and one or more of a buffer, a diluent, a filter, a needle, a syringe, and a package insert with instructions for performing the injections.

[0150] All methods described herein are performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Regarding any of the methods provided, the steps of the method may occur simultaneously or sequentially. When the steps of the method occur sequentially, the steps may occur in any order, unless noted otherwise.

[0151] In cases in which a method comprises a combination of steps, each and every combination or sub-combination of the steps is encompassed within the scope of the disclosure, unless otherwise noted herein.

[0152] It is to be understood that this invention is not limited to the particular molecules, constructs, 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 extent possible, in combination with and / or in the context of other particular aspects and embodiments of the invention, and in the invention generally.

[0153] All referenced patents, patent applications, book chapters, scientific publications, 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.

[0154] 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

[0155] As shown in the Examples, an AAV8-RK-hBBS10 vector demonstrated potency in restoring photoreceptor function and improving photoreceptor viability in a BBSlO-deficient animal model and showed a clean safety profile in mice. Taken together, this data supports the use of AAV8-RK-hBBS10 for the treatment of visual loss in patients with BBSlO-associated ciliopathy.

[0156] Example 1: Materials and Methods

[0157] Studies were conducted in BBS 10 mutant mice to show that the ocular failure in BBS10 ciliopathy is amenable to AAV-BBS10 gene therapy, as described below.

[0158] Promoters

[0159] Two different promoters for the expression of the human BBS10 transgene were tested. The rhodopsin kinase (RK) promoter drives expression specifically in photoreceptors, whilst the constitutive CAG promoter drives expression in all transduced cells. CAG consists of the CMV enhancer fused to the promoter and first intron of P-actin.

[0160] Electroretinogram (ERG) recordings

[0161] Electroretinography (ERG) measures the electrical activity of the retina in response to a light stimulus under dark- (scotopic) or light-adapted (photopic) conditions allowing assessment of retinal function. The a-wave (negative potential) and b-wave (positive potential) are the primary ERG components used for quantification of responses. As such, ERG is an electrophysiological method that measures retinal sensitivity. It is routinely used in patients and animal models to assess retinal function. FIG. 1 describes the experimental set up used to take measurements in mice at different light intensities and with conditions that focus on either rod function (dark adapted- scotopic) or cone function (light adapted-photopic). The indicated line graphs, also referred to as ERG traces, can be used to quantify photoreceptor sensitivity.

[0162] To perform ERG, mice were dark adapted overnight prior to examination. First, a scotopic ERG protocol assessing responses of rod photoreceptors was performed. Once completed, animals were tested using photopic ERG protocol and 5 step flicker protocol, both of which measure responses of cone photoreceptor cells. Details on the steps within each protocol used in this study can be found in Table 1.Table 1. Summary of ERG Protocols.Scotopic Intensity Frequency Sweeps Inter sweep delay Background cd.s.m2(ms) cd.s.m2Step 1 0.001 1 Hz 5 500 (0.5 s) —Step 2 0.01 1 Hz 5 2000 (2 s) —Step 3 0.1 0.5 Hz 3 15000 (15 s) —Step 4 1 0.1 Hz 3 20000 (20 s) —Step 5 10 0.05 Hz 3 60000 (60 s) —Step 6 30 0.04 Hz 3 90000 (90 s) — Phototopic intensity Frequency Sweeps Inter sweep delay Background cd.s.m2(ms) cd.s.m2Step 1 0.1 1 Hz 25 987 (0.987 s) 30Step 2 1 1 Hz 25 987 (0.987 s) 30Step 3 3.16 1 Hz 25 987 (0.987 s) 30Step 4 10 1 Hz 25 987 (0.987 s) 30Step 5 31.6 1 Hz 25 987 (0.987 s) 30Step 6 50 1 Hz 25 987 (0.987 s) 30 Flicker intensity Frequency Sweeps Inter sweep delay Background cd.s.m2(ms) cd.s.m2Step 1 1 5 Hz 20 — 30Step 2 3 5 Hz 20 — 30Step 3 5 5 Hz 20 — 30Step 4 10 5 Hz 20 — 30Step 5 30 5 Hz 20 — 30

[0163] Subretinal delivery

[0164] The route of administration in the in vivo studies was subretinal delivery, similar to a possible clinical route of administration. Following subretinal delivery as indicated in FIG.2, the vector was deposited in the subretinal space, where it can transduce the target photoreceptors.

[0165] Spectral Domain Optical Coherence Tomography (OCT)

[0166] Optical coherence tomography (OCT) is a noninvasive retinal imaging technique that allows assessment of retinal thickness.

[0167] Briefly, to assess retinal thickness, at least 9 scans were taken. Whole retina thickness measurements focused on the quantification of three horizontal scans per eye including one central scan through optic nerve head (area outside of injection site) and two scans superior of optic nerve head (mid-superior retina and peri-superior retina; treated area).

[0168] For central scans, 4 measurements covering position 0.5 mm, 1.5 mm, 3.5 mm and 4.5 mm for each image were taken and averaged. For superior-retina scans, 5 measurementscovering position 0.5 mm, 1.5 mm, 2.5 mm, 3.5 mm and 4.5 mm were collected and results were averaged.

[0169] Immunohistochemistry (IHC)

[0170] Mice were culled by cervical dislocation, the eyes were carefully removed using curved forceps and the cornea was pierced using a needle. Whole eyes were then fixed in 4% paraformaldehyde solution for 1 h, washed with PBS and stored in 20% sucrose at 4 °C until embedding. Each eye was embedded in OCT mounting media and frozen sections were cut using Leica cryostat (16 pm thickness, sections from each sample distributed across 6-8 slides). Slides containing frozen sections were stored at -20 °C. On the day of IHC staining, slides were removed from the freezer and allowed to defrost for a minimum of 10 minutes at room temperature. Sections were hydrated by brief administration of PBS followed by incubation with blocking solution containing 5% normal goat serum, 1% BSA, 0.01% Triton X-100 in PBS for 1 h at room temperature. Primary antibody working solution was prepared in 1% BSA, 0.01% Triton X-100 in PBS. Primary antibody incubation was performed overnight at 4 °C (mouse anti- Syntaxin-3 antibody diluted 1:300, rabbit anti-peripherin 2 antibody diluted 1 :200). Following primary antibody incubation slides were washed 3 times 5 minutes with PBS followed by addition of secondary antibody working solution for 2 h at room temperature (goat-anti-mouse AF488 used at 1:500; goat-anti-rabbit AF546 used at 1:500; both diluted in 1% BSA, 0.01% Triton X-100 in PBS). Final staining step consisted of nuclei staining with DAPI (1 :2000 in PBS), 2 minutes at room temperature.

