Recombinant adeno-associated virus vectors for multi-part gene delivery and treatment of Stargardt disease

Abstract

Recombinant AAV vectors, AAV vector genomes, AAV viral vectors, and capsid proteins for improving gene therapy, as well as methods for their manufacture and use in multi-part (e.g., bipart) delivery systems, are provided herein. The vectors provide transgene splitting segments, which direct their recombination within cells to provide complete transgenes. Regulatory sequences (e.g., enhancers, promoters) that result in high levels of expression of transgenes (e.g., ABCA4) useful for gene therapy (e.g., treatment of Stargardt disease) are also provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 63 / 500,134, filed on 4 May 2023, all of which are incorporated herein by reference in their entirety.

[0002] Reference to electronic sequence listings The contents of the electronic sequence listing (ABEO_010_01WO_SeqList_ST26.xml, size: 574,947 bytes, creation date: April 29, 2024) are incorporated herein by reference in their entirety. [Background technology]

[0003] Adeno-associated virus (AAV) vectors show promise as delivery vectors for gene therapy. However, their therapeutic efficacy is hampered by limitations in delivering genes exceeding a certain size. For example, Stargardt disease is a hereditary retinal disorder that causes juvenile macular degeneration. Autosomal recessive Stargardt disease is caused by mutations in the ABCA4 gene. The ORF of the human ABCA4 gene is approximately 6.8 kb long, which exceeds the typical packaging capacity of AAV vectors, which is approximately 4.7 kb. Therefore, there is an urgent need for AAV viral vector approaches to deliver large genes with regulatory sequences that enable optimal protein expression. [Overview of the project]

[0004] This disclosure generally relates to the field of gene therapy, and more specifically to recombinant adeno-associated virus (AAV) particles and their use for delivering transgenes in a multi-part (e.g., bipart) approach to treat or prevent diseases or disorders such as Stargardt disease.

[0005] In one embodiment, the present disclosure provides an AAV vector genome comprising, in the 5' to 3' direction, (a) a 5' AAV inverted terminal repeat, (b) an interphotoreceptor retinoid-binding protein (IRBP) enhancer, (c) an eye-specific promoter, (d) a 5' portion of a transgene, (e) a splice donor (SD) site, (f) a recombination site, (g) a polynucleotide encoding a recombinase, (h) a polyA site, and (i) a 3' AAV inverted terminal repeat. In the embodiment, the IRBP enhancer comprises or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 208.

[0006] In embodiments, the eye-specific promoter is the human rhodopsin kinase (hGRK1) promoter, the human rhodopsin (hRho) promoter, the human rod cGMP phosphodiesterase β-subunit promoter (βPDE) promoter, the human photoreceptor-retinoid-binding protein (IRBP) promoter, the human red / green opsin promoter, the human blue opsin (HB) promoter, the human vitiligo macular dystrophy 2 (VMD2) promoter, or the human RPE65 promoter. In embodiments, the eye-specific promoter is the human rod cGMP phosphodiesterase β-subunit promoter (βPDE) promoter, which contains or comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 200. In one embodiment, the eye-specific promoter is a human rhodopsin (hRho) promoter containing or comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 209. In another embodiment, the eye-specific promoter is a human rhodopsin kinase (hGRK1) promoter containing or comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 188.

[0007] In one embodiment, the disclosure provides an AAV vector genome comprising, in the 5' to 3' direction, (a) a 5' AAV inverted terminal repeat, (b) a human rhodopsin (hRho) promoter, (c) a 5' portion of a transgene, (d) a splice donor (SD) site, (e) a recombination site, (f) a polynucleotide encoding a recombinase, (g) a polyA site, and (h) a 3' AAV inverted terminal repeat. In embodiments, the human rhodopsin (hRho) promoter comprises or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 209. In embodiments, the AAV vector genome comprises an enhancer between the 5' AAV inverted terminal repeat and the human rhodopsin (hRho) promoter.

[0008] In one embodiment, the AAV vector genome includes a CBA-MVM hybrid sequence between the eye-specific promoter and the 5' portion of the transgene. In another embodiment, the CBA-MVM hybrid sequence includes or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 210.

[0009] In one embodiment, the present disclosure provides an AAV vector genome comprising, in the 5' to 3' direction, (a) a 5' AAV inverted terminal repeat, (b) a CMV enhancer, (c) a chicken beta-actin (CBA) promoter, (d) a 5' portion of a transgene, (e) a splice donor (SD) site, (f) a recombination site, (g) a polynucleotide encoding a recombinase, (h) a polyA site, and (i) a 3' AAV inverted terminal repeat. In the embodiment, the CMV enhancer comprises or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 218. In one embodiment, the CBA promoter includes or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 219. In another embodiment, the AAV vector genome includes a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 211 between the 5' AAV inverted terminal repeat and the 5' portion of the transgene.

[0010] In the embodiment, the 5' portion of the transgene is at least 0.5kb, at least 1kb, at least 1.5kb, at least 2kb, or at least 2.5kb in length. In the embodiment, the transgene encodes an ATP-binding cassette, subfamily A, member 4 (ABCA4) protein. In the embodiment, the ABCA4 protein contains or consists of a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 190. In the embodiment, the 5' portion of the transgene contains or consists of a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 212. In the embodiment, the 5' portion of the transgene encodes a polypeptide sequence containing amino acids 1-884 of the human ABCA4 protein (SEQ ID NO: 190). In the embodiment, the 5' portion of the transgene contains one or more of the following nucleotides: (i) nucleotide "G" at the position corresponding to nucleotide 813 of SEQ ID NO: 212, (ii) nucleotide "A" at the position corresponding to nucleotide 1311 of SEQ ID NO: 212, (iii) nucleotide "A" at the position corresponding to nucleotide 2274 of SEQ ID NO: 212, (iv) nucleotide "C" at the position corresponding to nucleotide 2277 of SEQ ID NO: 212, (v) nucleotide "T" at the position corresponding to nucleotide 2278 of SEQ ID NO: 212, and (vi) nucleotide "T" at the position corresponding to nucleotide 2568 of SEQ ID NO: 212. In the embodiment, the 5' portion of the transgene contains all of nucleotides (i) to (vi).

[0011] In the embodiment, the splice donor (SD) site includes or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with sequence number 186.

[0012] In the embodiment, the recombination site is a Lox71 site comprising or having sequence SEQ ID NO: 178, or a sequence having up to one, up to two, or up to three nucleotide mutations (deletions, insertions, and / or substitutions) thereto.

[0013] In this embodiment, the expression of the recombinase is operably linked to the promoter.

[0014] In one embodiment, the recombinase is Cre recombinase. In another embodiment, Cre recombinase contains a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 164. In another embodiment, the polynucleotide encoding the recombinase contains a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 214, or contains an intron consisting of such a sequence. In yet another embodiment, the polynucleotide encoding the recombinase contains a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 213.

[0015] In the embodiment, the recombinase includes or comprises an N-terminal nuclear localization sequence (NLS) consisting of SEQ ID NO: 165.

[0016] In the embodiment, the AAV vector genome includes a polynucleotide encoding an internally cleaved polypeptide located between the recombination site and the polynucleotide encoding the recombinase, wherein the 5' portion of the transgene, the polynucleotide encoding the internally cleaved polypeptide, and the polynucleotide encoding the recombinase are in the same reading frame. In the embodiment, the internally cleaved polypeptide is a self-cleaving peptide selected from the group consisting of T2A, P2A, E2A, and F2A. In the embodiment, the self-cleaving peptide is a P2A peptide containing or consisting of SEQ ID NO: 181.

[0017] In the embodiment, the polyA moiety includes or consists of sequence SEQ ID NO: 189, or a sequence having up to one, up to two, up to three, up to four, up to five, or up to six nucleotide mutations (deletions, insertions, and / or substitutions) therewith.

[0018] In the embodiment, the AAV vector genome includes an internal ribosome entry site (IRES) located between the recombination site and the polynucleotide encoding the recombinase, the IRES being operably ligated to the polynucleotide encoding the recombinase.

[0019] In one embodiment, the present disclosure provides an AAV vector genome comprising, in the 5' to 3' direction, (a) a 5' AAV inverted terminal repeat, (b) a Lox JTZ17 recombination site comprising, or consisting of, (b-1) sequence number 217 or up to one, up to two, or up to three nucleotide mutations (deletions, insertions, and / or substitutions) therefor, or (b-2) sequence number 179 or up to one, up to two, or up to three nucleotide mutations (deletions, insertions, and / or substitutions therefor, or consisting of, (c) a splice acceptor (SA) site, (d) a 3' portion of a transgene, (e) a poly-A site, and (f) a 3' AAV inverted terminal repeat. In the embodiment, the splice acceptor (SA) site contains or comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 187. In the embodiment, the transgene encodes the ABCA4 protein. In the embodiment, the 3' portion of the transgene contains or comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 193. In the embodiment, the polyA site contains or comprises a sequence having up to one, up to two, up to three, up to four, up to five, or up to six nucleotide mutations (deletions, insertions, and / or substitutions) with respect to SEQ ID NO: 189.

[0020] In one embodiment, the 5'AAV inverted terminal repeat contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with sequence number 215. In another embodiment, the 3'AAV inverted terminal repeat contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with sequence number 216.

[0021] In one aspect, the present disclosure provides AAV virus particles comprising (i) an AAV capsid comprising an AAV capsid protein and (ii) the AAV vector genome of the present disclosure. In embodiments, the AAV capsid protein comprises or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from SEQ ID NOs: 1-3, 67, 71, 196, 205, and 206. In embodiments, the AAV capsid protein comprises or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3. In embodiments, the AAV capsid protein comprises or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 67.

[0022] In one aspect, the present disclosure provides a pharmaceutical composition comprising the AAV vector genome of the present disclosure or the AAV virus particles of the present disclosure.

[0023] In one aspect, the present disclosure provides a pharmaceutical composition comprising (i) a first AAV virus particle comprising the first AAV vector genome of the present disclosure and (ii) a second AAV virus particle comprising the second AAV vector genome of the present disclosure.

[0024] In one aspect, the present disclosure provides a pharmaceutical composition comprising (i) a first AAV virus particle comprising the first AAV vector genome of the present disclosure and (ii) a second AAV virus particle comprising an AAV vector genome comprising, in the 5' to 3' direction, (a) a 5' AAV inverted terminal repeat, (b) a recombination site, (c) a splice acceptor (SA) site, (d) the 3' portion of the transgene, (e) a polyA site, and (f) a 3' AAV inverted terminal repeat.

[0025] In one embodiment, the present disclosure provides a method for expressing a protein encoded by a transgene in a cell, comprising: (1) transducing a cell with a first AAV virus particle containing a first AAV vector genome of the present disclosure; and (2) transducing a cell with a second AAV virus particle containing a second AAV vector genome of the present disclosure.

[0026] In one embodiment, the present disclosure provides a method for expressing a protein encoded by a transgene in a cell, comprising: (1) transducing a cell with a first AAV virus particle comprising a first AAV vector genome of the present disclosure; and (2) transducing a cell with a second AAV virus particle comprising an AAV vector genome comprising, in the 5' to 3' direction, (a) a 5' AAV inverted terminal repeat, (b) a recombination site, (c) a splice acceptor (SA) site, (d) a 3' portion of the transgene, (e) a poly-A site, and (f) a 3' AAV inverted terminal repeat.

[0027] In one embodiment, the present disclosure provides a method for treating a subject having a disease or disorder, comprising: (1) administering to the subject a first AAV virus particle comprising a first AAV vector genome of the present disclosure; and (2) administering to the subject a second AAV virus particle comprising a second AAV vector genome of the present disclosure.

[0028] In one embodiment, the present disclosure provides a method for treating a subject having a disease or disorder, comprising: (1) administering to the subject a first AAV virus particle comprising a first AAV vector genome of the present disclosure; and (2) administering to the subject a second AAV virus particle comprising an AAV vector genome comprising, in the 5' to 3' direction, (a) a 5' AAV inverted terminal repeat, (b) a recombination site, (c) a splice acceptor (SA) site, (d) a 3' portion of a transgene, (e) a poly-A site, and (f) a 3' AAV inverted terminal repeat.

[0029] In this embodiment, the recombination sites in the first AAV vector genome of the first AAV virus particle and the recombination sites in the AAV vector genome of the second AAV virus particle are configured to prevent reverse recombination.

[0030] In one embodiment, the AAV virus particle contains, or comprises an AAV capsid protein, a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs. 1-3, 67, 71, 196, 205, and 206. In another embodiment, the AAV capsid protein contains, or comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO. 3. In yet another embodiment, the AAV capsid protein contains, or comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO. 67.

[0031] In the embodiment, administration of the first AAV virus particle and the second AAV virus particle results in recombination of the first AAV vector genome and the second AAV vector genome via recombination sites in the AAV vector genome of the first AAV virus particle and the second AAV vector genome of the second AAV virus particle. In the embodiment, administration of the first AAV virus particle and the second AAV virus particle results in complete transgene expression.

[0032] In the embodiment, the protein encoded by the transgene is the ABCA4 protein. In the embodiment, the disease or disorder is Stargardt disease. In the embodiment, the disease or disorder is caused by ABCA4 gene deficiency. In the embodiment, ABCA4 gene deficiency results in one or more conditions selected from the group consisting of decreased expression of the ABCA4 protein, exclusion of ABCA4 protein expression, expression of a mutant ABCA4 protein, and decreased function of the ABCA4 protein.

[0033] In the embodiment, the first AAV virus particle and the second AAV virus particle are administered simultaneously. In the embodiment, the first AAV virus particle is administered before or after the second AAV virus particle. In the embodiment, the virus particles are administered by intravitreous, pararetinal, or subretinal injection. In the embodiment, the virus particles are administered by subretinal injection.

[0034] In the embodiment, stable expression of the recombinase is not detected in the transduced cells or subjects.

[0035] In one embodiment, the present disclosure provides a transdextrinsed cell comprising: (i) a genomic copy lacking an endogenous gene; and (ii) a recombinant nucleic acid comprising, in the 5' to 3' direction, (a) a 5'AAV inverted terminal repeat, (b) an interphotoreceptor retinoid-binding protein (IRBP) enhancer, (c) an eye-specific promoter, (d) a 5' portion of the transgene which is a working copy of the endogenous gene lacking the transgene, (e) a splice donor (SD) site, (f) a recombination site, (g) a splice acceptor (SA) site, (h) a 3' portion of the transgene, (i) a polyA site, and (j) a 3'AAV inverted terminal repeat.

[0036] In one embodiment, the present disclosure provides a transduction cell comprising: (i) a genomic copy lacking an endogenous gene; and (ii) a recombinant nucleic acid comprising, in the 5' to 3' direction, (a) a 5'AAV inverted terminal repeat; (b) optionally, an enhancer; (c) a human rhodopsin (hRho) promoter; (d) a 5' portion of the transgene, which is a working copy of the endogenous gene lacking the transgene; (e) a splice donor (SD) site; (f) a recombination site; (g) a splice acceptor (SA) site; (h) a 3' portion of the transgene; (i) a polyA site; and (j) a 3'AAV inverted terminal repeat.

[0037] In one embodiment, the present disclosure provides a transdextrinsed cell comprising: (i) a genomic copy lacking an endogenous gene; and (ii) a recombinant nucleic acid comprising, in the 5' to 3' direction, (a) a 5'AAV inverted terminal repeat, (b) a CMV enhancer, (c) a chicken beta-actin (CBA) promoter, (d) a 5' portion of the transgene which is a working copy of the endogenous gene lacking the transgene, (e) a splice donor (SD) site, (f) a recombination site, (g) a splice acceptor (SA) site, (h) a 3' portion of the transgene, (i) a poly-A site, and (j) a 3'AAV inverted terminal repeat.