[0171] Optomotor response (OMR)

[0172] OMR is a reflex behavior used to assess visual function. To evoke OMR, a mouse watches a defined pattern rotating within a cylinder. Stimulus-correlated head movements are then quantified. As such, the optomotor device tests functional vision with increasing difficulty in the stimulus presented.

[0173] Example 2: BBS10 Mutant Mouse Line Characterization

[0174] First, the kinetics of the retinal degeneration in BBS10 mutant mice were characterized. Both rod and cone responses were greatly diminished in the absence of BBS10.

[0175] Using ERG recordings, the kinetics of the retinal degeneration in BBS 10 KO mice for both rods and cones were characterized by measuring the electrical responses derived from cone cells (photopic A- wave, photopic B-wave and Pl flicker) and rod cells (scotopic A- wave and scotopic B-wave). Three timepoints were tested to assess the progression of disease in these mice (FIG. 3). Both rod and cone responses were greatly diminished from 10 wk of age.By 28wk of age, responses of BBS10 KO mice were extinct. Unaffected heterozygote (HET) mice and WT mice were graphed for comparison and underline the severe loss of retinal function in BBS10 KO mice over time.

[0176] In addition to measurement of ERG responses, the changes in the whole retinal thickness were assessed using OCT. The retinas of PBS-treated BBS 10 mutant mice become thinner over time when compared to WT mice (FIG. 4). Significantly thinner retinas were observed in mutant mice in all three areas in which measurements were taken (central retina, mid-superior retina and peri-superior retina). The central retina of / / / iS70-deficient mice was 15.34 % thinner than WT at the age of 4 months, which increased to 25.48 % at 7 months. In the mid-superior retina, a thinning of 13.76 % at 4 months and 27.62 % at 7 months was detected. In the peri-superior retina (the site of injection) those values increased to 16.97 % and 30.33 % accordingly.

[0177] Example 3: An AAV8-RK-BBS10 Vector Showed Superior Efficacy in Rescuing Loss of Retinal Function as Determined by ERG

[0178] Following BBS10 mutant mouse line characterization, different gene therapy vectors and their ability to rescue loss of retinal function were tested. Gene therapy vectors packaged with AAV8 capsids showed superior performance as compared to those packaged with AAV5 capsid (using identical AAV genomes). Furthermore, expression of the transgene driven by the photoreceptor-specific RK promoter led to significantly better and long-term therapeutic outcomes than the outcomes when expression of the transgene was driven by the constitutive CAG promoter.

[0179] Mutant mice were treated at the age of 4-5 wk and ERG recordings were performed at -6, -12 and 24-wk post injection (pi) (FIG. 5).

[0180] To determine the best vector for treatment, two AAV5 vectors with different promoters carrying the human BBS10 transgene were initially tested. The RK promoter drives expression specifically in photoreceptors, whilst the constitutive CMV early enhancer / chicken beta actin (CAG) promoter drives expression in all transduced cells. Sequences for the constructs can be found in Table 2.

[0181] Mutant mice were treated with a vector high dose (2 x 1010viral genomes (VG) / eye) of AAV5-CAG-hBBS10 or AAV5-RK-hBBS10. Only AAV5-RK-hBBS10 treatment resulted in amplitudes higher than sham treatment with significantly increased photopic B wave (at 50 cd.sm2p<0.05), increased scotopic A (at 1 cd.sm2p<0.001; at 30 cd.sm2p<0.01)and B waves (at 1 cd.sm2p<0.05, at 10 cd.sm2p<0.05, at 30 cd.sm2p<0.001) and increased flicker responses(at 5Hz 10 cd.sm2p<0.05) at 6- wk pi (FIG. 6). This effect, however, was not sustained at 12 and 24 wk pi.

[0182] Subsequently, both promoter constructs were packaged into a vector comprising AAV8 capsid and tested again to compare their efficacy versus AAV5 counterparts. AAV8-RK-hBBSlO rescued both cone and rod responses following subretinal delivery when compared with sham-treated controls (FIG. 7). At 6 wk pi, significantly increased amplitudes were observed in the photopic b-wave, scotopic a-wave and flicker. The therapeutic effect was preserved in the photopic and scotopic b-waves until the last assessed timepoint of 25 wk after treatment. In comparison, treatment with AAV8-CAG-hBBS10 did not result in sustained therapeutic effects. Significant differences could only be observed in photopic A and B-wave as well as in flicker at 6 wk pi.

[0183] In sum, vector AAV8-RK-hBBS10 showed superior efficacy in its ability to rescue loss of retinal function as compared to vectors using an AAV5 capsid or a CAG promoter.

[0184] Example 4: An AAV8-RK-hBBS10 Vector (Expressing Human BBS10) Showed a Dose-Dependent Effect of Retinal Sensitivity Rescue in BBS10 KO Mice

[0185] To examine a dose-dependent effect of retinal sensitivity rescue in BBS10 KO mice, a 5-fold lower dose of the AAV8 vectors was employed than the one used in FIG. 7, referred to as mid dose (4 x 109VG / eye = 1 x 1012VG / ml). Mid dose treatment with AAV8-RK-hBBSlO resulted in only a modest increase in retinal function at individual light intensities compared to sham treated control group, whilst no effect was observed following treatment with AAV8-CAG-hBBS10 (Fig 8). No significant differences were recorded at 6wk pi. AAV8-RK-hBBSlO showed significantly higher amplitudes in photopic B-wave at 31.6 cd.sm2(p<0.05) and in 5Hz 5 cd.sm2Flicker (p<0.05) at 12wk pi as well as in photopic B-wave at 50 cd.sm2(p<0.05) at 24wk.