[0038] In this embodiment, the deleted endogenous gene is the ABCA4 gene, and the transgene encodes the ABCA4 protein.

[0039] In the embodiment, the transduced cell or recombinant nucleic acid comprises a nucleic acid sequence in the 5'-3' direction, including a 5'AAV inverted terminal repeat, a recombination site, a polynucleotide encoding a recombinase, and a 3'AAV inverted terminal repeat, wherein the second recombinant nucleic acid sequence lacks a promoter. In the embodiment, the nucleic acid sequence is located downstream of the 3' end of (ii)(j)3'AAV inverted terminal repeat.

[0040] In the embodiment, the cells are ex vivo cells. In the embodiment, the cells are photoreceptor cells.

[0041] In one embodiment, the disclosure provides a nucleic acid comprising the 5' portion of the ABCA4 gene, wherein the 5' portion of ABCA4 comprises one or more of the following: (i) nucleotide "G" at the position corresponding to nucleotide 813 of SEQ ID NO: 212, (ii) nucleotide "A" at the position corresponding to nucleotide 1311 of SEQ ID NO: 212, (iii) nucleotide "A" at the position corresponding to nucleotide 2274 of SEQ ID NO: 212, (iv) nucleotide "C" at the position corresponding to nucleotide 2277 of SEQ ID NO: 212, (v) nucleotide "T" at the position corresponding to nucleotide 2278 of SEQ ID NO: 212, and (vi) nucleotide "T" at the position corresponding to nucleotide 2568 of SEQ ID NO: 212. In embodiments, the nucleic acid comprises two or more, three or more, four or more, five or more, or all six of nucleotides (i) to (vi). In embodiments, the 5' portion of the ABCA4 gene is at least 1 kb, at least 1.5 kb, at least 2 kb, or at least 2.5 kb in length. In the embodiment, the 5' portion of the ABCA4 gene contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 212. In the embodiment, the 5' portion of the transgene encodes a polypeptide sequence containing amino acids 1-884 of the human ABCA4 protein (SEQ ID NO: 190).

[0042] In one embodiment, the Disclosure provides an AAV vector genome comprising the nucleic acid of the Disclosure.

[0043] In one embodiment, the present disclosure provides an AAV virus particle comprising (i) an AAV capsid, the AAV capsid comprising an AAV capsid protein, and (ii) an AAV vector genome of the present disclosure.

[0044] In one embodiment, the Disclosure provides a pharmaceutical composition comprising the AAV vector genome or the AAV virus particles of the Disclosure.

[0045] In one embodiment, the present disclosure provides a method for treating a subject having a disease or disorder, comprising administering to the subject AAV virus particles of the present disclosure. In the embodiment, the disease or disorder is Stargardt disease.

[0046] In one embodiment, the present disclosure provides transduced cells comprising the nucleic acids of the present disclosure. [Brief explanation of the drawing]

[0047] [Figure 1A] Figure 1A shows an example AAV vector genome for multi-part gene delivery. "ITR" stands for "inverted terminal repeat," "SD" stands for "splice donor," "RS" stands for "recombination site," "IC" stands for "internal cleavage polypeptide," "Rec" stands for "recombinase," "NLS" stands for "nuclear localization sequence," "pA" stands for "polyA site," and "SA" stands for "splice acceptor." Figure 1B shows an example AAV vector genome for multi-part gene delivery of a ternarily divided transgene. "ITR" stands for "inverted terminal repeat," "SD" stands for "splice donor," "RS" stands for "recombination site," "IC" stands for "internal cleavage polypeptide," "Rec" stands for "recombinase," "NLS" stands for "nuclear localization sequence," "pA" stands for "polyA site," and "SA" stands for "splice acceptor." [Figure 1B]Figure 1A shows an example AAV vector genome for multi-part gene delivery. "ITR" stands for "inverted terminal repeat," "SD" stands for "splice donor," "RS" stands for "recombination site," "IC" stands for "internal cleavage polypeptide," "Rec" stands for "recombinase," "NLS" stands for "nuclear localization sequence," "pA" stands for "polyA site," and "SA" stands for "splice acceptor." Figure 1B shows an example AAV vector genome for multi-part gene delivery of a ternarily divided transgene. "ITR" stands for "inverted terminal repeat," "SD" stands for "splice donor," "RS" stands for "recombination site," "IC" stands for "internal cleavage polypeptide," "Rec" stands for "recombinase," "NLS" stands for "nuclear localization sequence," "pA" stands for "polyA site," and "SA" stands for "splice acceptor." [Figure 2] The ABCA4 construct used in tissue culture tests is shown. [Figure 3A] This study demonstrates the need for Cre recombinase for the expression of full-length ABCA4 protein mediated by recombination of a partially transfected ABCA4 construct in HEK293 cells. [Figure 3B] This shows the expression levels of recombinant full-length ABCA4 protein in HEK293 cells transfected with various ABCA4 constructs. [Figure 4] This shows a significant decrease in Cre recombinase expression in HEK293 cells after partial ABCA4 construct recombination. [Figure 5] This shows the expression levels of full-length ABCA4 mRNA in HEK293 cells transfected with various ABCA4 constructs. [Figure 6] This shows the time course of expression levels of full-length ABCA4 mRNA in cells transduced with the specified AAV9 virus particles. [Figure 7]A representative diagram of the bipartite delivery of the ABCA4 gene via an AAV vector genome is shown. A shows the construction of the first AAV vector genome (5'ABCA4+Cre), which includes the human GRK1 promoter, the 5' portion of the ABCA4 gene, the Cre recombinase gene, and other genomic elements. B shows the construction of the second AAV vector genome (3'ABCA4), which includes the 3' portion of the ABCA4 gene and other genomic elements. [Figure 8] Includes charts showing gene expression levels for mouse ABCA4 (mABCA4), human ABCA4 (hABCA4), and Cre recombinase. [Figure 9] Western blotting analysis of the protein expression levels of mABCA4, hABCA4, and Cre recombinase is shown. [Figure 10] This paper presents an in vivo animal study design for a novel bipartite AAV system. [Figure 11A] A schematic diagram of the vector design for the new two-part AAV system is shown. [Figure 11B] Cre-mediated recombination of the first and second AAV vector genomes is shown. [Figure 12A] The results of Western blotting analysis of hABCA4 protein expression in mouse retinal samples are shown. [Figure 12B] The results of Western blotting analysis of hABCA4 protein expression in mouse retinal samples are shown. [Figure 13A] The results of Western blotting analysis of Cre recombinase protein expression levels, hABCA4 protein expression levels, and Brn3a protein expression levels are shown. [Figure 13B] This shows a correlation analysis comparing Cre recombinase protein expression levels and hABCA4 protein expression levels. [Modes for carrying out the invention]

[0048] Several embodiments of this disclosure will be described in more detail below. However, aspects of this disclosure may be embodied in different forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided so as to make this disclosure complete and comprehensive, and to fully communicate the scope of the invention to those skilled in the art. The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit them.

[0049] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as those generally understood by those skilled in the art to which this invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having the meaning consistent with their meaning in the context of this application and related art, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0050] Unless otherwise indicated in the context, it is specifically intended that the various features of the present invention described herein may be used in any combination. Furthermore, this disclosure also intends that, in embodiments, any feature or combination of features described herein may be excluded or omitted.

[0051] Unless otherwise explicitly indicated, all embodiments, features, and terms are intended to include both the listed embodiments, features, or terms, as well as their biological equivalents.

[0052] Embedding by reference All references, articles, publications, patents, patent gazettes, and patent applications cited herein are incorporated in their entirety by reference for all purposes. However, no reference to any reference, article, publication, patent, patent gazette, or patent application cited herein should be construed as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of common general knowledge in any country worldwide.

[0053] definition The implementation of this technology will, unless otherwise specified, utilize conventional techniques within the technical scope of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology, and recombinant DNA. For example, see Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989), Current Protocols in Molecular Biology (FMAusubel, et al. eds., (1987)), the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, B.D. Hames and G.T. Taylor eds., (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (RI. Freshney, ed., (1987)).

[0054] When used in the description of this invention and the appended claims, the singular forms "a," "an," and "the" are intended to also include the plural forms unless the context explicitly indicates otherwise.

[0055] As used herein, the term “comprising” is intended to mean that a composition and method includes the enumerated elements (e.g., materials or steps) but does not exclude other elements. As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by.” As used herein, the transitional phrase “essentially consisting of” (and its grammatical variations) should be interpreted as encompassing the enumerated elements (e.g., materials or steps) and not substantially affecting the basic and novel characteristics of the enumerated embodiments. Therefore, as used herein, the term “essentially consisting of” should not be interpreted as equivalent to “comprising.” As used herein, the transitional phrase “consisting of” excludes any elements (e.g., materials or steps) not specified in the composition or method. Any enumeration of the term “comprising” or its equivalents herein is understood to encompass compositions and methods that are essentially consisting of and comprise the enumerated elements (e.g., materials or steps).

[0056] When referring to measurable values ​​such as quantities or concentrations and similars, the term “approximately” as used herein is intended to encompass a variation of ±10% of the specified quantity unless it is evident from the context that it is impossible to extend the boundary beyond a certain point (e.g., less than 0%).

[0057] When used to describe the selection of any component, range, dosage form, etc. disclosed herein, the terms “acceptable,” “effective,” or “sufficient” are intended to indicate that such component, range, dosage form, etc. is suitable for the purposes disclosed herein.

[0058] Furthermore, as used herein, “and / or” refers to and encompasses not only every possible combination of one or more of the listed items in question, but also, when interpreted as an alternative ("or"), the absence of any combination.

[0059] Unless specifically listed, the term “host cell” includes eukaryotic host cells, such as fungal cells, yeast cells, higher plant cells, insect cells, and mammalian cells. Non-limiting examples of eukaryotic host cells include monkeys, cattle, pigs, mice, rats, birds, reptiles, and humans, such as HEK293 cells and 293T cells.

[0060] As used herein, the term “isolated” refers to molecules, or biological or cellular materials, that are substantially free from other materials.

[0061] As used herein, the terms “nucleic acid sequence” and “polynucleotide” are used interchangeably to refer to polymeric forms of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. Therefore, the terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers consisting of, or essentially composed of, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or purine bases and pyrimidine bases or other native bases, chemically or biochemically modified bases, non-native bases, or derivatized nucleotide bases.

[0062] A “gene” refers to a polynucleotide containing at least one open reading frame (ORF) capable of coding for a specific polypeptide or protein. A “gene product,” or alternatively, a “gene expression product,” refers to the amino acid sequence (e.g., a peptide or polypeptide) produced when a gene is transcribed and translated.

[0063] As used herein, “expression” refers to the two-step process in which a polynucleotide is transcribed into mRNA, and / or the process in which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may also include the splicing of mRNA in eukaryotic cells.

[0064] "Transcriptionally regulated" is a term well understood in the art and indicates that the transcription of a polynucleotide sequence, typically a DNA sequence, depends on it being operably ligated to an element that contributes to the initiation of transcription or promotes transcription. "Operatably ligated" means that the polynucleotide is arranged in a manner that enables it to function within a cell. In some embodiments, the disclosure provides a promoter operably ligated to a downstream sequence.

[0065] When applied to polynucleotides, the term “coding” refers to a polynucleotide that, in its natural state or when manipulated by methods well known to those skilled in the art, can be transcribed to produce mRNA for a polypeptide and / or its fragments. The antisense strand is the complement of such nucleic acid, and the coding sequence can be inferred from it.

[0066] As used herein, the term “promoter” means a regulatory sequence, which is a region of polynucleotide sequence that controls the initiation and transcription rate of a coding sequence, such as a gene or transgene. Promoters may be, for example, constitutive, inductive, repressive, or tissue-specific. Promoters may contain genetic elements to which regulatory proteins and molecules, such as RNA polymerases and transcription factors, can bind. Non-limiting exemplary promoters include the human rhodopsin kinase (hGRK1) promoter, the human photoreceptor-retinoid-binding protein promoter (IRBP), the human red / green opsin promoter (pR2.1), the human blue opsin promoter (HB), the mouse opsin promoter (mOP), the mouse shortwave opsin promoter (mBP) (the shortwave opsin promoter may also be referred to as the S-opsin motor or blue opsin promoter), the human rhodopsin cGMP phosphodiesterase β-subunit promoter (βPDE), and the Rouss sarcoma virus (RSV) LTR promoter (optionally, the RSV enhancer). Examples include the cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, U6 promoter, H1 promoter, ubiquitous chicken β-actin hybrid (CBh) promoter, nuclear small RNA (U1a or U1b) promoter, MeCP2 promoter, MeP418 promoter, MeP426 promoter, minimal MeCP2 promoter, VMD2 promoter, mRho promoter, or elongation factor-1 alpha (EF1 alpha) promoter.

[0067] Additional, non-exclusive, exemplary promoters provided herein include, but are not limited to, EFla, Ubc, human β-actin, CAG, TRE, Ac5, polyhedrin, CaMKIIa, Gal1, TEF1, GDS, ADH1, Ubi, and α-1-antitrypsin (hAAT). It is known in the art that the nucleotide sequences of such promoters may be modified to increase or decrease the efficiency of mRNA transcription. See, for example, Gao et al. (2018) Mol. Ther.: Nucleic Acids 12:135-145 (modifying TATA box of 7SK, U6 and H1 promoters to abolish RNA polymerase III transcription and stimulate RNA polymerase II-dependent mRNA transcription). Synthetic promoters may be used for ubiquitous or tissue-specific expression. Furthermore, viral promoters (some of which are described above) may be useful in the methods disclosed herein, e.g., CMV, HIV, adenovirus, and AAV promoters. In embodiments, the promoter is used in conjunction with an enhancer to increase transcription efficiency. Non-limiting examples of enhancers include photoreceptor-retinoid-binding protein (IRBP) enhancers, RSV enhancers, or CMV enhancers.

[0068] An enhancer is a regulatory element that increases the expression of a target sequence. A “promoter / enhancer” is a polynucleotide containing a sequence that can provide both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. An enhancer / promoter may be “endogenous” or “exogenous” and may be “heterogeneous.” An “endogenous” enhancer / promoter is one that is naturally linked to a given gene in the genome. An “exogenous” or “heterogeneous” enhancer / promoter is one that is juxtaposed with a gene by its genetic engineering (i.e., molecular biological techniques) so that the transcription of that gene is directed by the linked enhancer / promoter. Non-limiting examples of linked enhancer / promoters for use in the methods, compositions, and constructs provided herein include the PDE promoter + IRBP enhancer or the CMV enhancer + U1a promoter. In the art, it is understood that enhancers can operate from a certain distance and regardless of their orientation relative to the location of an endogenous or heterologous promoter. Therefore, it is further understood that an enhancer operating at a certain distance from a promoter is therefore "operably coupled" to that promoter, regardless of its location within the vector or its orientation relative to the promoter's location.

[0069] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably and in their broadest sense to refer to compounds of two or more subunits of an amino acid, an amino acid analog, or a peptide mimetic. The subunits may be linked by peptide bonds. In other embodiments, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that constitute, essentially constitute, or can constitute a protein or peptide sequence. As used herein, the term “amino acid” refers to any natural and / or unnatural or synthetic amino acid, including glycine, as well as both D and L optical isomers, amino acid analogs, and peptide mimetic.

[0070] As used herein, the terms “signal peptide” or “signal polypeptide” typically refer to an amino acid sequence that is normally present at the N-terminus of a newly synthesized secretory polypeptide or membrane polypeptide or protein. This acts, for example, to direct a specific cellular site across the cell membrane into the cell membrane or into the nucleus. In embodiments, the signal peptide is removed after localization. Examples of signal peptides are well known in the art. Non-limiting examples are described in U.S. Patents 8,853,381, 5,958,736, and 8,795,965. In embodiments, the signal peptide may be an IDUA signal peptide.

[0071] The terms “equivalent” or “biological equivalent” are used synonymously when referring to a particular molecule, biological substance, or cellular substance, intended to have minimal homology while still maintaining the desired structure or functionality. Non-limiting examples of equivalent polypeptides include polypeptides having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity with a reference polypeptide (e.g., wild-type polypeptide), or polypeptides encoded by polynucleotides having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity with a reference polynucleotide (e.g., wild-type polynucleotide).

[0072] "Homologie," "identity," or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. The percentage of identity can be determined by comparing the positions within each sequence, which may be aligned for comparison purposes. If the positions within the compared sequences are occupied by the same base or amino acid, the molecules are identical at that position. The degree of identity between sequences is a function of the number of matching positions shared by the sequences. "Unrelated" or "non-homologous" sequences share less than 40% identity and less than 25% identity with any of the sequences in this disclosure. Alignment and sequence identity percentages may be determined for nucleic acid sequences or amino acid sequences by importing the nucleic acid sequences or amino acid sequences provided herein into and using the Basic Local Alignment Search Tool (BLAST). Unless otherwise stated, sequence alignment and identity calculations are performed by BLAST version 2.11.0 using default parameters.

[0073] As used herein with respect to the position(s) of an amino acid or nucleic acid, the term "corresponding to" refers to the position(s) in the first polypeptide / polynucleotide sequence that aligns with a given amino acid / nucleic acid in the reference polypeptide / polynucleotide sequence when the first polypeptide / polynucleotide sequence is aligned with the reference polypeptide / polynucleotide sequence.

[0074] As used herein, amino acid modifications may be substitutions, deletions, or insertions. Amino acid substitutions may be conservative or non-conservative. A conservative substitution (also called a conservative mutation, conservative substitution, or conservative variation) is the replacement of an amino acid in a protein that changes a given amino acid to a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, or size). As used herein, a "conservative variation" refers to the substitution of an amino acid residue by another biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another, or the substitution of one charged or polar residue with the other, for example, lysine by arginine, aspartic acid by glutamic acid, or asparagine by glutamine. Other examples of conservative substitutions include the substitution of alanine to serine, asparagine to glutamine or histidine, aspartic acid to glutamic acid, cysteine ​​to serine, glycine to proline, histidine to asparagine or glutamine, lysine to arginine, glutamine, or glutamate, phenylalanine to tyrosine, serine to threonine, threonine to serine, tryptophan to tyrosine, tyrosine to tryptophan or phenylalanine, and similar substitutions.

[0075] As used herein, the term “vector” means a nucleic acid containing, essentially consisting of, or comprising an intact replicon, which may be replicated, for example, by processes of transfection, infection, or transformation, once immobilized within a cell. Once inside a cell, vectors may be replicated as extrachromosomal (episome) elements or incorporated into the host cell chromosome, as is understood in the art. Vectors may include retroviruses, adenoviruses, herpesviruses, baculoviruses, modified baculoviruses, papovaviruses, AAV virus vectors, lentiviral vectors, adenovirus vectors, alphavirus vectors, or nucleic acids derived from otherwise modified viruses or naturally occurring viruses. Examples of non-viral vectors for delivering nucleic acids include naked DNA; DNA complexed with cationic lipids, DNA alone, or DNA combined with cationic polymers; anionic liposomes and cationic liposomes; DNA-protein complexes and particles containing, essentially consisting of, or comprising, heterologous polylysines, length-defined oligopeptides, and cationic polymers such as polyethyleneimine, which are sometimes contained within liposomes; and the use of triple complexes containing, essentially consisting of, or comprising viruses and polylysine-DNA.

[0076] Regarding general recombination techniques, vectors containing both a promoter and a cloning site into which polynucleotides can be operably ligated are well known in the art. Such vectors have the ability to transcribe RNA in vitro or in vivo and are commercially available from suppliers such as Agilent Technologies (Santa Clara, Calif) and Promega Biotech (Madison, Wis.). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the cloned transgene to remove excessive, potentially inappropriate alternative translation start codons or other sequences that may interfere with or reduce expression at either the transcriptional or translational level. Alternatively, a consensus ribosome binding site can be immediately inserted into the 5' start codon to enhance expression.

[0077] A "viral vector" is defined as a recombinant virus or viral particle containing polynucleotides that is delivered to a host cell either in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, AAV virus vectors, lentiviral vectors, adenovirus vectors, alphavirus vectors, and similar types.

[0078] As used herein, the terms “recombinant expression system” or “recombinant vector” refer to a genetic construct(s) formed by recombination for the expression of a particular genetic substance.

[0079] A “gene delivery vehicle” is defined as any molecule capable of delivering an inserted polynucleotide into a host cell. Examples of gene delivery vehicles include liposomes, micelle biocompatible polymers including natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metallic particles; bacteria; viruses such as baculoviruses, adeno-associated viruses, adenoviruses, and retroviruses; bacteriophages, cosmids, plasmids, and fungal vectors; and other recombinant vehicles that are typically used in the art described for expression in various eukaryotic and prokaryotic hosts and can be used for gene therapy and simple protein expression. Additionally, targeted antibodies or fragments thereof, or liposomes consisting of them, can be used in the manner disclosed herein. In addition to the delivery of polynucleotides to cells or cell populations, the direct introduction of the proteins described herein into cells or cell populations can be carried out by non-limiting techniques of protein transfection, and alternatively, cultivating conditions that can enhance and / or promote the expression and / or activity of the proteins disclosed herein are other non-limiting techniques.

[0080] The polynucleotides disclosed herein can be delivered to cells or tissues using gene delivery vehicles. “Genetic delivery,” “gene transfer,” “transduction,” and similar terms, as used herein, refer to the introduction of exogenous polynucleotides into host cells, regardless of the method used for introduction (sometimes also referred to as “transgenes”). Such methods include vector-mediated gene transfer (e.g., viral infection / transfection, or by various other protein-based or lipid-based gene delivery complexes), as well as various well-known techniques such as techniques that facilitate the delivery of “naked” polynucleotides (electroporation, “gene gun” delivery, and various other techniques used for the introduction of polynucleotides). The introduced polynucleotides may be maintained stably or transiently within the host cell. Stable maintenance typically requires that the introduced polynucleotides contain a replicative origin compatible with the host cell, or be incorporated into an extrachromosomal replicon (e.g., a plasmid) or a host cell replicon such as a nuclear or mitochondrial chromosome. Numerous vectors are known in the art and, as described herein, are capable of mediating the transfer of genes into mammalian cells.

[0081] A plasmid is a DNA molecule that is typically separate from chromosomal DNA and capable of replicating independently of chromosomal DNA. Often, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a microbial population, typically offering a selective advantage under given environmental conditions. Plasmids may carry genes that provide resistance to spontaneously occurring antibiotics in competing environmental niches, or alternatively, the produced protein may act as a toxin under similar conditions. While plasmid vectors often exist as extrachromosomal circular DNA molecules, they may be designed to integrate stably into host chromosomes in either a random or targeted manner, and such integration may be achieved using either circular plasmids or plasmids linearized before introduction into host cells, as is known in the art.

[0082] Plasmids used in genetic engineering are called plasmid vectors. Many plasmids are commercially available for this purpose. The gene to be replicated is inserted into multiple cloning sites (MCS, or polylinkers), which are short regions containing a copy of the plasmid that gives cells resistance to a particular antibiotic, and several commonly used restriction sites that allow for the easy insertion of DNA fragments at these sites. Another major use of plasmids is to produce large quantities of protein. In this case, researchers can grow bacteria or eukaryotic cells containing a plasmid carrying the target gene and induce them to produce large quantities of protein from the inserted gene.

[0083] As used herein, the terms “adeno-associated virus” or “AAV” refer to a class of viruses related to this name and belonging to the genus Dependoparvovirus and family Parvoviridae. Adeno-associated viruses are single-stranded DNA viruses that grow only in cells where certain functions are provided by co-infecting helper viruses. General information and reviews of AAVs can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). Since it is well known that various serotypes are structurally and functionally very closely related, even at the genetic level, it is quite expected that the same principles described in these reviews will also be applicable to additional AAV serotypes characterized after the publication date of the reviews. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, JR Pattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes exhibit very similar replication characteristics mediated by homologous rep genes, and all possess three related capsid proteins, such as those expressed in AAV2. The degree of relevance is further suggested by heteroduplex analysis revealing extensive cross-hybridization between serotypes along genome length, and by the presence of similar self-annealing segments at the terminals corresponding to "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that replication function in each serotype is under similar regulatory control. Multiple serotypes of this virus are known to be favored for gene delivery, and all known serotypes can infect cells from various tissue types. In the art, at least 11 consecutively numbered AAV serotypes are known.Non-limiting exemplary serotypes useful for the methods disclosed herein include any of 11 serotypes, e.g., AAV2, AAV8, AAV9, or any of the variant serotypes, e.g., AAV-DJ and AAV PHP.B. AAV particles typically contain three major viral proteins: VP1, VP2, and VP3. In embodiments, AAV refers to serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74, AAV 110, AAV 204, AAV 214, AAV 214A, AAV 214e, AAV 214e8, AAV 214e9, AAV 214e10, AAV ITB102_45, or AAV 214AB.

[0084] As used herein, “AAV vector” refers to a vector comprising one or more heterogeneous nucleic acid (HNA) sequences and one or more AAV inverted terminal repeat sequences (ITRs). Such AAV vectors can replicate when present in a host cell that provides functionality for rep and cap gene products, and can enable the ITRs and inter-ITR nucleic acids to be packaged in infectious viral particles. In embodiments, an AAV vector comprises a promoter, at least one nucleic acid capable of encoding at least one protein or RNA, and / or enhancers and / or terminators in adjacent ITRs that are packaged in infectious AAV particles. The ITRs and inter-ITR nucleic acids can be encapsidated into an AAV capsid, and this capsidated nucleic acid portion may be referred to as the “AAV vector genome.” In addition to the capsid portion, an AAV vector may contain elements, such as antibiotic resistance genes or other elements known in the art, which are contained in a plasmid for manufacturing purposes but are not packaged in AAV particles.

[0085] As used herein, the terms “viral capsid” or “capsid” refer to the proteinaceous shell or coat of a viral particle. The capsid functions to enclose, protect, transport, and / or release the viral genome into a host cell. Generally, a capsid consists of oligomeric structural subunits of a protein ("capsid proteins"). As used herein, the term “capsid-enclosed” means enclosed within a viral capsid. The viral capsid of AAV consists of a mixture of three viral capsid proteins: VP1, VP2, and VP3. The mixture of VP1, VP2, and VP3 contains 60 monomers arranged in a T=1 icosahedral symmetry with a ratio of 1:1:10 (VP1:VP2:VP3) or 1:1:20 (VP1:VP2:VP3), as described in Sonntag F et al., (June 2010) “A viral assembly factor promotes AAV2 capsid formation in the nucleolus”. Proceedings of the National Academy of Sciences of the United States of America. 107(22):10220-5, and Rabinowitz JE, Samulski RJ (December 2000). “Building a better vector: the manipulation of AAV virions”. Virology. 278(2):301-8, respectively.

[0086] "AAV virion," "AAV virus particle," "AAV virus vector," "AAV vector particle," or "AAV particle" refers to a viral particle composed of at least one AAV capsid protein and a capsid-formed AAV vector genome.

[0087] As used herein, the term “helper” with respect to a virus or plasmid refers to a virus or plasmid used to provide additional components necessary for the replication and packaging of any one of the AAV vector genomes disclosed herein. Components encoded by a helper virus or plasmid may include any genes required for virion assembly, capsid formation, genome replication, and / or packaging. For example, a helper virus or plasmid may encode enzymes or other factors necessary for the replication of the viral genome in a host cell. Non-limiting examples of helper viruses and plasmids suitable for use with AAV constructs include pHELP (plasmid), adenovirus (virus), or herpesvirus (virus).

[0088] As used herein, packaging cells (or helper cells) are cells used to produce a viral vector. The production of a recombinant AAV viral vector requires the Rep and Cap proteins provided in trans, as well as adenovirus-derived gene sequences that assist in AAV replication. In some embodiments, the packaging / helper cells contain plasmids that are stably incorporated into the cell's genome. In other embodiments, the packaging cells may be transiently transfected. Typically, the packaging cells are eukaryotic cells, such as mammalian cells or insect cells.

[0089] As used herein, a reporter protein is operably ligated to a promoter to control promoter expression (e.g., tissue specificity and / or). In some embodiments, the reporter protein may be operably ligated to a polypeptide. In some embodiments, the reporter protein may be used for monitoring DNA delivery methods, functional identification and characterization of promoter and enhancer elements, translation and transcription regulation, mRNA processing, and protein-protein interactions. Non-limiting examples of reporter proteins include β-galactosidase, fluorescent proteins such as green fluorescent protein (GFP) or red fluorescent protein (RFP), luciferase, glutathione S-transferase (GST), and maltose-binding protein (MBP).

[0090] A "pharmaceutical composition" comprises a combination of an active ingredient, such as a polypeptide, polynucleotide, antibody, or viral vector, and an inert or inactive support, such as a solid support, and is intended to be suitable for diagnostic or therapeutic use in vitro, in vivo, or ex vivo.

[0091] As used herein, the term “pharmaceutically acceptable carrier” includes any of the following: standard pharmaceutical carriers such as phosphate-buffered saline and water, as well as emulsions such as oil / water emulsions or water / oil emulsions, and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0092] The “subject” of diagnosis or treatment is a cell, or an animal such as a mammal, or a human. The subject includes non-human animals that are the subject of diagnosis or treatment, and non-human animals that are the subject of infection or animal models, including but not limited to monkeys, mice, rats, dogs, or rabbits, as well as other livestock, sporting animals, or pets. In embodiments, the subject is a human.

[0093] The term “tissue” is used herein to mean any tissue of a living or dead organism, or any tissue derived from or designed to mimic a living or dead organism. Tissues may be healthy or diseased, and may have genetic mutations. Biological tissue may include any single tissue (e.g., a collection of cells that can be interconnected) or a group of tissues that constitute an organ or part or region of the body of an organism. Tissues may include homogeneous cellular material, consist of essentially homogeneous cellular material, or consist of homogeneous cellular material, and may be complex structures, such as those found in regions of the body including the rib cage, which may include lung tissue, skeletal tissue, and / or muscle tissue. Exemplary tissues include, but are not limited to, those derived from the eye, liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidney, brain, bile duct tree, duodenum, abdominal aorta, iliac vein, heart, and intestine (including any combination thereof).

[0094] As used herein, “treating” a disease in a subject, or “treatment” of a disease, means (1) preventing the onset of symptoms or disease in a subject that is susceptible to the disease or does not yet show symptoms of the disease, (2) inhibiting the disease or halting its onset, or (3) causing improvement or regression of the disease or symptoms of the disease. As understood in the art, “treatment” is an approach to obtain a beneficial or desired outcome, including clinical outcomes. For the purposes of this art, beneficial or desired outcomes may include, but are not limited to, one or more of the following, whether detectable or undetectable: reduction or improvement of one or more symptoms, reduction of the severity of a condition (including disease), stabilization (i.e., no worsening) of a condition (including disease), delay or slowing of the progression of a condition (including disease), improvement or mitigation of the condition (including disease), and remission (whether partial or complete).

[0095] As used herein, the term “effective dose” is intended to mean an amount sufficient to achieve the desired effect. In the context of therapeutic or preventive uses, the effective dose will depend on the type and severity of the condition in question, as well as the characteristics of the individual subject, such as overall health, age, sex, weight, and tolerance to the pharmaceutical composition. In the context of gene therapy, in embodiments, the effective dose is an amount sufficient to bring about the partial or complete regaining of the function of a deficient gene in the subject. In embodiments, the effective dose of AAV virus particles is an amount sufficient to bring about gene expression in the subject. Those skilled in the art will be able to determine an appropriate dose in accordance with these and other factors.