[0186] The mid dose used in these cohorts has been previously used in various inherited retinal gene supplementation studies successfully. See, e.g., Georgiadis et al., Development of an optimized AAV2 / 5 gene therapy vector for Leber congenital amaurosis owing to defects in RPE65, Gene Ther. 2016 Dec;23(12):857-862. Erratum in: Gene Ther. 2024 Sep;31(9-10):524; Pawlyk et al., Photoreceptor rescue by an abbreviated human RPGR gene in a murine model of X-linked retinitis pigmentosa, Gene Ther. 2016 Feb;23(2): 196-204; Mihelec et al., Longterm preservation of cones and improvement in visual function following gene therapy in a mouse model of leber congenital amaurosis caused by guanylate cyclase- 1 deficiency, Hum Gene Ther. 2011 Oct;22(10): 1179-90; Sun et al., Gene therapy with a promoter targeting bothrods and cones rescues retinal degeneration caused by AIPL1 mutations, Gene Ther. 2010 Jan; 17(1): 117-31, all of which are incorporated herein in their entireties. As such, it is possible that the modest treatment effect observed here was associated with the nature of the transgene and the physiological impact of inter-species interactions. More specifically, the modest treatment effect might be the result of introducing the human BBS 10 protein in the mouse BBSome.

[0187] As discussed herein, the BBSome is a hetero-octameric protein complex that plays a central role in primary cilia homeostasis and is comprised of a number of BBS-related proteins. BBS proteins are not acting individually, but are dependent on their associations with other BBSome counterparts. While the human and mouse BBS 10 proteins share approximately 70% homology, the lack of complete homology may prevent close structural association of the human BBS10 protein with mouse Bbs proteins of the BBScc in 7> / iS7d-deficient mice, which is required for a fully functional BBSome. In order to test the hypothesis, the therapeutic effect of a gene therapy vector was assessed when employing the mouse BBS10 (mBBSlO) transgene.

[0188] The vector AAV8-RK-mBBS10 was constructed to be identical to the AAV8-RK-hBBSlO (same capsid, same promoter) except for the transgene sequence, for which the mouse orthologue was used. AAV8-RK-mBBS10 provided ERG rescue across all timepoints using both high and mid doses (FIG. 9). In particular, the mid dose provided higher retinal function when compared to the effect attained with the mid-dose of AAV8-RK-hBBS10 (see FIG. 8), confirming the hypothesis that the mechanism of action of the BBSome and its multicomponent nature is a driver for higher efficacy in a same species platform.

[0189] In sum, the potency of AAV8-RK-hBBS10 appears to be underestimated when assessed in the BBS10 KO mouse due to this species difference. However, as discussed in Example 9, when applying optimization techniques for AAV vector production, also the middose of AAV8-RK-hBBS10 is effective in rescuing retinal sensitivity in BBS10 KO mice.

[0190] Example 5: The Efficacy of the AAV8-RK-BBS10 Vector In Vivo Was Further Confirmed by OCT

[0191] In addition to the ERG recordings, OCT measurements confirmed statistically significant rescue of retinal thickness in eyes treated with the AAV8-RK-BBS10 vector.

[0192] Following final ERG examination at 24 ± 1 wk after vector administration, OCT was used to quantify the retinal thickness in BBS 10 KO mice treated with either sham injections or AAV8-RK vector (FIG. 10). Statistically significant rescue of retinal thickness was observed in eyes treated with the high dose of AAV8-RK-mBBS10, showing increased whole retinathickness by 10.7% in central retina, 9.2% in mid-superior retina and by 9.1% in per-superior retina, compared to PBS-treated BbslO KO mice (p<0.01). Although high dose of AAV8-RK-hBBSlO increased whole retina thickness in all three areas, a statistically significant difference was only detected in mid-superior retina (thickness increased by 11.4% compared to PBS-treated BBS10 KO mice; p<0.01), which may be due to the low number of eyes used for this assessment. In contrast to the ERG data, no difference was discernible between the mid doses of the human or the mouse vector and sham-treated eyes when measuring whole retinal thickness. A closer examination of the outer retinal thickness (e.g. photoreceptor outer segments to outer nuclear layer) may be required to uncover potential structural differences between the two constructs and sham treatment.

[0193] In sum, statistically significant rescue of retinal thickness was observed in the conditions that previously demonstrated robust ERG rescue, further strengthening the potency data following gene supplementation.

[0194] Example 6: AAV8-RK-hBBS10 Treatment Partly Corrects STX3 MisLocalization

[0195] Treatment of BBS10 mutant mice with AAV8-RK-hBBS10 also led to partial correction of syntaxin 3 localization in the inner photoreceptor segments, as opposed to mislocalization and trafficking to outer photoreceptor segments in untreated BBS10 KO mice.

[0196] Eyes of treated mice were collected for immunohistochemical analysis 4 weeks post administration of high dose AAV8-RK-hBBS10 or AAV8-RK-mBBS10. All eyes were stained with antibodies targeting Peripherin 2 (PRPH2), a marker for rod-photoreceptor outer segments (OS) and syntaxin-3 (STX3). STX3 is a photoreceptor protein that under healthy conditions is trafficked to the inner segments (IS) (see WT panel). In the BBS10 KO, STX3 mis-localizes to the OS of photoreceptors (see KO panel) (FIG. 11). Following treatment (high dose), a partial correction of its localization was observed (see AAV8-RK-h / -mBBS10 panels) in comparison to untreated KO controls. White arrows indicate the IS area in each panel.

[0197] In sum, in conjunction with the functional and structural rescue data, these molecular data demonstrates that the ERG and retinal thickness improvements are directly associated with the correction of the subcellular localization of BB Some partner proteins following treatment with AAV8-RK-hBBS10.

[0198] Example 7: Treatment of WT mice with Mid-Dose of AAV8-RK-hBBS10 is Safe

[0199] To probe for any detrimental effects of AAV8-RK-hBBS10 in the murine retina, WT mice received subretinal injections with AAV8-RK-hBBS10. ERG recordings were taken at 1, 3, and 6 months after dosing (FIG. 12). In safety studies, toxicity is denoted as the statistically significant drop in either retinal function or structure at two consecutive timepoints.

[0200] At all timepoints, there was no reduction in ERG recordings between treated and sham-treated eyes. In addition, histological assessment confirms good safety profile and no detrimental effect on retinal thickness was observed (FIG. 13A, FIG. 13B, FIG. 13C, and FIG. 13D)

[0201] In sum, the vector AAV8-RK-hBBS10 demonstrated a clean safety profile.

[0202] Example 8: An AAV8-RK-BBS10 Vector Shows Superior Efficacy as Compared to a Known hBBSlO Vector

[0203] The AAV8-RK-BBS10 disclosed herein was directly compared to AAVAnc80-CMV-mBBSlO and AAVAnc80-CMV-hBBS10 vectors. Vector AAVAnc80-CMV-mBBS10 had been previously demonstrated to be effective in restoring the ERG phenotype in BbslO-deficient mice. See Hsu et al., Subretinal gene therapy delays vision loss in a Bardet-Biedl Syndrome type 10 mouse model, Mol Ther Nucleic Acids. 2022 Dec 12;31:164-181.

[0204] A statistically significant increase in ERG responses could be observed for cohorts treated with a high dose of AAVAnc80-CMV-mBBS10 (expressing murine BBS10) at all time points (FIG. 14). No therapeutic effect was detected following treatment with the same dose of AAVAnc80-CMV-hBBS10 expressing human BBS10.

[0205] In sum, the combination of promoter and transgene species can play an important role in rescuing the retinal phenotype in / / / iS7d-deficient mice.

[0206] Example 9: MSAT-grade AAV8-RK-hBBS10 Promotes Sustained ERG Rescue Using Both Mid- and High-Dose

[0207] The AAV vectors used in Examples 2-8 used research and development (R&D) grade AAV vectors. R&D grade vectors can be manufactured relatively quickly and are usually produced in smaller volumes.

[0208] The AAV vectors used in Examples 9 and 10 were produced using a Manufacturing Science and Technology (MS AT) process, which more closely mirrors the GMP process to be used at later stages of development and for drug product manufacturing ahead of clinical development. The MSAT process is a optimization process for vector production in which a number of parameters can be varied, including the selection of a specific producer cell line,cultivation medium and conditions, vessels for cultivation, cultivation volume and other parameters of the vector production process. Methods of producing AAV vectors and optimizing the production of AAV vectors are known to a person skilled in the art.

[0209] MSAT-grade AAV8-RK-hBbslO provides long-term rescue of retinal sensitivity following treatment of BBS10 KO mice, both at mid and high dose (FIG. 15).

[0210] Example 10: AAV8-RK-hBBS10 Preserves Functional Vision

[0211] Treatment with an AAV8-RK-hBBS10 vector not only resulted in increased retinal sensitivity, but also translated to functional vision.

[0212] OMR is a reflex behavior that can be used to assess visual function (FIG. 16A).During this test, the test subject watches a defined pattern rotating within a cylinder. Stimulus-correlated head movements are then quantified.

[0213] In FIG. 16B, the Y axis represents the success score. 1 represents the failure threshold at which the test subject has a 50 / 50 chance of scoring right. A higher value indicates improved functional vision. In FIG. 16B, the X axis represents the difficulty. Higher values indicate an increased difficulty. At the rightmost, the test becomes very difficult for all mice, including healthy mice. As such, data for intermediate difficulty are most useful for assessing differences between different treatment conditions.