[0096] In embodiments, the effective amount will depend on the size and nature of the application in question, as well as the nature and sensitivity of the target object and the method of use. A person skilled in the art will be able to determine the effective amount based on these and other considerations. Depending on the embodiment, the effective amount may consist of, essentially consist of, or be composed of one or more doses of the composition.

[0097] As used herein, the terms “administer” or “dosage” are intended to mean the delivery of a substance to an object such as an animal or a human. Administration can be carried out in a single dose, continuously or intermittently, throughout the course of treatment. Methods for determining the most effective means and dosage of administration are known to those skilled in the art, and single or multiple doses, which will vary depending not only on the composition used for treatment and the purpose of treatment, but also on the age, health, or sex of the object being treated, can be carried out in dose levels and patterns selected by the treating physician, or in the case of pets and other animals, by the treating veterinarian.

[0098] As used herein, the term “substantially retains” with respect to the function of a biological sequence (e.g., the promoter function of a promoter sequence) means retaining at least 70% of the function of a reference sequence, e.g., at least 80%, at least 90%, at least 95%, or at least 100% of the function of the reference sequence. For example, a promoter sequence may contain some mutations compared to a reference promoter sequence, but substantially retains the promoter function of the reference promoter sequence (i.e., retains at least 70%). Unless otherwise indicated, this disclosure includes sequences that have a certain level of identity (e.g., at least 90% identity) with a reference sequence disclosed herein (e.g., an enhancer sequence or a promoter sequence) and substantially retain the function of that reference sequence. Unless otherwise indicated, the function of a biological sequence can be measured using assays known in the art, e.g., the function of an enhancer or promoter sequence can be determined based on RNA transcription levels measured by quantitative RT-PCR.

[0099] Structure and Function of AAV AAV is a replication-deficient parvovirus with a single-stranded DNA genome approximately 4.7 kb long, containing two approximately 145-nucleotide inverted terminal repeats (ITRs). Multiple serotypes of AAV exist. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is available under GenBank accession number NC_002077, the complete genome of AAV-2 is available under GenBank accession number NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983), the complete genome of AAV-3 is available under GenBank accession number NC_001729, the complete genome of AAV-4 is available under GenBank accession number NC_001829, the genome of AAV-5 is available under GenBank accession number NC_006152, the complete genome of AAV-6 is available under GenBank accession number NC_001862, the complete genome of AAV-7 is available under GenBank accession number NC_006260, the complete genome of AAV-8 is available under GenBank accession number NC_006261, and the genome of AAV-9 is available under Gao et al. The genome for AAV-10 is available in al., J. Virol., 78:6381-6388 (2004), the genome for AAV-11 is available in Mol. Ther., 13(1):67-76 (2006), and the genome for AAV-11 is available in Virology, 330(2):375-383 (2004). The sequence for the AAV rh.74 genome is available in U.S. Patent No. 9,434,928, which is incorporated herein by reference in its entirety. U.S. Patent No. 9,434,928 also provides sequences for the capsid protein and the self-complementary genome. In one embodiment, the genome is a self-complementary genome. The AAV ITR contains cis-acting sequences that direct viral DNA replication (rep), capsidation / packaging, and integration into the host cell chromosome. Three AAV promoters (named p5, p19, and p40 based on their relative map locations) drive the expression of two AAV internal open reading frames that encode the rep and cap genes.Two rep promoters (p5 and p19), along with differential splicing of a single AAV intron (nucleotides 2107 and 2227), result in the generation of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. These rep proteins possess multiple enzymatic properties that ultimately contribute to the replication of the viral genome.

[0100] The cap gene is expressed from the p40 promoter and encodes three capsid proteins: VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three related capsid proteins. More specifically, after the transcription of a single mRNA into which each of the VP1, VP2, and VP3 proteins is translated, it can be spliced ​​in two different ways. That is, either a longer or shorter intron can be excised to form two mRNA pools (a 2.3kb mRNA pool and a 2.6kb mRNA pool). The longer intron is often preferred, and therefore the 2.3kb mRNA can be called the major splice variant. This form lacks the initial AUG codon that initiates the synthesis of the VP1 protein, resulting in a reduced overall level of VP1 protein synthesis. The initial AUG codon remaining in the major splice variant is the start codon for the VP3 protein. However, upstream of this codon within the same open reading frame, there is an ACG sequence (encoding threonine) surrounded by an optimal Kozak (translation start) context.This contributes to the low-level synthesis of the VP2 protein, which is actually the VP3 protein, similar to VP1 but with an additional N-terminal residue, and is incorporated herein by reference to: Becerra SP et al., (December 1985), “Direct mapping of adeno-associated virus capsid proteins B and C: a possible ACG initiation codon”, Proceedings of the National Academy of Sciences of the United States of America, 82(23):7919-23; Cassinotti P et al., (November 1988), “Organization of the adeno-associated virus (AAV) capsid gene: mapping of a minor spliced ​​mRNA coding for virus capsid protein 1”, Virology, 167(1):176-84; Muralidhar S et al., (January 1994), “Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation This is described in "codons: effects on regulation of synthesis and biological activity", Journal of Virology, 68(1):170-6, and Trempe JP, Carter BJ (September 1988), "Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein", Journal of Virology, 62(9):3356-63. A single consensus polyadenylation signal (poly-A site) is located at map position 95 of the AAV genome.The life cycle and genetics of AAVs are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992). Non-exclusive exemplary capsid proteins include AAV 110, AAV 204, AAV 214, AAV 214A, AAV 214e, AAV 214e8, AAV 214e9, AAV 214e10, AAV ITB102_45, AAV 214AB, AAV6, AAV8, AAV9, and AAV2 capsid proteins, as shown in Tables 1-3 of this disclosure. Additional non-exclusive exemplary capsid proteins include those disclosed in WO2016081811, which are incorporated entirely herein by reference, specifically the AAV capsid protein sequence.

[0101] Each VP1 protein contains a VP1 moiety, a VP2 moiety, and a VP3 moiety. The VP1 moiety is the N-terminal portion of the VP1 protein, which is unique to the VP1 protein. The VP2 moiety is the amino acid sequence present in the VP1 protein, which can also be found in the N-terminal portion of the VP2 protein. The VP3 moiety and the VP3 protein have the same sequence. The VP3 moiety is the C-terminal portion of the VP1 protein, which is shared with both the VP1 and VP2 proteins.

[0102] The VP3 protein can be further classified into individual variable surface regions I to IX (VR-I to IX). Each variable surface region (VR) contains, or may contain, a specific amino acid sequence, either alone or in combination with the specific amino acid sequence of each of the other VRs, which can confer a distinctive infectious phenotype (e.g., reduced antigenicity, improved transduction, and / or tissue-specific targeting compared to other AAV serotypes) to a particular serotype, as described in DiMatta et al., “Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9” J.Virol., Vol.86(12):6947-6958, June 2012, the contents of which are incorporated herein by reference in their entirety.

[0103] AAV possesses unique characteristics that make it attractive as a viral vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is non-cellular, and natural infection in humans and other animals is asymptomatic. Furthermore, AAV infects many mammalian cell types, enabling the potential to target many different tissues in vivo. Additionally, AAV can transduce slow-dividing and non-dividing cells and persist essentially throughout the lifespan of those cells as a transcriptionally active nuclear episome (extrachromosomal factor). Moreover, because signals directing AAV replication and genomic capsidation are contained within the ITR of the AAV genome, a portion or all of approximately 4.3 kb of the genome's interior (rep-cap, encoding the replication and structural capsid proteins) can be replaced with foreign DNA to generate the AAV vector genome. The rep and cap proteins may be supplied in trans. Another important characteristic of AAV is that it is an extremely stable and robust virus. This is because AAV readily withstands the conditions used to inactivate adenoviruses (56°C to 65°C for several hours), making low-temperature storage of AAV less important. AAV can even be freeze-dried. Finally, AAV-infected cells are not resistant to co-infection.

[0104] Multiple studies have demonstrated long-term (over 1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther, 8:659-669 (1997), Kessler et al., Proc Nat. Acad Sc. USA, 93:14082-14087 (1996), and Xiao et al., J Virol, 70:8098-8108 (1996). Also see Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). Furthermore, as described by Herzog et al., Proc Natl Acad Sci USA, 94:5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94:13921-13926 (1997), muscle is highly angiogenic, so recombinant AAV transduction allows the transgene product to appear in the systemic circulation after intramuscular injection. In addition, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the cellular factors necessary for correct antibody glycosylation, folding, and secretion, showing that muscle can stably express secreted protein therapeutics. The recombinant AAV (rAAV) genome of the present invention comprises, essentially consists of, or consists of, a nucleic acid molecule encoding a therapeutic protein or a portion thereof (e.g., ABCA4) and one or more AAV ITRs adjacent to the nucleic acid molecule. The AAV DNA in the rAAV genome may originate from any AAV serotype capable of inducing recombinant viruses, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV PHP.B, and AAV rh74. The generation of pseudotype rAAV is disclosed, for example, in WO2001 / 083692. Other types of rAAV variants, such as rAAV with capsid mutations, are also being considered.For example, see Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). Nucleotide sequences of various AAV serotype genomes are known in the art.

[0105] AAV vector particles, capsid proteins, and AAV vectors This specification provides AAV vector particles, AAV vectors, and capsid proteins useful for delivering a variety of therapeutic payloads, including nucleic acids and proteins useful for treating diseases. The methods and compositions disclosed herein may be used to deliver transgenes larger than the typical packaging capacity of a single AAV vector genome, approximately 4.7 kb. Delivery of larger transgenes may be achieved by delivering portions of the transgene in two or more AAV vector particles and recombining those portions in the cell to provide the completed transgene (e.g., as shown in Figures 1A and 1B). In the novel preferred approach described herein, portions of the transgene are joined by Cre-lox-mediated recombination. Advantageously, the design of the AAV vector genome ensures that the 5' portion of the transgene is located upstream of the 3' portion of the transgene after recombination. The recombination site may also indicate the direction of the recombination event (e.g., to reduce or eliminate reverse recombination). A further benefit is that the recombination event may remove the promoter operably linked to the expression of the Cre recombinase protein, leaving behind a promoter-less vector region encoding Cre recombinase, thereby potentially reducing or preventing further expression of Cre recombinase in host cells.

[0106] Further descriptions of multipart (e.g., bipart) AAV delivery systems can be found, for example, in International Publication No. WO2022 / 015788, the entirety of which is incorporated by reference.

[0107] AAV Capsid Protein This disclosure provides AAV particles and AAV vector genomes for multi-part (e.g., bipart) delivery of large genes. In embodiments, two AAV particles are used for bipartite delivery, with the first AAV particle containing the 5' portion of the gene and the second AAV particle containing the 3' portion of the gene. The first and second AAV particles may have the same capsid protein or different capsid proteins.

[0108] In the embodiments, the VP1 capsid protein includes a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that are mutated, deleted, or added to any one of the amino acid sequences listed in Table 1. In the embodiments, up to 15, 20, 30, or 40 amino acids may be mutated, deleted, or added compared to these sequences. In the embodiments, the VP1 capsid protein is encoded by a sequence having up to 5, 10, 30, or 60 nucleotide changes compared to any one of the nucleic acid sequences listed in Table 1.

[0109] [Table 1]

[0110] In embodiments, the AAV VP1 protein comprises, essentially comprises, or comprises the amino acid sequences of SEQ ID NOs: 1-3, 30-34, 49, 63, 67, 71, 84, or 196, or sequences having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different amino acids from SEQ ID NOs: 1-3, 30-34, 49, 63, 67, 71, 84, or 196. Polynucleotides encoding these VP1 proteins are also provided. In embodiments, the polynucleotides encoding the VP1 protein comprises, essentially comprises, or comprises the sequences of SEQ ID NOs: 15, 18-23, 47, 66, 70, 82, 98, or 197, or sequences having up to 5, up to 10, or up to 30 nucleotide changes from SEQ ID NOs: 15, 18-23, 47, 66, 70, 82, 98, or 197.

[0111] In some embodiments, the AAV capsid protein is AAV-110 capsid protein (SEQ ID NO: 1), AAV204 capsid protein (SEQ ID NO: 2), AAV214 capsid protein (SEQ ID NO: 3), or AAV ITB102_45 capsid protein (SEQ ID NO: 49). In some embodiments, the AAV capsid protein is a variant of the AAV214 capsid protein. In some embodiments, the AAV capsid protein is AAV214A (SEQ ID NO: 30), AAV-214-AB (SEQ ID NO: 84), AAV214e (SEQ ID NO: 31), AAV214e8 (SEQ ID NO: 32), AAV214e9 (SEQ ID NO: 33), or AAV214e10 (SEQ ID NO: 34). In some embodiments, the AAV capsid protein is AAV9 capsid protein (SEQ ID NO: 71).

[0112] Exemplary sequences of the VP2 and VP3 proteins are provided in Tables 2 and 3. Given the VP2 and VP3 sequences, the VP1 portion can be determined by alignment with the complete VP1 protein sequence.

[0113] [Table 2]

[0114] Exemplary nucleic acids of other capsid VP2 moieties may originate from the corresponding moieties of VP1 capsid protein nucleic acids.

[0115] [Table 3]

[0116] The VP3 proteins AAV214, AAV214e, AAV214e8, AAV214e9, and AAV214e10 have the same amino acid (SEQ ID NO: 41) and nucleic acid (SEQ ID NO: 24) sequences.

[0117] In embodiments, the AAV VP2 protein comprises, essentially comprises, or comprises one of the amino acid sequences from SEQ ID NOs. 35-40, 50, or 85, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different amino acids from SEQ ID NOs. 35-40, 50, or 85. Polynucleotides encoding these VP2 proteins are also provided. In embodiments, the polynucleotide encoding the VP2 protein comprises, essentially comprises, or comprises the sequence of SEQ ID NOs. 47, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes from SEQ ID NOs. 47.

[0118] In embodiments, the AAV VP3 protein comprises, essentially comprises, or comprises the amino acid sequence of SEQ ID NOs: 17, 41-46, 51, or 86, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different amino acids from SEQ ID NOs: 17, 41-46, 51, or 86. Polynucleotides encoding these VP3 proteins are also provided. In embodiments, the polynucleotide encoding the VP3 protein comprises, essentially comprises, or comprises one of SEQ ID NOs: 16, 24-29, 48, and 83, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes from one of SEQ ID NOs: 16, 24-29, 48, and 83.

[0119] In embodiments, the AAV capsid protein is a chimeric protein. In embodiments, the VP1, VP2, or VP3 portion of the AAV capsid protein disclosed herein may be replaced with the VP1, VP2, or VP3 portion from a different AAV capsid protein disclosed herein.

[0120] In some embodiments, an AAV capsid protein is provided herein that contains a leucine residue at amino acid position 129, an asparagine residue at amino acid position 586, and a glutamic acid residue at amino acid position 723, where the amino acid positions in the AAV capsid protein are numbered relative to the amino acid positions in the amino acid sequence of SEQ ID NO: 2. In some cases, the protein contains the amino acid sequence of SEQ ID NO: 2. In other cases, these amino acids may be introduced into other capsid proteins.

[0121] In embodiments, an AAV VP1 capsid protein comprising a VP1 subunit, a VP2 subunit, and a VP3 subunit is provided herein, wherein the VP1 subunit comprises a leucine (L) residue at amino acid position 129, the VP2 subunit comprises a threonine (T) or asparagine (N) residue at amino acid position 157 and a lysine (K) or serine (S) residue at amino acid position 162, and the VP3 subunit comprises an asparagine (N) residue at amino acid position 223, an alanine (A) residue at amino acid position 224, a histidine (H) residue at amino acid position 272, a threonine (T) residue at amino acid position 410, a histidine (H) residue at amino acid position 724 and a proline (P) residue at amino acid position 734, and the amino acid positions in the AAV capsid protein are numbered relative to the amino acid positions in the amino acid sequence of Sequence ID No. 3 (i.e., VP1 capsid subunit numbering).