[0214] Treatment with MSAT-grade AAV8-RK-hBBS10 vector increases visual function in BbslO KO mice in a dose-dependent manner until 38-41 weeks post-delivery (FIG. 16B).Table 2. Nucleic Acid SequencesElement SEQ ID SequenceNO AAV2 1 CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGC ITR (5’) CCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCC GGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGG CCAACTCCATCACTAGGGGTTCCT AAV2 6 AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCG ITR (3’) CGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGC CCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGA GCGAGCGCGCAGCTGCCTGCAGG CAG 2 CTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTA promoter CGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCA ACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCC ATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGT GGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGAElement SEQ ID SequenceNO CGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCT TATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTC ATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTT CACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGT ATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGG GGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGG GGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGC GGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTT ATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGA AGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGC CCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCC GGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGC GGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGT TTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCC TTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCG GCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCG CCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCT GCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGC GCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCG GGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGT GTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCG GTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTG CTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGG GGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGC GGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGG CCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGG AGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCAT TGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTC CTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCG CCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTG CGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCG TGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCT CGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGA CGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGC GGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTT TTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGT CTCATCATTTTGGCAAAGAATTGhRK 3 TGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTACA promoter TTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATT GATTATTGACTAGAATTCGCTAGCAAGATCCAAGCTCAGAT CTCGATCGAGTTGGGCCCCAGAAGCCTGGTGGTTGTTTGT CCTTCTCAGGGGAAAAGTGAGGCGGCCCCTTGGAGGAAG GGGCCGGGCAGAATGATCTAATCGGATTCCAAGCAGCTCA GGGGATTGTCTTTTTCTAGCACCTTCTTGCCACTCCTAAGC GTCCTCCGTGACCCCGGCTGGGATTTAGCCTGGTGCTGTG TCAGCCCCGGTCTCCCAGGGGCTTCCCAGTGGTCCCCAGG AACCCTCGACAGGGCCCGGTCTCTCTCGTCCAGCAAGGGCAGGGACGGGCCACAGGCCAAGGGCCCTCGATCGAGGAAElement SEQ ID SequenceNO CTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTCTTTTT GTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATC AAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGG CCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAA TTGTACCCGCGGCCGChBBSlO 4 ATGTTAAGTTCTATGGCCGCTGCAGGGTCTGTGAAGGCGG gene CGTTGCAGGTGGCCGAGGTGCTGGAAGCCATCGTGAGCT (w / o stop GCTGCGTGGGGCCCGAGGGACGGCAAGTTTTGTGTACGA codon) AGCCCACTGGCGAGGTGCTTCTCAGCCGGAATGGAGGCC GCCTCCTGGAGGCGCTACACTTAGAGCATCCCATAGCCAG GATGATAGTGGACTGTGTTTCCAGTCATCTCAAAAAAACA GGAGATGGTGCAAAAACATTTATTATCTTTCTTTGCCATTT GCTTAGAGGACTTCATGCAATCACAGACAGAGAAAAGGA TCCTTTGATGTGTGAAAACATTCAAACCCATGGAAGGCAT TGGAAAAATTGTTCTCGGTGGAAATTTATTTCCCAGGCTCT CCTAACGTTTCAGACACAAATATTAGACGGTATTATGGACC AGTACCTAAGTAGACACTTTTTGTCTATCTTTTCGTCTGCT AAAGAGAGAACATTGTGTAGGAGCTCTTTAGAGTTGCTCT TAGAAGCATACTTTTGTGGAAGAGTGGGAAGAAATAATCA TAAATTTATTTCACAGTTGATGTGTGACTACTTTTTCAAGT GTATGACTTGTAAAAGTGGGATTGGTGTATTTGAGTTAGTG GATGACCATTTTGTAGAGTTGAATGTTGGTGTCACTGGCCT TCCTGTTTCAGATTCCAGGATCATAGCTGGTCTTGTGCTTC AGAAAGATTTTTCTGTGTACCGCCCAGCAGATGGTGACAT GCGAATGGTGATAGTAACAGAAACCATTCAGCCTCTTTTTT CCACTTCTGGATCAGAGTTTATTCTAAATTCAGAAGCACAG TTTCAGACATCTCAATTTTGGATTATGGAAAAGACAAAAG CAATAATGAAACATCTACATAGTCAGAATGTAAAATTGCTC ATATCTAGTGTGAAACAACCAGATTTAGTTAGTTATTATGC AGGGGTGAATGGCATATCAGTGGTTGAGTGTTTATCATCAG AAGAAGTTTCTCTTATCCGGAGGATCATTGGTCTTTCTCCA TTTGTACCACCACAGGCCTTTTCGCAGTGTGAAATACCTA ACACTGCTTTGGTGAAATTTTGTAAACCTCTTATCCTTAGA TCCAAAAGATATGTTCATCTAGGCTTGATAAGCACATGTGC ATTTATACCACACTCTATAGTTCTTTGTGGACCAGTGCATG GTCTCATTGAACAACATGAGGATGCTTTACATGGAGCACT TAAAATGCTTCGGCAATTATTTAAAGACCTTGATCTAAATT ACATGACACAAACCAATGACCAAAATGGCACTTCAAGTCT TTTTATTTATAAGAACAGTGGAGAAAGTTATCAAGCACCA GATCCTGGTAATGGCTCAATACAAAGGCCTTATCAGGACA CAGTTGCAGAGAACAAAGATGCATTGGAAAAAACTCAAA CATATTTAAAAGTACATTCTAATTTGGTAATTCCAGATGTAG AATTAGAAACATATATTCCGTATTCAACCCCCACACTGACA CCAACAGATACATTCCAAACAGTTGAAACGCTGACATGTT TGTCTTTGGAAAGAAACAGGCTAACTGATTATTATGAACC ATTACTCAAGAACAATTCCACTGCTTATTCAACAAGGGGA AATAGAATAGAAATTTCTTACGAAAATTTACAGGTCACAAATATTACTAGAAAGGGAAGCATGTTACCAGTGAGCTGTAAElement SEQ ID SequenceNO GTTACCGAATATGGGTACTTCCCAGAGTTACCTTTCCTCAT CTATGCCAGCTGGTTGTGTTTTGCCAGTAGGTGGTAATTTT GAGATCTTGTTACATTACTATCTTCTCAATTATGCCAAAAA ATGCCATCAATCAGAAGAAACCATGGTTAGTATGATAATAG CTAATGCACTTTTAGGCATTCCCAAAGTCCTTTATAAATCTA AAACAGGAAAGTACAGCTTTCCACATACATATATAAGAGC TGTCCATGCACTGCAAACCAATCAACCCTTGGTAAGCAGT CAGACAGGTTTGGAATCAGTAATGGGTAAATACCAGCTAC TAACTTCAGTTCTTCAGTGTTTGACAAAAATATTAACCATT GACATGGTAATCACTGTTAAGAGACACCCTCAGAAAGTTC ACAATCAAGATTCAGAAGATGAACTAbGH 5 CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCC poly(A) CCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTG signal TCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTG AGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGG ACAGCAAGGGGGAGGATTGGGAAGAGAATAGCAGGCATG CTGGGGA AAV2 7 CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGC ITR (5’) CGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGT CAG CGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGhBBSlO GGAGTGGCCAACTCCATCACTAGGGGTTCCTTCTAGAC bGH AACTTTGTATAGAAAAGTTGCTCGACATTGATTATTGACTA poly(A) GTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCC signal CATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGC AAV2 CCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT ITR (3’) CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACT TTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC(portion CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGC of CCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCAT pAAV- TATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCA CAG- GTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGT BBS10- GAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCT BGHpA CCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTG plasmid) TGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGC CAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCG AGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCG CTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGC GGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCG CTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGC CTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTC CCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGC TGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCT GTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCC TTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTG TGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCC CGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTG TGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAElement SEQ ID SequenceNO AGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGG GGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCA CCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGT GCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTG CCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGG GGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGG GGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCG CGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGA GAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAG CCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGG CGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAAT GGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCC CCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGAC GGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGC TTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACC ATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAAC GTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATT GCAAGmGJACAGCCACCATGTTAAGTTCTATGGCCGCTG CAGGGTCTGTGAAGGCGGCGTTGCAGGTGGCCGAGGTGCT GGAAGCCATCGTGAGCTGCTGCGTGGGGCCCGAGGGACG GCAAGTTTTGTGTACGAAGCCCACTGGCGAGGTGCTTCTC AGCCGGAATGGAGGCCGCCTCCTGGAGGCGCTACACTTAG AGCATCCCATAGCCAGGATGATAGTGGACTGTGTTTCCAGT CATCTCAAAAAAACAGGAGATGGTGCAAAAACATTTATTAT CTTTCTTTGCCATTTGCTTAGAGGACTTCATGCAATCACAG ACAGAGAAAAGGATCCTTTGATGTGTGAAAACATTCAAACC CATGGAAGGCATTGGAAAAATTGTTCTCGGTGGAAATTTAT TTCCCAGGCTCTCCTAACGTTTCAGACACAAATATTAGACG GTATTATGGACCAGTACCTAAGTAGACACTTTTTGTCTATCT TTTCGTCTGCTAAAGAGAGAACATTGTGTAGGAGCTCTTTA GAGTTGCTCTTAGAAGCATACTTTTGTGGAAGAGTGGGAAG AAATAATCATAAATTTATTTCACAGTTGATGTGTGACTACTT TTTCAA GTGTATGA CTTGTAAAA GTGGGATTGGTGTATTTG AGTTAGTGGATGACCATTTTGTAGAGTTGAATGTTGGTGTC ACTGGCCTTCCTGTTTCAGATTCCAGGATCATAGCTGGTCT TGTGCTTCAGAAAGATTTTTCTGTGTACCGCCCAGCAGATG GTGACATGCGAATGGTGATAGTAACAGAAACCATTCAGCCT CTTTTTTCCACTTCTGGATCAGAGTTTATTCTAAATTCAGAA GCACAGTTTCAGACATCTCAATTTTGGATTATGGAAAAGAC AAAAGCAATAATGAAACATCTACATAGTCAGAATGTAAAATT GCTCATATCTAGTGTGAAACAACCAGATTTAGTTAGTTATTA TGCAGGGGTGAATGGCATATCAGTGGTTGAGTGTTTATCAT CAGAAGAAGTTTCTCTTATCCGGAGGATCATTGGTCTTTCT CCATTTGTACCACCACAGGCCTTTTCGCAGTGTGAAATACC TAACACTGCTTTGGTGAAATTTTGTAAACCTCTTATCCTTAG ATCCAAAAGATATGTTCATCTAGGCTTGATAAGCACATGTGC ATTTATACCACACTCTATAGTTCTTTGTGGACCAGTGCATGGTCTCATTGAACAACATGAGGATGCTTTACATGGAGCACTTAElement SEQ ID SequenceNO AAATGCTTCGGCAATTATTTAAAGACCTTGATCTAAATTACA TGACACAAACCAATGACCAAAATGGCACTTCAAGTCTTTTT ATTTATAA GAA CA GTGGA GAAA GTTATCAA GCA CCA GATCC TGGTAATGGCTCAATACAAAGGCCTTATCAGGACACAGTTG CAGAGAACAAAGATGCATTGGAAAAAACTCAAACATATTTA AAAGTACATTCTAATTTGGTAATTCCAGATGTAGAATTAGAA ACATATATTCCGTATTCAACCCCCACACTGACACCAACAGAT ACATTCCAAACAGTTGAAACGCTGACATGTTTGTCTTTGGA AAGAAACAGGCTAACTGATTATTATGAACCATTACTCAAGA ACAATTCCACTGCTTATTCAACAAGGGGAAATAGAATAGAA ATTTCTTACGAAAATTTACAGGTCACAAATATTACTAGAAAG GGAAGCATGTTACCAGTGAGCTGTAAGTTACCGAATATGGG TACTTCCCAGAGTTACCTTTCCTCATCTATGCCAGCTGGTTG TGTTTTGCCAGTAGGTGGTAATTTTGAGATCTTGTTACATTA CTATCTTCTCAATTATGCCAAAAAATGCCATCAATCAGAAGA AACCATGGTTAGTATGATAATAGCTAATGCACTTTTAGGCAT TCCCAAAGTCCTTTATAAATCTAAAACAGGAAAGTACAGCT TTCCACATACATATATAAGAGCTGTCCATGCACTGCAAACCA ATCAACCCTTGGTAAGCAGTCAGACAGGTTTGGAATCAGTA ATGGGTAAATACCAGCTACTAACTTCAGTTCTTCAGTGTTTG ACAAAAATATTAACCATTGACATGGTAATCACTGTTAAGAGA CACCCTCAGAAAGTTCACAATCAAGATTCAGAAGATGAACT AZ4AGATATCGAATTCCTAGAGCTCGCTGATCAGCCTCGAC TGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCC GTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTT TCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGT GTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAG GGGGAGGATTGGGAAGAGAATAGCAGGCATGCTGGGGAGGG CCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTC TCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCA AAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCT CAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG AAV2 8 CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGC ITR (5’) CGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGT hRK CGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAG hBBSlO GGAGTGGCCAACTCCATCACTAGGGGTTCCTTCTAGAC bGH AACTTTGTATAGAAAAGTTGTGTAGTTAATGATTAACCCGC poly(A) CATGCTACTTATCTACGTACATTTATATTGGCTCATGTCCAA signal CATTACCGCCATGTTGACATTGATTATTGACTAGAATTCGCT AAV2 AGCAAGATCCAAGCTCAGATCTCGATCGAGTTGGGCCCCA ITR (3’) GAAGCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGTGA GGCGGCCCCTTGGAGGAAGGGGCCGGGCAGAATGATCTA(portion ATCGGATTCCAAGCAGCTCAGGGGATTGTCTTTTTCTAGCA of CCTTCTTGCCACTCCTAAGCGTCCTCCGTGACCCCGGCTG pAAV- GGATTTAGCCTGGTGCTGTGTCAGCCCCGGTCTCCCAGGG hRK- GCTTCCCAGTGGTCCCCAGGAACCCTCGACAGGGCCCGG BBS10- TCTCTCTCGTCCAGCAAGGGCAGGGACGGGCCACAGGCCAAGGGCCCTCGATCGAGGAACTGAAAAACCAGAAAGTTAElement SEQ ID SequenceNO BGHpA ACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCG plasmid) GATCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTG GATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTT CTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGCCAAG TTTGTACAGCCACCATGTTAAGTTCTATGGCCGCTGCAGGG TCTGTGAAGGCGGCGTTGCAGGTGGCCGAGGTGCTGGAAG CCATCGTGAGCTGCTGCGTGGGGCCCGAGGGACGGCAAGT TTTGTGTACGAAGCCCACTGGCGAGGTGCTTCTCAGCCGG AATGGAGGCCGCCTCCTGGAGGCGCTACACTTAGAGCATC CCATAGCCAGGATGATAGTGGACTGTGTTTCCAGTCATCTC AAAAAAACAGGAGATGGTGCAAAAACATTTATTATCTTTCTT TGCCATTTGCTTAGAGGACTTCATGCAATCACAGACAGAGA AAAGGATCCTTTGATGTGTGAAAACATTCAAACCCATGGAA GGCATTGGAAAAATTGTTCTCGGTGGAAATTTATTTCCCAG GCTCTCCTAACGTTTCAGACACAAATATTAGACGGTATTATG GACCAGTACCTAAGTAGACACTTTTTGTCTATCTTTTCGTCT GCTAAAGAGAGAACATTGTGTAGGAGCTCTTTAGAGTTGCT CTTAGAAGCATACTTTTGTGGAAGAGTGGGAAGAAATAATC ATAAATTTATTTCACAGTTGATGTGTGACTACTTTTTCAAGT GTATGACTTGTAAAAGTGGGATTGGTGTATTTGAGTTAGTG GATGACCATTTTGTAGAGTTGAATGTTGGTGTCACTGGCCT TCCTGTTTCAGATTCCAGGATCATAGCTGGTCTTGTGCTTC AGAAAGATTTTTCTGTGTACCGCCCAGCAGATGGTGACATG CGAATGGTGATA GTAA CA GAAA CCATTCA GCCTCTTTTTTC CACTTCTGGATCAGAGTTTATTCTAAATTCAGAAGCACAGT TTCAGACATCTCAATTTTGGATTATGGAAAAGACAAAAGCA ATAATGAAACATCTACATAGTCAGAATGTAAAATTGCTCATA TCTAGTGTGAAACAACCAGATTTAGTTAGTTATTATGCAGG GGTGAATGGCATATCAGTGGTTGAGTGTTTATCATCAGAAG AAGTTTCTCTTATCCGGAGGATCATTGGTCTTTCTCCATTTG TACCACCACAGGCCTTTTCGCAGTGTGAAATACCTAACACT GCTTTGGTGAAATTTTGTAAACCTCTTATCCTTAGATCCAAA AGATATGTTCATCTAGGCTTGATAAGCACATGTGCATTTATA CCACACTCTATAGTTCTTTGTGGACCAGTGCATGGTCTCAT TGAACAACATGAGGATGCTTTACATGGAGCACTTAAAATGC TTCGGCAATTATTTAAAGACCTTGATCTAAATTACATGACAC AAACCAATGACCAAAATGGCACTTCAAGTCTTTTTATTTATA AGAACAGTGGAGAAAGTTATCAAGCACCAGATCCTGGTAAT GGCTCAATACAAAGGCCTTATCAGGACACAGTTGCAGAGAA CAAAGATGCATTGGAAAAAACTCAAACATATTTAAAAGTAC ATTCTAATTTGGTAATTCCAGATGTAGAATTAGAAACATATA TTCCGTATTCAACCCCCACACTGACACCAACAGATACATTC CAAACAGTTGAAACGCTGACATGTTTGTCTTTGGAAAGAAA CAGGCTAACTGATTATTATGAACCATTACTCAAGAACAATTC CACTGCTTATTCAACAAGGGGAAATAGAATAGAAATTTCTTA CGAAAATTTACAGGTCACAAATATTACTAGAAAGGGAAGCA TGTTACCAGTGAGCTGTAAGTTACCGAATATGGGTACTTCCCAGAGTTACCTTTCCTCATCTATGCCAGCTGGTTGTGTTTTElement SEQ ID SequenceNO GCCAGTAGGTGGTAATTTTGAGATCTTGTTACATTACTATCT TCTCAATTATGCCAAAAAATGCCATCAATCAGAAGAAACCA TGGTTAGTATGATAATAGCTAATGCACTTTTAGGCATTCCCA AAGTCCTTTATAAATCTAAAACAGGAAAGTACAGCTTTCCA CATACATATATAAGAGCTGTCCATGCACTGCAAACCAATCAA CCCTTGGTAAGCAGTCAGACAGGTTTGGAATCAGTAATGGG TAAATACCAGCTACTAACTTCAGTTCTTCAGTGTTTGACAAA AATATTAACCATTGACATGGTAATCACTGTTAAGAGACACCC TCA GAAA GTTCA CAATCAA GATTCA GAA GATGAA CTATAAG AJCICGCIGAICAGCCICGACTGTGCCTTCTAGTTGCCAGCC ATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGA AGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATT GCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG GGGTGGGGCAGGA CAGCAA GGGGGAGGA TTGGGAAGA GAA TAGCAGGCATGCTGGGGAGGGCCGCAGGAACCCCTAGTG ATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGC TCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCC GGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGC GCGCAGCTGCCTGCAGG WPRE25 10 AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTG 0 GTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACG CTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGG CTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTC TTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGT GGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGG GGCA WPRE3 11 AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTG GTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACG CTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGG CTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTG CCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTG GACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTG TT WPREm 12 AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTG ut6 GTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACG CTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGG CTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTC TTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGT GGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGG GGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCG CTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCC TGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCA CTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTT CCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCG GGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGC GGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCT CTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCElement SEQ ID SequenceNO WPRE 13 CGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGA CTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGAT ACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTA TGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGT CTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGG CGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGT TGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTT TCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCC GCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGG GCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTC CTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGC GCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCC AGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGG CCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGGTable 3. Amino Acid SequencesElement SEQ ID SequenceNOhBBSlO 9 MLSSMAAAGSVKAALQVAEVLEAIVSCCVGPEGRQVLCTKP TGE VLLSRNGGRLLE ALHLEHP I ARMI VDC VS SHLKKTGDG AKTFIIFLCHLLRGLHAITDREKDPLMCENIQTHGRHWKNCS RWKFISQ ALLTFQTQILDGIMDQYLSRHFLSIF S S AKERTLCR SSLELLLEAYFCGRVGRNNHKFISQLMCDYFFKCMTCKSGIG VFELVDDHFVELNVGVTGLPVSDSRIIAGLVLQKDFSVYRPA DGDMRMVIVTETIQPLFSTSGSEFILNSEAQFQTSQFWIMEKT KAIMKHLHSQNVKLLIS S VKQPDLVS YYAGVNGIS VVECLS S EEVSLIRRIIGLSPFVPPQAFSQCEIPNTALVKFCKPLILRSKRY VHLGLISTCAFIPHSIVLCGPVHGLIEQHEDALHGALKMLRQ LFKDLDLNYMTQTNDQNGT S SLFIYKNSGES YQAPDPGNGS IQRPYQDTVAENKDALEKTQTYLKVHSNLVIPDVELETYIPY STPTLTPTDTFQTVETLTCLSLERNRLTDYYEPLLKNNSTAYS TRGNRIEISYENLQVTNITRKGSMLPVSCKLPNMGTSQSYLS SSMPAGCVLPVGGNFEILLHYYLLNYAKKCHQSEETMVSMII ANALLGIPKVLYKSKTGKYSFPHTYIRAVHALQTNQPLVSSQ TGLESVMGKYQLLTSVLQCLTKILTIDMVITVKRHPQKVHNQDSEDEL