[0122] In the embodiment, the VP1 portion further contains an aspartic acid (D) residue or an alanine (A) residue at amino acid position 24, and the amino acid positions in the AAV capsid protein are numbered relative to the amino acid positions in the amino acid sequence of SEQ ID NO: 3. In the embodiment, the VP2 portion further contains one or more of the following: (i) a proline (P) residue at amino acid position 148, (ii) an inserted arginine (R) residue at amino acid position 152, (iii) an arginine (R) residue at amino acid position 168, (iv) an isoleucine (I) residue at amino acid position 189, and (v) a serine (S) residue at amino acid position 200, and the amino acid positions in the AAV capsid protein are numbered relative to the amino acid positions in the amino acid sequence of SEQ ID NO: 3.

[0123] In embodiments, one or more variable regions I-IX in the disclosed VP3 partial capsid protein may be removed and replaced with alternative regions. Preferred alternatives are identified in Table 4 below. These locations, as well as the identity of additional alternatives, may be identified by alignment with SEQ ID NO: 41. In embodiments, one or more VRs may have insertions of one, two, or three amino acids. In embodiments, one or more VRs may have deletions of one, two, or three amino acids.

[0124] [Table 4]

[0125] This disclosure provides nucleic acids encoding any one of the AAV capsid proteins disclosed herein. This disclosure also provides vectors comprising any one of the nucleic acids disclosed herein. The AAV may be AAV2, AAV8, or AAV9 serotype.

[0126] AAV Vector An AAV vector supplies a nucleic acid to be capsided onto an AAV vector particle containing elements involved in regulating nucleic acid expression in a target, as well as an ITR to facilitate capsidation. In embodiments, the AAV vector comprises the AAV vector genome of this disclosure. In embodiments, the AAV vector disclosed herein comprises at least one heterologous nucleic acid (HNA) sequence or a portion thereof, which is expressed in a target cell and, when recombined with the remainder of the HNA sequence, encodes a transgene that treats a disease or disorder. Thus, the HNA sequence comprises the transgene or a portion thereof. In embodiments, the AAV vector comprises at least one ITR sequence and at least one transgene or a portion thereof. In embodiments, the AAV vector comprises at least one ITR sequence and a portion of one transgene. In embodiments, the transgene encodes a therapeutic protein or therapeutic RNA.

[0127] In embodiments, the regulation of transgene expression in host cells can be controlled by regulatory elements contained within the AAV vector, including a promoter sequence and a polyadenylation signal. In embodiments, the AAV vector also encodes a signal peptide. In embodiments, the AAV vector has a 5' inverted terminal repeat (ITR) and a 3' ITR. In embodiments, the 5' ITR and 3' ITR have the same sequence. In embodiments, they have different sequences.

[0128] In one embodiment, the 5'AAV inverted terminal repeat comprises or consists of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with sequence number 215.

[0129] In this embodiment, the 3'AAV inverted terminal repeat comprises or consists of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with sequence number 216.

[0130] This disclosure provides a method for expressing transgenes encoded by genes exceeding a size limit. The size limit is determined by the package capacity of the viral particle carrying the heterologous nucleic acid sequence, and for most AAV viral vectors, the size limit of the capsid-forming vector genome is typically about 4.7 kb. This disclosure provides an approach using AAV viral particles to deliver to host cells and express transgenes up to approximately 8 kb, 7.9 kb, 7.8 kb, 7.7 kb, 7.6 kb, 7.5 kb, or 7.4 kb. Suitable transgene lengths for a bipartite gene delivery approach are approximately 4.0 kb to approximately 8.0 kb, for example, approximately 4.1 kb to approximately 7.9 kb, approximately 4.2 kb to approximately 7.8 kb, approximately 4.3 kb to approximately 7.7 kb, approximately 4.4 kb to approximately 7.7 kb, approximately 4.5 kb to approximately 7.7 kb, or approximately 4.6 kb to approximately 7.7 kb. In embodiments, the disclosure provides a multipartite gene delivery approach for delivering a transgene to a host cell and expressing a transgene of up to approximately 8 kb, approximately 9 kb, approximately 10 kb, approximately 11 kb, approximately 12 kb, approximately 13 kb, approximately 14 kb, approximately 15 kb, approximately 16 kb, approximately 17 kb, approximately 18 kb, approximately 19 kb, approximately 20 kb, or any value in between.

[0131] Conventional approaches to delivering transgenes that exceed the size limitations of the AAV system involve using homologous recombination mediated by the host cell's own cellular mechanisms. However, the efficiency of homologous recombination varies greatly between organisms and cell types, and the endogenous homologous recombination process is prone to errors.

[0132] The novel method described herein mediates efficient and precise recombination between AAV vector genomes encoding different portions of a transgene using an exogenous recombinase, thereby overcoming problems associated with endogenous homologous recombination. In embodiments, the exogenous recombinase is Cre recombinase. Cre recombinase belongs to the integrase family of site-specific recombinases. It catalyzes recombination between two recognition sites called Lox sites. The Lox site is typically a 34 bp polynucleotide sequence consisting of an 8 bp core spacer region and two 13 bp adjacent sequences (recognition regions). The asymmetric core sequence defines orientation to the Lox site. By using the Cre recombination approach method disclosed herein, we have achieved highly efficient recombination regardless of the cellular environment and in the absence of additional cofactors. Other exogenous recombinases, such as yeast flippase (Flp), may be used.

[0133] The multipart viral delivery system of this disclosure uses an exogenous recombinase (e.g., Cre recombinase) to mediate the recombination and integration of different portions of a target transgene via specific recombination sites in the AAV vector genome delivered to host cells within the viral vector. In embodiments, three or more portions of a target transgene can be delivered using the multipart viral delivery system and the same strategy can be used to form a complete transgene in host cells.

[0134] Figure 1A illustrates an exemplary approach for delivering a transgene to a host cell and expressing a transgene that exceeds the size limitations of a single AAV vector genome. The transgene is divided into two parts: a 5' portion and a 3' portion. The 5' portion is integrated into the first AAV vector genome, and the 3' portion is integrated into the second AAV vector genome. The first AAV vector genome also contains an HNA sequence encoding a recombinase (Rec), e.g., Cre recombinase. Once expressed in cells, the recombinase mediates recombination between the first and second AAV vector genomes via recombination sites (RS) in both genomes. After recombination, the recombinant nucleic acid, upon transcription, contains both the 5' and 3' portions of the transgene, which, due to the presence of splice donor (SD) and splice acceptor (SA) sites, can be bound together during RNA splicing, resulting in the expression of the full-length transgene.

[0135] The first AAV vector genome is oriented from 5' to 3'. a.5'AAV inverted terminal repeat (ITR), b. Promoter, c. The 5' portion of the introduced gene, d. Splice donor (SD) site, e. Recombination site (RS), f. Optionally, a nucleic acid sequence encoding an internal cleavage (IC) polypeptide or an internal ribosome entry site (IRES), g. A nucleic acid sequence encoding a recombinase, optionally containing a nuclear localization sequence (NLS). h. Poly-A portion (pA), and i.3'AAV inverted terminal repeat may be included.

[0136] Typically, there is no stop codon between the 5' portion of the transgene and the recombinase nucleic acid; that is, the HNA region containing the 5' portion of the transgene and the gene encoding the recombinase (which optionally has a nuclear localization sequence) form a contiguous ORF (i.e., they are in the same reading frame).

[0137] In this embodiment, the first AAV vector genome is oriented from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Enhancers (e.g., photoreceptor-retinoid-binding protein (IRBP) promoters), (c) Promoter (e.g., eye-specific promoter), (d) The 5' portion of the transgene (for example, a transgene encoding the ABCA4 protein), (e) Splice donor (SD) site, (f) Recombination site, (g) Polynucleotide encoding a recombinase, (h) Poly A portion, and (i) Includes 3'AAV inverted terminal repeat.

[0138] In this embodiment, the first AAV vector genome is oriented from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Human rhodopsin (hRho) promoter, (c) The 5' portion of the transgene (for example, a transgene encoding the ABCA4 protein), (d) Splice donor (SD) site, (e) Recombination site, (f) Polynucleotide encoding a recombinase, (g) PolyA portion, and (h) Includes 3'AAV inverted terminal repeat. In the embodiment, the first AAV vector genome includes an enhancer (e.g., an interphotoreceptor retinoid-binding protein (IRBP) promoter) between the 5' AAV inverted terminal repeat and the human rhodopsin (hRho) promoter.

[0139] In this embodiment, the first AAV vector genome is oriented from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) CMV enhancer, (c) Promoter (e.g., chicken beta-actin (CBA) promoter), (d) The 5' portion of the transgene (for example, a transgene encoding the ABCA4 protein), (e) Splice donor (SD) site, (f) Recombination site, (g) Polynucleotide encoding a recombinase, (h) Poly A portion, and (i) Includes 3'AAV inverted terminal repeat.

[0140] The second AAV vector genome is oriented from 5' to 3'. a. 5'AAV inverted terminal repetition, b. Recombination site, c. Splice acceptor (SA) site, d. The 3' portion of the transgene ORF, e. Poly A area, This may include f. and 3'AAV inverted terminal repeats. In the embodiment, the recombination site of the second AAV vector genome is the Lox 66 recombination site. In the embodiment, the Lox 66 recombination site contains or consists of a sequence having up to one, up to two, up to three, up to four, up to five, or up to six nucleotide mutations (deletions, insertions, and / or substitutions) of SEQ ID NO: 179. In the embodiment, the recombination site of the second AAV vector genome is the Lox JTZ17 recombination site. In the embodiment, the Lox JTZ17 recombination site contains or consists of a sequence having up to one, up to two, up to three, up to four, up to five, or up to six nucleotide mutations (deletions, insertions, and / or substitutions) of SEQ ID NO: 217. In the embodiment, the mutated recombination site sequence(s) substantially retains the recombination function of the reference sequence (e.g., SEQ ID NO: 179 or 217). In the embodiment, the mutated recombinant site sequence(s) retain at least 80%, at least 90%, at least 95%, or at least 100% of the recombinant function of the reference sequence(essentially sequence number 179 or 217).

[0141] Elements of the first and second AAV vector genomes promote the production of full-length transgenes. When both the first and second AAV vector genomes are present in the same cell, the recombinase is expressed under the control of the promoter in the first AAV vector genome. The recombinase mediates recombination between the first and second AAV vector genomes via recombination sites in the first and second AAV vector genomes, resulting in the formation of new recombinant nucleic acids. The recombinant nucleic acid contains both the 5' and 3' portions of the transgene, separated by splice donor and acceptor sites. After transcription and RNA maturation, full-size mRNA is produced from the first recombinant nucleic acid.

[0142] Three or more portions of a transgene can be delivered to host cells using a multipart viral delivery system and recombined to enable complete transgene expression. An exemplary approach for delivering three portions of a transgene is shown in Figure 1B, and the same strategy can be used to deliver four or more portions of the transgene. Briefly, in Figure 1B, the first portion of the transgene is incorporated into the first AAV vector genome, the second portion into the second AAV vector genome, and the third portion into the third AAV vector genome. The AAV vector genomes are then capsided to produce AAV vector particles that are delivered to host cells and introduce the AAV vector genome. Similar to the design in Figure 1A, the first AAV vector genome in Figure 1B includes a promoter, a first splice donor (SD1) site, a recombination site RS1, optionally a nucleic acid sequence encoding an internal cleavage (IC) polypeptide or internal ribosome entry site (IRES), optionally a nucleic acid sequence encoding a recombinase (Rec) with a nuclear localization sequence (NLS), and a poly-A site (pA). The second AAV vector genome also includes a recombination site RS1', a first splice acceptor (SA1) site, a second splice donor (SD2) site, and a recombination site RS2. The third AAV vector genome includes a recombination site RS2', a second splice acceptor (SA2) site, and a poly-A site.

[0143] When three AAV vector genomes are delivered to the same host cell, the expression of a recombinase (e.g., Cre recombinase) mediates recombination between the RS1 and RS1' sites and between the RS2 and RS2' sites. In embodiments, the RS1 and RS1' sites are orthogonal to the RS2 and RS2' sites (i.e., minimal or no recombination between RS1 / RS1' and RS2 / RS2'). As a non-limiting example, if the recombinase is Cre recombinase, RS1 and RS1' can be LoxP sites, and RS2 and RS2' can be LoxN sites. After recombination, an HNA containing all three parts of the transgene is formed, and its precursor mRNA transcript can undergo RNA splicing mediated by SD1 / SA1 splicing pairs and SD2 / SA2 splicing pairs to form a mature mRNA transcript containing the integrated transgene but not any of the recombination sites. In this embodiment, the SD1 / SA1 splicing pairs and the SD2 / SA2 splicing pairs are orthogonal to each other (i.e., no splicing occurs between SD1 / SA2 pairs or between SD2 / SA1 pairs), thereby ensuring that all parts of the transgene are retained in their correct positions within the mature mRNA transcript.

[0144] In the embodiment, the recombinase is cleaved from the N-terminal region of the protein via an internal cleavage polypeptide. In the embodiment, the expression of the recombinase may be driven by an internal ribosome entry site (IRES).

[0145] The recombinant nucleic acid may include, in the 5' to 3' direction, a 5' AAV inverted terminal repeat (ITR), a promoter, the 5' portion of the transgene, a splice donor (SD) site, a recombination site (RS) formed by the recombination process, a splice acceptor (SA) site, the 3' portion of the transgene, a poly(A) site (pA), and a 3' AAV inverted terminal repeat. The promoter drives the efficient transcription of the HNA sequence containing the transgene, and the 5' and 3' portions of the transgene are joined together after splicing the RNA transcript via the splice donor and acceptor sites, thereby resulting in the formation of a full-length transgene mRNA transcript.

[0146] In the embodiment, the gene encoding the recombinase located in the recombinant nucleic acid cannot be efficiently transcribed due to the lack of a promoter. As a result, in the embodiment, stable expression of the recombinase is little to no detectable in cells transduced with both the first and second AAV vector genomes.

[0147] The recombinase may be Cre recombinase. Cre recombinase may contain, essentially consist of, or otherwise consist of the amino acid sequence of SEQ ID NO: 164, or may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity with SEQ ID NO: 164. In embodiments, Cre recombinase may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 different amino acids from SEQ ID NO: 164. In embodiments, Cre recombinase substantially retains the recombinase function of SEQ ID NO: 164. In embodiments, Cre recombinase retains at least 80%, at least 90%, at least 95%, or at least 100% of the recombinase function of SEQ ID NO: 164.

[0148] Cre recombinase may be fused to a nuclear localization sequence (NLS). The NLS may be located at the N-terminus, C-terminus, or in the center of the Cre recombinase. The NLS may have the amino acid sequence of PKKKRKV (SEQ ID NO: 165), or up to one, two, or three amino acid mutations relative to SEQ ID NO: 165.

[0149] Cre recombinases having NLS may contain, essentially consist of, or be essentially the amino acid sequence of SEQ ID NO: 166, or may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity with SEQ ID NO: 166. In embodiments, the polynucleotide encoding Cre recombinase having NLS may contain, essentially consist of, or be essentially the same as, a sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% identity with SEQ ID NO: 167.

[0150] In embodiments, the polynucleotide encoding the recombinase (e.g., Cre recombinase) includes an intron. In embodiments, the intron includes or consists of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 214.

[0151] In one embodiment, the polynucleotide encoding Cre recombinase (and associated introns) comprises or consists of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 213.

[0152] The recombination site recognized by Cre recombinase may contain one or more LoxP (X-over P1 locus) sequences. Examples of LoxP sequences are listed in Table 5 below. A typical LoxP sequence is 34 bp long and contains an asymmetric 8 bp spacer region between two sets of 13 bp recognition regions. In embodiments, the LoxP sequence contains a spacer region having the nucleotide sequence ATGTATGC. The pairing of the recombination sites (one in the first AAV vector genome and the other in the second AAV vector genome) may promote unidirectional recombination and / or prevent reverse recombination.

[0153] The recombination site recognized by Cre recombinase may contain, be essentially, or may contain one or more variant Lox sites containing spacer region sequences different from the standard sequence (ATGTATGC). In embodiments, the variant Lox site is orthogonal to the LoxP site (i.e., no cross-recombination between the variant Lox site and the LoxP site). In embodiments, the Lox site is the loxN site containing the spacer region sequence of GTATACCT. In embodiments, the Lox site is the lox2272 site containing the spacer region sequence of AAGTATCC. In embodiments, the Lox site is the lox511 site containing the spacer region sequence of ATGTATAC. In embodiments, two or more Lox sites containing different spacer region sequences are used, and these Lox sites are orthogonal to each other. In embodiments, the Lox site undergoes recombination with Lox sites containing the same spacer region sequence, but without cross-compatibility. Further discussions regarding orthogonal Lox sites can be found in Livet et al. Nature. 2007 Nov 1,450(7166):56-62 and Missirlis et al. BMC Genomics, 2006 Apr 4,7:73, respectively, which are incorporated herein by reference in their entirety. Exemplary variant Lox site sequences are provided in Table 5 below.

[0154] [Table 5]

[0155] In embodiments, the recombination site in the first AAV vector genome may, essentially be, or may be, a Lox71 sequence, e.g., SEQ ID NO: 178, or a sequence having up to one, up to two, up to three, up to four, up to five, or up to six nucleotide mutations (deletions, insertions, and / or substitutions) thereto. In embodiments, the mutated Lox71 sequence substantially retains the recombination function of SEQ ID NO: 178. In embodiments, the mutated Lox71 sequence retains at least 80%, at least 90%, at least 95%, or at least 100% of the recombination function of SEQ ID NO: 178.

[0156] In embodiments, the recombination site in the second AAV vector genome may, essentially be, or may be, a Lox JTZ17 sequence, e.g., SEQ ID NO: 217, or a sequence having up to one, up to two, up to three, up to four, up to five, or up to six nucleotide mutations (deletions, insertions, and / or substitutions) thereto. In embodiments, the mutated Lox JTZ17 sequence substantially retains the recombination function of SEQ ID NO: 217. In embodiments, the mutated Lox JTZ17 sequence retains at least 80%, at least 90%, at least 95%, or at least 100% of the recombination function of SEQ ID NO: 217.

[0157] In embodiments, the recombination site in the second AAV vector genome may, essentially be, or may be, a Lox 66 sequence, e.g., SEQ ID NO: 179, or a sequence having up to one, up to two, up to three, up to four, up to five, or up to six nucleotide mutations (deletions, insertions, and / or substitutions) thereto. In embodiments, the mutated Lox 66 sequence substantially retains the recombination function of SEQ ID NO: 179. In embodiments, the mutated Lox 66 sequence retains at least 80%, at least 90%, at least 95%, or at least 100% of the recombination function of SEQ ID NO: 179.

[0158] The first AAV vector genome may encode an internally cleaved polypeptide to facilitate the cleavage of the recombinase from the rest of the polypeptide. The nucleic acid encoding the internally cleaved polypeptide may be located approximately 1 to 1000 bp upstream of the 5' end of the nucleic acid portion encoding the recombinase, for example, approximately 1 to 500 bp, 1 to 300 bp, 1 to 200 bp, 1 to 150 bp, 1 to 100 bp, 1 to 50 bp, 1 to 30 bp, 1 to 20 bp, or 1 to 10 bp upstream of the 5' end of the nucleic acid portion encoding the recombinase.

[0159] The internally cleaved polypeptide may be a 2A self-cleaving peptide. Preferred examples of 2A self-cleaving peptides are listed in Table 6 below. In embodiments, the 2A self-cleaving peptide contains or consists of P2A (SEQ ID NO: 181). In embodiments, the 2A self-cleaving peptide further contains three amino acid "GSG" directly adjacent to the N-terminus of one of SEQ ID NOs: 180-183. A preferred example is an internally cleaved peptide having the amino acid sequence of SEQ ID NO: 184, encoded by a nucleic acid having the sequence of SEQ ID NO: 185. The C-terminal residue of the internally cleaved peptide may overlap with the N-terminal residue of a Cre recombinase having an NLS.

[0160] [Table 6]

[0161] In the embodiments, stable expression of recombinase is little to no detectable in cells transduced with both the first and second AAV vector genomes. In the embodiments, recombinase expression is undetectable by Western blotting in cells approximately 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 1 month after transduction of both AAV vector genomes. In the embodiments, recombinase expression is undetectable by Western blotting in cells approximately 48 hours after transduction of both AAV vector genomes. Western blotting analysis of recombinase can be performed by those skilled in the art using commercially available antibodies. For example, the expression level of Cre recombinase can be analyzed by Western blotting using rabbit anti-Cre mAb (Cell signaling technology, no. 15036) at a 1:10,000 dilution, according to the protocol described in Example 3.

[0162] In the embodiment, stable expression of recombinase-encoding mRNA is little to no detectable in cells transduced with both the first and second AAV vector genomes. In the embodiment, expression of recombinase-encoding mRNA is undetectable by quantitative PCR (qPCR) in cells approximately 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 1 month after transduction of both AAV vector genomes. In the embodiment, expression of recombinase-encoding mRNA is undetectable by qPCR in cells approximately 1 week after transduction of both AAV vector genomes.

[0163] The first AAV vector genome may include an internal ribosome entry site (IRES) to facilitate the expression of downstream recombinases.

[0164] Splice donor (SD) sites and splice acceptor (SA) sites can be used to remove a portion of the mRNA transcript. The splice donor (SD) site may be located downstream of the 5' portion of the transgene in the AAV vector genome. For example, the splice donor (SD) site may be located approximately 1–500 bp, 1–300 bp, 1–200 bp, 1–150 bp, 1–100 bp, 1–50 bp, 1–30 bp, 1–20 bp, or 1–10 bp downstream of the 5' portion of the transgene.

[0165] The splice acceptor (SA) site may be located upstream of the 3' portion of the transgene in the AAV vector genome. For example, approximately 1-500 bp, 1-300 bp, 1-200 bp, 1-150 bp, 1-100 bp, 1-50 bp, 1-30 bp, 1-20 bp, or 1-10 bp upstream of the 3' portion of the transgene.

[0166] The splice donor (SD) and splice acceptor (SA) sites may be located in different AAV vector genomes, respectively, so that after recombination between the first and second AAV vector genomes, the mRNA transcripts containing the 5' and 3' portions of the transgene can be processed to form a mature mRNA transcript containing a continuous full-length transgene. See Figure 1A.

[0167] The splice donor (SD) site may essentially consist of, or be composed of, the nucleotide sequence of SEQ ID NO: 186, or a sequence having up to 1, up to 3, up to 5, or up to 10 nucleotide changes relative to SEQ ID NO: 186. The splice acceptor (SA) site may essentially consist of, or be composed of, the nucleotide sequence of SEQ ID NO: 187, or a sequence having up to 1, up to 3, up to 5, or up to 10 nucleotide changes relative to SEQ ID NO: 187. In embodiments, the mutated splice donor site and / or splice acceptor site substantially retain the splicing function of SEQ ID NO: 186 or 187. In embodiments, the mutated splice donor site and / or splice acceptor site retain at least 80%, at least 90%, at least 95%, or at least 100% of the splicing function of SEQ ID NO: 186 or 187.

[0168] The regulation of transgene expression within host cells may be regulated by regulatory elements contained within the AAV vector genome, including a promoter sequence and a poly-A region. The AAV vector may also encode a signal peptide. In embodiments, the AAV vector genome has a 5' inverted terminal repeat (ITR) and a 3' ITR. The 5' ITR is located upstream of the promoter, which is also upstream of the transgene. In embodiments, the 5' ITR and 3' ITR have the same sequence. In embodiments, they have different sequences. In embodiments, the AAV vector genome of the present disclosure may include, in the 5' to 3' direction, a first (5') ITR, a promoter, a transgene, a poly-A region, and a second (3') ITR.

[0169] An HNA (e.g., an HNA containing a transgene or a portion thereof) can be operably linked to one or more regulatory elements (e.g., promoters). In embodiments, the HNA is operably linked to a promoter.

[0170] In embodiments, the HNA is operably linked to a constitutive promoter. The constitutive promoter may be any constitutive promoter known in the art and / or provided herein. In embodiments, the constitutive promoter may include, essentially consist of, or consist of, the Roussarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, the U6 promoter, the H1 promoter, the hybrid chicken beta-actin promoter, the MeCP2 promoter, the H1 promoter, the U1a promoter, the mMeP418 promoter, the mMeP426 promoter, the minimal MeCP2 promoter, the CAG promoter, or the EF1 promoter. It is known in the art that the nucleotide sequences of such promoters may be modified to increase or decrease the efficiency of mRNA transcription. For example, see Gao et al. (2018) Mol.Ther.:Nucleic Acids 12:135-145 (modifying TATA box of 7SK,U6 and H1 promoters to abolish RNA polymerase III transcription and stimulate RNA polymerase II-dependent mRNA transcription).

[0171] In embodiments, the promoters used herein include, essentially consist of, or consist of, a polynucleotide having the sequence of SEQ ID NO: 96 (mouse U1 promoter) or SEQ ID NO: 97 (H1 promoter). In embodiments, the promoter may be a U1a or U1b promoter, an EF1 promoter, or a chicken beta-actin (CBA) promoter.

[0172] In embodiments, the promoter is a chicken beta-actin (CBA) promoter. In embodiments, the CBA promoter contains, essentially consists of, or comprises a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 219. In embodiments, the CBA promoter substantially retains the promoter function of SEQ ID NO: 219. In embodiments, the CBA promoter retains at least 80%, at least 90%, at least 95%, or at least 100% of the promoter function of SEQ ID NO: 219.

[0173] In the embodiments, the HNA sequence is operably linked to a tissue-specific promoter or an inducible promoter. In the embodiments, the tissue-specific promoter is a central nervous system (CNS) cell-specific promoter, a lung-specific promoter, a skin-specific promoter, a muscle-specific promoter, a liver-specific promoter, or an eye-specific promoter.

[0174] In embodiments, the HNA sequence is operably linked to an eye-specific promoter. In embodiments, the eye-specific regulatory promoter is selected from the human rhodopsin kinase (hGRK1) promoter, the human rhodopsin (hRho) promoter, the human rhodopsin cGMP phosphodiesterase β-subunit promoter (βPDE) promoter, the human photoreceptor-retinoid-binding protein (IRBP) promoter, the human red / green opsin (pR2.1) promoter, the human blue opsin (HB) promoter, the human vitiligo macular dystrophy 2 (VMD2) promoter, the human RPE65 promoter, the mouse opsin (mOP) promoter, the mouse short-wavelength opsin promoter (mBP) (the short-wavelength opsin promoter may also be referred to as the S-opsin or blue opsin promoter), and the mouse rhodopsin (mRho) promoter.

[0175] In embodiments, the promoter is a human rod cGMP phosphodiesterase β-subunit promoter (βPDE) promoter. In embodiments, the βPDE promoter contains, essentially consists of, or comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 200. In embodiments, the βPDE promoter substantially retains the promoter function of SEQ ID NO: 200. In embodiments, the βPDE promoter retains at least 80%, at least 90%, at least 95%, or at least 100% of the promoter function of SEQ ID NO: 200.

[0176] In embodiments, the promoter is a human rhodopsin (hRho) promoter. In embodiments, the hRho promoter contains, essentially consists of, or comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 209. In embodiments, the hRho promoter substantially retains the promoter function of SEQ ID NO: 209. In embodiments, the hRho promoter retains at least 80%, at least 90%, at least 95%, or at least 100% of the promoter function of SEQ ID NO: 209.

[0177] In embodiments, the promoter is a human rhodopsin kinase (hGRK1) promoter. In embodiments, the hGRK1 promoter contains, essentially consists of, or comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 188. In embodiments, the hGRK1 promoter substantially retains the promoter function of SEQ ID NO: 188. In embodiments, the hGRK1 promoter retains at least 80%, at least 90%, at least 95%, or at least 100% of the promoter function of SEQ ID NO: 188.

[0178] In embodiments, the promoter includes, essentially consists of, or comprises any one of the nucleic acid sequences listed in Table 7, or any sequence having up to 5, up to 10, or up to 30 nucleotide changes with respect to any one of the nucleic acid sequences listed in Table 7. In embodiments, the promoter includes, essentially consists of, or comprises any nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of the nucleic acid sequences listed in Table 7. In embodiments, the promoter substantially retains the promoter function of any one of the sequences listed in Table 7. In embodiments, the promoter retains at least 80%, at least 90%, at least 95%, or at least 100% of the promoter function of any one of the sequences listed in Table 7.

[0179] [Table 7]

[0180] In one embodiment, the AAV vector includes a CBA-MVM hybrid sequence between the promoter and the 5' portion of the transgene. In another embodiment, the CBA-MVM hybrid sequence includes or comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 210.

[0181] In the embodiment, the AAV vector includes an enhancer. In the embodiment, the HNA sequence is operably ligated to the enhancer. In the embodiment, the enhancer is located upstream of the promoter. In the embodiment, the enhancer is located immediately upstream of the promoter without any additional nucleotides in between.

[0182] In embodiments, the enhancer is a photoreceptor-retinoid-binding protein (IRBP) enhancer. In embodiments, the IRBP enhancer comprises or consists of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 208. In embodiments, the IRBP enhancer substantially retains the enhancer function of SEQ ID NO: 208. In embodiments, the IRBP enhancer retains at least 80%, at least 90%, at least 95%, or at least 100% of the enhancer function of SEQ ID NO: 208.

[0183] In the embodiment, the enhancer is a CMV enhancer. In the embodiment, the CMV enhancer contains or consists of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with sequence number 218. In the embodiment, the CMV enhancer substantially retains the enhancer function of sequence number 218. In the embodiment, the CMV enhancer retains at least 80%, at least 90%, at least 95%, or at least 100% of the enhancer function of sequence number 218.

[0184] In embodiments, the AAV vector genome includes a sequence comprising a CMV enhancer and a CBA promoter, which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 211. In embodiments, the CMV enhancer / CBA promoter sequence is located between the 5' AAV inverted terminal repeat and the 5' portion of the transgene. In embodiments, the CMV enhancer / CBA promoter sequence substantially retains the enhancer and promoter functions of SEQ ID NO: 211. In embodiments, the CMV enhancer / CBA promoter sequence retains at least 80%, at least 90%, at least 95%, or at least 100% of the enhancer and promoter functions of SEQ ID NO: 211.

[0185] In the embodiment, the HNA sequence is operably linked to an additional regulatory element. The additional regulatory element may be a woodchuck hepatitis virus post-transcriptional regulator (WPRE). In the embodiment, the AAV vector may contain regulatory components suitable for vector growth and culture in a bacterial host for vector production purposes. For example, the vector may contain genes for antibiotic resistance and plasmid maintenance within the bacterium, as well as relevant regulatory elements for controlling protein expression in the bacterium.