Claims

CLAIMSWe claim:

1. An expression construct comprising:(a) a rhodopsin kinase promoter;(b) a nucleic acid sequence encoding a Bardet-Biedl syndrome 10 protein (BBS 10 protein), wherein the nucleic acid sequence is operatively linked to the promoter; and(c) a polyadenylation signal.

2. The expression construct of claim 1, wherein the rhodopsin kinase promoter 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:

33. The expression construct of claim 2, wherein the rhodopsin kinase promoter comprises SEQ IDN0:3.

4. The expression construct of any one of claims 1-3, wherein the nucleic sequence encoding the BBS10 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:4.

5. The expression construct of claim 4, wherein the nucleic sequence encoding the BBS10 protein comprises SEQ ID NO:4.

6. The expression construct of any one of claims 1-3, wherein the BBS10 protein 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:9.

7. The expression construct of claim 6, wherein the BBS10 protein comprises SEQ ID NO:9.

8. The expression construct of any one of the preceding claims, wherein the polyadenylation signal comprises a sequence that is 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: 5.

9. The expression construct of claim 8, wherein the polyadenylation signal comprises SEQ ID NO:5.

10. The expression construct of any of the preceding claims, wherein the expression construct further comprises a post-transcriptional regulatory element.

11. A vector comprising the expression construct of any one of the preceding claims.

12. The vector of claim 11, wherein the vector is a viral vector.

13. The vector of claim 12, wherein the vector is an adeno-associated viral (AAV) vector.

14. The vector of claim 13, wherein the vector comprises a nucleic acid sequence comprising (i) the expression construct of any one of claims 1-10 and (ii) one or more inverted terminal repeats (ITR).

15. The vector of claim 14, wherein the nucleic acid sequence comprises a 5' ITR and a 3' ITR.

16. The vector of claim 15, wherein the 5' ITR and the 3' ITR are derived from AAV serotype AAV2.

17. The vector of claim 16, wherein (a) the 5' ITR comprises a sequence that is 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 (b) the 3' ITR 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:6.

18. The vector of claim 17, wherein (a) the 5' ITR comprises SEQ ID NO: 1 and (b) the 3' ITR comprises SEQ IDN0:6.

19. The vector of claim 15, wherein the vector comprises a nucleic acid sequence that is 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:8.

20. The vector of claim 19, wherein the vector comprises a nucleic acid sequence comprising SEQ IDN0:8.

21. A cell comprising the expression construct of any one of claims 1-10 or the vector of any one of claims 11-20.

22. A pharmaceutical composition comprising (i) the expression construct of any one of claims 1-10 or the vector of any one of claims 11-20 and (ii) a pharmaceutically acceptable carrier.

23. A method of treating Bardet-Biedl syndrome 10 (BBS 10) in a subject in need thereof, the method comprising administering to the subject the vector of any one of claims 11-20 or the pharmaceutical composition of claim 22.

24. A method of treating reducing vision loss in a subject in need thereof, the method comprising administering to the subject the vector of any one of claims 11-20 or the pharmaceutical composition of claim 22, wherein the subject has a BBSlO-deficiency.

25. A method of restoring photoreceptor function in a subject in need thereof, the method comprising administering to the subject the vector of any one of claims 11-20 or the pharmaceutical composition of claim 22, wherein the subject has a BBSlO-deficiency.

26. A method of restoring vision in a subject in need thereof, the method comprising administering to the subject the vector of any one of claims 11-20 or the pharmaceutical composition of claim 22, wherein the subject has a BBSlO-deficiency.

27. The method of any one of claims 23-26, wherein the vector or the pharmaceutical composition is administered via retinal injection, subretinal injection, intravitreal injection, or suprachoroidal injection.

28. The method of claim 27, wherein the vector or the pharmaceutical composition is administered via subretinal injection.

29. The method of any one of claims 23-26, wherein the subject is a human.

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