[0186] In embodiments, the HNA sequence is operably linked to a polyadenylation (polyA) signal, also known as a polyA site. The polyA site includes, essentially consists of, or consists of, the MeCP2 polyA site, the retinol dehydrogenase 1 (RDH1) polyA site, the bovine growth hormone (BGH) polyA site, the SV40 polyA site, the SPA49 polyA site (SEQ ID NO: 189), the sNRP-TK65 polyA site, the sNRP polyA site, or the TK65 polyA site. An exemplary SPA49 polyA sequence is described by Ostedgaard et al., Proc. Nat'l Acad. Sci. USA (Feb. 22, 2005) 102:2952-2957, which is incorporated herein by reference.

[0187] In embodiments, the polyadenylation signal (poly-A site) contains, essentially consists of, or consists of, a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 189. In embodiments, the polyadenylation signal (poly-A site) contains, essentially consists of, or consists of, a sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotide mutations (deletions, insertions, and / or substitutions) with respect to SEQ ID NO: 189. In embodiments, the poly-A site substantially retains the mRNA transport and / or stabilization function of SEQ ID NO: 189. In embodiments, the poly-A site retains at least 80%, at least 90%, at least 95%, or at least 100% of the mRNA transport and / or stabilization function of SEQ ID NO: 189.

[0188] Heterologous nucleic acid (HNA) The AAV viral vectors disclosed herein infect and deliver one or more heterologous nucleic acids (HNAs) to a target tissue. The HNA sequences of the first and second AAV vector genomes are recombined in the cell to form a new HNA sequence. The HNA sequence is transcribed in the target tissue cell and optionally translated. In embodiments, the new recombinant HNA sequence includes a protein-coding transgene. In embodiments, the HNA is operably ligated to a promoter.

[0189] In embodiments, the cells of the disclosure contain a genomic copy lacking an endogenous gene, and the transgene is a working copy of the lacking endogenous gene, thereby compensating for the function of the lacking endogenous gene. In embodiments, both alleles of the endogenous gene are lacking in the cell. In embodiments, the “working copy” of the gene substantially retains the function of the wild-type version of the endogenous gene. In embodiments, the transgene retains at least 80%, at least 90%, at least 95%, or at least 100% of the function of the wild-type gene. In embodiments, the transgene codes for a protein, and the protein substantially retains the function of the wild-type protein encoded by the wild-type endogenous gene. In embodiments, the transgene codes for a protein that retains at least 80%, at least 90%, at least 95%, or at least 100% of the function of the wild-type protein encoded by the wild-type endogenous gene. In embodiments, the transgene codes for an intact wild-type protein. In embodiments, the transgene codes for a fragment of the wild-type protein that substantially retains the function of the wild-type protein.

[0190] In the case of a protein-coding transgene, a full-length protein-coding transgene is divided into two parts: a 5' portion and a 3' portion. The length of each portion is less than the length of the complete transgene. The 5' portion of the transgene codes for the N-terminal portion of the protein, and the 3' portion codes for the C-terminal portion of the protein. Those skilled in the art will understand that, after recombination of the first and second AAV vector genomes, these two portions can be rejoined via mRNA splicing to form a full-length transgene, so the 5' and / or 3' portions of the transgene do not need to have nucleic acid lengths that are multiples of 3 (i.e., the split point of the transgene does not need to be between two codons). Therefore, the portions of the transgene and the corresponding amino acid sequences they encode may be a perfect match or may have a small mismatch of one or two nucleotides at the split point. Regarding size limitations, the 5' portion of the transgene in the first AAV vector genome may have a size limitation of approximately 3.5kb, 3.4kb, 3.3kb, or 3.2kb at most. The 3' portion of the transgene in the second AAV vector genome may have size limitations of approximately 4.6kb, 4.5kb, 4.4kb, or 4.3kb.

[0191] In embodiments, the 5' portion of the transgene in the first AAV vector genome is at least 0.5kb, at least 1kb, at least 1.5kb, at least 2kb, at least 2.5kb, at least 3kb, at least 3.2kb, or at least 3.5kb long. In embodiments, the 5' portion of the transgene in the first AAV vector genome is 0.5-1kb, 1-1.5kb, 1.5-2kb, 2-2.5kb, 2.5-3kb, 3-3.2kb, 3.2-3.5kb, 0.5-1.5kb, 1-2kb, 1.5-2.5kb, 2-3kb, 2.5-3.2kb, 3-3.5kb, 0 The lengths are 0.5-2kb, 1-2.5kb, 1.5-3kb, 2-3.2kb, 2.5-3.5kb, 0.5-2.5kb, 1-3kb, 1.5-3.2kb, 2-3.5kb, 0.5-3kb, 1-3.2kb, 1.5-3.5kb, 0.5-3.2kb, 1-3.5kb, or 0.5-3.5kb (including the endpoint).

[0192] In embodiments, the 3' portion of the transgene in the second AAV vector genome is at least 0.5kb, at least 1kb, at least 1.5kb, at least 2kb, at least 2.5kb, at least 3kb, at least 3.5kb, at least 4kb, or at least 4.5kb in length. In embodiments, the 3' portion of the transgene in the second AAV vector genome is 0.5-1kb, 1-1.5kb, 1.5-2kb, 2-2.5kb, 2.5-3kb, 3-3.5kb, 3.5-4kb, 4-4.7kb, 0.5-1.5kb, 1-2kb, 1.5-2.5kb, 2-3kb, 2.5-3.5kb, 3-4kb, 3.5-4.7kb, 0.5-2kb, 1-2.5kb, The lengths are 1.5-3kb, 2-3.5kb, 2.5-4kb, 3-4.7kb, 0.5-2.5kb, 1-3kb, 1.5-3.5kb, 2-4kb, 2.5-4.7kb, 0.5-3kb, 1-3.5kb, 1.5-4kb, 2-4.7kb, 0.5-3.5kb, 1-4kb, 1.5-4.7kb, 0.5-4kb, 1-4.7kb, or 0.5-4.7kb (including the endpoint).

[0193] A representative example of a protein encoded by a transgene is ABCA4 (ATP-binding cassette, subfamily A, member 4). Mutations in the ABCA4 protein can cause Stargardt disease. In humans, the wild-type ABCA4 protein is encoded by the ABCA4 gene and has an amino acid sequence following Uniprot accession number P78363 (sequence number 190). An exemplary nucleic acid sequence encoding the ABCA4 protein is sequence number 191. The functions of the ABCA4 protein include removing potentially toxic retinoid compounds from photoreceptors and actively inverting N-retynidene-phosphatidylethanolamine (N-Ret-PE) and phosphatidylethanolamine from the lumen to the cytoplasmic lateral layer of the disc membrane. Various mutations reduce the transport activity of ABCA4 and cause Stargardt disease. Further descriptions of assays for measuring the function of the ABCA4 protein and ABCA4 transport function can be found, for example, in Quazi et al., Nat Commun. 2012 Jun 26:3:925, where the content is incorporated in its entirety by reference for all purposes.

[0194] In embodiments, the ABCA4 protein contains or consists of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 190. In embodiments, the ABCA4 protein is identical to SEQ ID NO: 190, or has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different amino acids from SEQ ID NO: 190. In embodiments, the ABCA4 protein substantially retains the N-Ret-PE transport function of SEQ ID NO: 190. In embodiments, the ABCA4 protein retains at least 80%, at least 90%, at least 95%, or at least 100% of the N-Ret-PE transport function of SEQ ID NO: 190.

[0195] In this embodiment, the ABCA4 transgene is divided into a 5' portion and a 3' portion, which are then placed into separate AAV vector genomes.

[0196] In the embodiment, the 5' portion of the ABCA4 transgene contains or consists of a nucleotide sequence having at least 70%, at least 75%, about 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 192. In the embodiment, the 5' portion of the ABCA4 transgene contains or consists of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 212. In the embodiment, the 5' portion of the ABCA4 transgene contains the following nucleotides: (i) The nucleotide "G" at the position corresponding to nucleotide 813 of sequence number 212, (ii) Nucleotide "A" at the position corresponding to nucleotide 1311 of sequence number 212, (iii) Nucleotide "A" at the position corresponding to nucleotide 2274 of sequence number 212, (iv) The nucleotide "C" at the position corresponding to nucleotide 2277 of sequence number 212, (v) The nucleotide "T" at the position corresponding to nucleotide 2278 of sequence number 212, (vi) Contains one or more nucleotides "T" at the position corresponding to nucleotide 2568 of sequence number 212. In the embodiment, the 5' portion of the ABCA4 transgene contains two or more, three or more, four or more, five or more, or all six of these nucleotides. In the embodiment, the 5' portion of the ABCA4 transgene does not contain any BglII restriction sites. In the embodiment, the 5' portion of the ABCA4 transgene does not contain any BamHI restriction sites. In the embodiment, the 5' portion of the ABCA4 transgene does not contain any KpnI restriction sites. In the embodiment, the 5' portion of the ABCA4 transgene does not contain any BglII restriction site or KpnI restriction site. In the embodiment, the 5' portion of the ABCA4 transgene contains a HindIII restriction site (5'-AAGCTT-3') (for example, at the position corresponding to nucleotides 2274-2279 of SEQ ID NO: 212).

[0197] In this embodiment, the 3' portion of the ABCA4 transgene contains or comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity with SEQ ID NO: 193.

[0198] Examples of additional transgenes suitable for bipartite gene delivery include MYO7A and CEP290.

[0199] MYO7A encodes the myosin VIIa protein, and mutations in MYO7A are associated with Usher syndrome. In humans, the myosin VIIa protein has an amino acid sequence that follows SEQ ID NO: 201. An exemplary nucleic acid sequence of the MYO7A gene is SEQ ID NO: 202. A suitable myosin VIIa protein may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity with SEQ ID NO: 201. The myosin VIIa protein may be identical to SEQ ID NO: 201, or it may have up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different amino acids from SEQ ID NO: 201.

[0200] The CEP290 gene encodes a centrosome protein localized to photoreceptor-binding cilia, involved in both ciliation and ciliary transport, and mutations in CEP290 are associated with Leber congenital amaurosis (LCA). In humans, the CEP290 protein (also known as the 290 kDa centrosome protein) has an amino acid sequence following SEQ ID NO: 203. An exemplary nucleic acid sequence of the CEP290 gene is SEQ ID NO: 204. A suitable CEP290 protein may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity with SEQ ID NO: 203. The CEP290 protein may be identical to SEQ ID NO: 203, or it may have up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different amino acids from SEQ ID NO: 203.

[0201] Method for producing an AAV virus vector Various approaches can be used to produce AAV viral vectors. In one embodiment, packaging is achieved by using a helper virus or helper plasmid and a cell line. The helper virus or helper plasmid contains elements and sequences that facilitate viral vector production. In another embodiment, the helper plasmid is stably incorporated into the genome of the packaging cell line, thereby eliminating the need for additional transfection with the helper plasmid in the packaging cell line.

[0202] In the embodiments, the cells are a packaging cell line or a helper cell line. In the embodiments, the helper cell line is a eukaryotic cell, for example, HEK293 cells or 293T cells. In the embodiments, the helper cells are yeast cells or insect cells.

[0203] The helper plasmid may, for example, include at least one viral helper DNA sequence for producing virion proteins that can package non-replicating AAVs at high titers, derived from a non-replicating viral genome that trans-encodes all the virion proteins necessary to package non-replicating AAVs without generating non-replicating AAVs.

[0204] Helper plasmids for packaging AAV are known in the art; see, for example, U.S. Patent Publication 2004 / 0235174A1, which is incorporated entirely herein by reference. As described herein, an AAV helper plasmid may, in non-limiting examples, contain the Ad5 genes E2A, E4, and VA as helper viral DNA sequences, controlled by their respective original or heterologous promoters. The AAV helper plasmid may additionally contain an expression cassette for the expression of a marker protein, such as a fluorescent protein, to enable simple detection of transfection of desired target cells.

[0205] This disclosure provides a method for producing AAV particles, comprising transfecting a packaging cell line with one of the AAV helper plasmids and one of the AAV vectors disclosed herein. In embodiments, the AAV helper plasmid and the AAV vector are co-transfected into the packaging cell line. In embodiments, the cell line is a mammalian cell line, for example, the human embryonic kidney (HEK) 293 cell line. This disclosure provides cells comprising one of the AAV vectors, AAV vector genomes, and / or AAV particles disclosed herein.

[0206] Pharmaceutical composition This disclosure provides a pharmaceutical composition comprising one of the AAV vectors, AAV vector genomes, AAV capsids, and / or AAV particles described herein. Typically, AAV particles are administered for therapeutic purposes.

[0207] The pharmaceutical compositions described herein may be formulated by either methods known or developed in the field of pharmaceutical development, which include, but are not limited to, contacting an active ingredient (e.g., a viral particle or recombinant vector) with an excipient or other accessory, and dividing or packaging the product into dose units. In embodiments, the pharmaceutical compositions are suitable for ocular injection. The viral particles of this disclosure may be formulated to have desirable characteristics, such as increased stability, increased cell transfection, sustained or delayed release, in vivo distribution or directionality, regulated or enhanced translation of the encoded protein in vivo, and an in vivo release profile of the encoded protein.

[0208] In embodiments, the pharmaceutical composition may further include saline, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transduced with viral vectors (e.g., for transplantation into a subject), nanoparticle mimics, or combinations thereof. In embodiments, the pharmaceutical composition is formulated as nanoparticles. In embodiments, the pharmaceutical composition is formulated as a sterile, substantially isotonic solution.

[0209] The pharmaceutical compositions according to this disclosure may be prepared, packaged, and / or sold in bulk as single-dose units and / or as multiple single-dose units. The amount of the active ingredient is approximately equal to the dose of the active ingredient to be administered to a subject, and / or a favorable proportion of such dose, for example, half or one-third of such dose. The formulations of the present invention may contain one or more excipients in amounts that collectively increase the stability of the viral vector, increase cell transfection or transduction by the viral vector, increase the expression of the protein encoded by the vector genome, and / or modify the release profile of the protein encoded by the vector genome. In embodiments, the pharmaceutical composition includes excipients. Non-limiting examples of excipients include solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, or combinations thereof.

[0210] In embodiments, the pharmaceutical composition includes an antifreeze agent. The term “antifreeze agent” refers to an agent that can reduce or eliminate damage to a substance during freezing. Non-limiting examples of antifreeze agents include sucrose, trehalose, lactose, glycerol, dextrose, raffinose, and / or mannitol.

[0211] Treatment method This disclosure provides a method for preventing or treating a genetic disorder, which comprises, essentially consists of, or consists of administering to a subject a therapeutically effective amount of one of the pharmaceutical compositions disclosed herein.

[0212] In the embodiment, the hereditary disorder is an eye disorder, CNS disorder, skin disorder, lung disorder, muscle disorder, liver disorder, digestive disorder, blood or lymphatic disorder, inflammatory disorder, or cancer. In the embodiment, the disorder is Stargardt disease.

[0213] In embodiments, the disorders include hypophosphatasia, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), recessive dystrophy of epidermolysis bullosa (RDEB), lysosomal storage disorders (including Duchenne muscular dystrophy and Becker muscular dystrophy), juvenile Batten disease, infantile Batten disease, autosomal dominant disorders, muscular dystrophy, Vietti crystalline dystrophy, and retinal schizophrenia (e.g., degenerative, hereditary, traction). Hemophilia (exudative, visceral), hemophilia A, hemophilia B, multiple sclerosis, diabetes mellitus, Fabry disease, Pompe disease, neuronal ceroid lipofuscinosis 1 (CLN1), CLN3 disease (or juvenile neuronal ceroid lipofuscinosis), Gaucher disease, cancer, arthritis, muscle wasting, heart disease, intimal hyperplasia, Rett syndrome, epilepsy, Huntington's disease, Parkinson's disease, Alzheimer's disease, autoimmune diseases, cystic fibrosis, thalassemia, Harler syndrome (MPS) These include IH), Sly syndrome, Schaye syndrome, Haller-Scheye syndrome, Hunter syndrome, Sanfilippo syndrome A (mucopolysaccharidosis IIIA or MPS IIIA), Sanfilippo syndrome B (mucopolysaccharidosis IIIB or MPS IIIB), Sanfilippo syndrome C, Sanfilippo syndrome D, Morquio syndrome, Maloto-Lamy syndrome, Krabbe disease, phenylketonuria, spinal ataxia, LDL receptor deficiency, hyperammonemia, anemia, arthritis, or adenosine deaminase deficiency.

[0214] In addition to the specific transgenes disclosed herein, known active enzymes, structural proteins, or RNA sequences may be used as transgenes to deliver functional activity.

[0215] The disorder may be an eye disease. The eye is an immune-privileged tissue, and low doses of the virus may provide therapeutic benefits. Eye diseases may affect photoreceptors and / or RPE cells. For example, eye diseases include retinitis pigmentosa (e.g., autosomal recessive (SPATA7 gene, LRAT gene, TULP1 gene), autosomal dominant (AIPL1 gene), and X-linked (RPGR gene)), eye disorders associated with mutations in the bethroffin-1 (BEST-1) gene (e.g., vitiligo macular dystrophy, age-related macular degeneration, autosomal dominant vitreoretinopathy, glaucoma, cataract), and Leber congenital amaurosis (LCA, aryl-hydrocarbon interacting protein-like 1 (AIPL1) gene). The child may have cone-rod dystrophy (CRD, ABCA4 gene), Stargard syndrome (ABCA4 gene), congenital choroidal absence (CHM gene), Usher syndrome (MYO7A gene, CDH23 gene, USH2A gene, CLRN1 gene), retinoschisis (RS1 gene), Vietti crystalline dystrophy (CYP4V2 gene), or color blindness (CNGA3 gene, CNGB3 gene, GNAT2 gene, PDE6C gene, or PDE6H gene).

[0216] Preferred treatment targets for the methods and compositions of this disclosure include Usher syndrome (USH) and Leber congenital amaurosis (LCA). Usher syndrome is an autosomal recessive disorder characterized by hearing impairment associated with retinitis pigmentosa and, in some cases, vestibular dysfunction. Usher syndrome type I (USH1) is the most severe type and is characterized by severe to profound congenital sensorineural hearing loss, balance deficits, and pre-pubescent onset of retinitis pigmentosa leading to blindness. Six loci of USH1 (USH1B to USH1G) have been mapped. Mutations in the MYO7A gene have been found to be associated with USH1B, the most common subtype of USH1. The MYO7A gene encodes the myosin VIIA protein. In the human retina, myosin VIIA is active in the migration of RPE melanosomes, phagocytosis of the extracellular segment tips of photoreceptors, and opsin transport of these photoreceptors through cilia. The coding sequence for MYO7A is approximately 6.6 kb in size, and the encoded myosin VIIA has an amino acid sequence that follows Uniprot accession number Q13402-1 (www.uniprot.org / uniprot / Q13402#Q13402-1). Leber congenital amaurosis (LCA) refers to a group of severe hereditary retinal disorders characterized by decreased visual acuity, nystagmus, and unrecordable electroretinogram within the first year of life. Mutations in CEP290 are one of the most common contributing factors to LCA. The CEP290 gene codes for a centrosome protein that is localized to photoreceptor-binding cilia and is involved in both ciliation and ciliary transport. CEP290 plays a crucial role in primary ciliation and ciliary protein transport. Patients with CEP290-associated LCA have several cone nuclei in the foveal region, where cones are concentrated in the center. However, these cone photoreceptors have abnormal medial and lateral segments that result in severe vision loss in most patients. The coding sequence of the CEP290 gene is approximately 7.4 kb in size and encodes a protein sequence according to Uniprot accession number O15078-1 (www.uniprot.org / uniprot / O15078#O15078-1).

[0217] In embodiments, cancer is a solid tumor, such as a tumor of the bladder, breast, cervix, colon, rectum, endometrium, kidney, lip, oral cavity, liver, melanoma, mesothelioma, non-small cell lung, non-melanoma skin, ovary, pancreas, prostate, sarcoma, small cell lung, or thyroid.

[0218] In the embodiments, the subject is a mammal, for example, a human. In the embodiments, the human is an infant, for example, under 3 years old, under 2 years old, or under 1 year old.

[0219] The therapeutic and preventive methods disclosed herein may be combined with appropriate diagnostic techniques for identifying and selecting patients for treatment or prevention. For example, a method for treating or preventing a disorder disclosed herein, such as Stargardt disease, may further include the step of performing genetic testing to identify gene mutations or deletions associated with the disorder in a subject. In embodiments, a method for treating or preventing a disorder, such as Stargardt disease, includes administration to a subject previously identified as having a mutation associated with the disorder or being at high risk of developing the disorder (e.g., based on genetic factors).

[0220] This disclosure provides a method for increasing the level of a protein in a host cell, comprising contacting the host cell with two AAV virus particles disclosed herein, wherein the first AAV virus particle comprises a first AAV vector genome and the second AAV virus particle comprises a second AAV vector genome. Cre-lox-mediated recombination of the first and second AAV vector genomes yields an HNA sequence encoding the protein. In embodiments, the protein is a therapeutic protein. In embodiments, the host cell is in vitro, in vivo, or ex vivo. In embodiments, the host cell is derived from a subject. In embodiments, the subject suffers from a disorder that results in a decrease in the level and / or functionality of the protein compared to the level and / or functionality of the protein in a normal subject.

[0221] The present disclosure provides a method for preventing or treating a disorder of a subject by introducing a transgene into the subject. In embodiments, the method comprises co-administering an effective amount of two AAV viral particles, wherein the first AAV viral particle comprises a first AAV vector genome comprising a 5' portion of the transgene, and the second AAV viral particle comprises a second AAV vector genome comprising a 3' portion of the transgene. For example, the present disclosure provides a method for treating Stargardt disease in a subject. In embodiments, the method comprises co-administering two AAV viral particles, wherein the first AAV viral particle comprises a first AAV vector genome comprising a 5' portion of the transgene (e.g., the ABCA4 gene), and the second AAV viral particle comprises a second AAV vector genome comprising a 3' portion of the transgene (e.g., the ABCA4 gene).

[0222] Dosage and Administration Methods for determining the most effective means of administration and dosage are known to those of skill in the art and vary depending on the composition used for treatment, the purpose of the treatment, and the subject being treated. Single or multiple administrations can be carried out using dosage levels and patterns selected by the attending physician. It should be noted that the dosage can be affected by the route of administration. Suitable dosage forms and methods of administration of the agent are known in the art. Non-limiting examples of such suitable dosages are at least 10 9 vector genomes per administration and at most 10 17 vector genomes.

[0223] In embodiments of the methods described herein, the number of viral particles (e.g., AAV) administered to the subject ranges from about 10 9 to about 10 17 In embodiments, about 10 10 to about 10 12 , about 10 11 to about 10 13 , about 10 11 to about 10 12 , about 10 11 to about 10 14 , about 5×10 11 to about 5×10 12 , or about 10 12~about 10 13 A number of virus particles are administered to the target. When administered to the human eye, approximately 1 x 10⁶ particles are administered. 10 The total dose of vg / eye can be used for mouse eyes. 5 × 10 9 The total dose of the vg / ocular vector genome may be used. Efficacy / safety in animals can be monitored using non-invasive in vivo imaging techniques, which include, but are not limited to, scanning laser ophthalmography (SLO), optical coherence tomography (OCT), multiphoton microscopy, and fluorescein angiography.

[0224] In embodiments, viral particles are delivered to a target intravenously, intrathecally, intracerebrally, intraventricularly, intranasally, intratracheally, intraaurally, intraocularly, periocularly, orally, rectally, transmucosally, by inhalation, percutaneously, parenterally, subcutaneously, intradermally, intramuscularly, intrathoracically, topically, intralymphatically, or into the cisterna magna. In embodiments, such delivery may be intraarterial, intracardiac, subventricular, epidural, intracerebral, intraventricular, subretinal, intravitreous, intraarticular, intraperitoneal, intrauterine, or any combination thereof. In embodiments, viral particles are delivered to a desired target tissue, e.g., the lungs, eyes, or CNS. In embodiments, delivery of viral particles is systemic. The cisterna magna administration route involves direct administration of the drug into the cerebrospinal fluid of the ventricles. This can be done by direct injection into the cisterna magna or through a permanently placed tube.

[0225] For intraocular treatment of eye diseases (ocular disorders), there are several modes of administration known to those skilled in the art, including, but not limited to, lacrimal gland (LG) administration, topical eye drops, intramatriate administration into the cornea, anterior chamber administration, intravitreous administration, subretinal administration, systemic administration, or combinations thereof. Approximately 80% of hereditary ocular disorders occur in photoreceptors. Intravitreous delivery of small amounts of gene therapy can be performed in an outpatient clinic.

[0226] Some AAV particles exhibit brain and cervical spine directivity and may cross the blood-brain barrier (BBB). Some AAV particles exhibit high retinal directivity via subretinal, pararetinal, and / or intravitreous injection. AAV particles may target multiple ophthalmic cell types, e.g., cones, rods, and retinal pigment epithelium (RPE). Advantageously, AAV particles may evade neutralizing antibodies against native serotypes, thus potentially allowing for re-administration. In further embodiments, AAV particles and compositions may be administered in combination with other known treatments for the disorder being treated.

[0227] AAV particles may be administered via subretinal injection. Subretinal injection routes include (1) a transcorneal route through the pupil, lens, vitreous humor, and retina; (2) a transdural route entering the ciliary body squamous region or corneal margin region, traversing the opposite side of the vitreous humor and retina to enter the subretinal space; and (3) a transscleral route through the choroid and Bruch's membrane without penetrating the retina. Subretinal injection is described in Peng et al. Ophthalmic Res. 2017;58(4):217-226, which is incorporated herein by reference in its entirety.

[0228] AAV particles may be administered via pararetinal injection. In embodiments, pararetinal injection includes injection at a distance of 0–13 mm, 0–10 mm, 0–5 mm, or 0–3 mm from the surface of the retina in the posterior vitreous cavity of the eye. In embodiments, pararetinal injection includes injection at a distance of 0–13 mm from the surface of the retina in the posterior vitreous cavity. In embodiments, pararetinal injection includes injection at a distance of 0–10 mm from the surface of the retina in the posterior vitreous cavity. In embodiments, pararetinal injection includes injection at a distance of 0–5 mm from the surface of the retina in the posterior vitreous cavity. In embodiments, pararetinal injection includes injection at a distance of 0–3 mm from the surface of the retina in the posterior vitreous cavity.

[0229] As used herein, the term “pararetinal administration” refers to a form of intravitreous administration in which a therapeutic agent (e.g., AAV particles) is injected into the vitreous cavity in close proximity to a desired area of ​​the retina (i.e., targeted delivery). In embodiments, the desired area of ​​the retina is near the foveal region of the retina. In contrast to conventional intravitreous administration, which is performed using a short needle designed to deposit the product in the intermediate vitreous cavity and does not require direct visualization, pararetinal injection is typically performed under direct visualization with a longer needle that can deliver the product into the posterior vitreous cavity closer to the retina. In embodiments, the therapeutic agent is deposited in the vitreous cavity at a distance of 0-13 mm from the retinal surface, 0-10 mm from the retinal surface, 0-5 mm from the retinal surface, or 0-3 mm from the retinal surface. In the embodiment, the therapeutic agent is deposited in the vitreous cavity at a distance of 0-13 mm, 0-12 mm, 0-11 mm, 0-10 mm, 0-9 mm, 0-8 mm, 0-7 mm, 0-6 mm, 0-5 mm, 0-4 mm, 0-3 mm, 0-2 mm, or 0-1 mm from the surface of the retina.

[0230] In embodiments, pararetinal administration is used in situations where subretinal injection is not appropriate. In embodiments, pararetinal administration is used for targeted transduction of the optic nerve. In embodiments, pararetinal administration is used to treat diseases or disorders associated with optic nerve dysfunction. In embodiments, pararetinal administration is used to treat dominant optic atrophy or retinoschiosis.

[0231] In embodiments, pararetinal administration involves the use of a small gauge needle (30 gauge or equivalent) having sufficient length (25 mm or equivalent) to reach the posterior pole of the human eye, external or internal irradiation and visualization using a microscope, and the use of a corneal contact lens that allows focusing on the posterior vitreous cavity and retina. This is typically performed after appropriate analgesia and disinfection, at which point the corneal contact lens is fitted to the eye, the microscope is positioned, and the posterior retina is visualized. The needle is inserted through the ocular wall in the ciliary body squamous region, and its tip is visualized. Under direct visualization, the tip of the needle is advanced to the desired position close to the retinal surface. The syringe plunger is advanced to slowly deposit the viral vector (which may be contained in any suitable composition or formulation). The needle is withdrawn, and the eye is examined. The port may be closed with sutures, but in the case of sufficiently small diameter needles (such as 30 gauge), suturing to close the needle pathway is not necessary. Ointments and eye shields may be applied, and if desired, the subject may be kept in a supine position for a period of time postoperatively to further facilitate the increase in pararetinal density of the product. Variations of the delivery device include the creation of a scleral incision with or without the use of a vitrectomy port to allow the use of a blunt cannula, and / or a cannula design with tapered and / or flexible extendable tip or lateral ports to optimize accessibility and safety to the retinal surface, and / or the use of a pneumatic system instead of a simple syringe plunger. Additional descriptions of pararetinal administration are disclosed, for example, in WO2020 / 018766, WO2022 / 245919, and Zeng et al., Mol Ther Methods Clin Dev. 2020 Sep 11;18:422-427, the contents of which are incorporated herein in whole by reference for all purposes.

[0232] Each of the AAV vectors, AAV virus particles, or compositions of this disclosure may be administered as a single, continuous, or intermittent dose throughout the course of treatment. In embodiments, the AAV vectors, AAV particles, or compositions of this disclosure are administered parenterally by injection, infusion, or implantation.

[0233] AAV virus particles containing the first AAV vector genome and AAV virus particles containing the second AAV vector genome may be administered simultaneously or sequentially. "Simultaneous administration" means administering the first AAV virus particle and the second AAV virus particle together (i.e., both AAV virus particles may be administered in the same composition). "Sequential administration" means that the two AAV virus particles are administered separately, but within a time interval that allows them to act together on target cells to achieve a physiological effect (i.e., transduce the same cells into the target, recombinate the transgene portion, and produce the full-length transgene). For example, the administration of one virus particle may precede the administration of the other virus particle by up to approximately 10 minutes, 20 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, 2 days, 4 days, 1 week, 2 weeks, or 4 weeks.

[0234] AAV virus particles containing a first AAV vector genome and AAV virus particles containing a second AAV vector genome may be administered in a specific ratio or within a specific range of ratios. In embodiments, the ratio of AAV virus particles containing a first AAV vector genome to AAV virus particles containing a second AAV vector genome is approximately 0.1:1, approximately 0.2:1, approximately 0.3:1, approximately 0.4:1, approximately 0.5:1, approximately 0.6:1, approximately 0.7:1, approximately 0.8:1, approximately 0.9:1, approximately 1:1, approximately 1.1:1, approximately 1.2:1, approximately 1.3:1, approximately 1.5:1, approximately 1.7:1, approximately 2:1, approximately 3:1, approximately 4:1, approximately 5:1, approximately 6:1, approximately 7:1, approximately 8:1, approximately 9:1, or approximately 10:1. In the embodiments, the ratio of AAV virus particles containing the first AAV vector genome to AAV virus particles containing the second AAV vector genome is within the range of 0.1:1 to 10:1, 0.2:1 to 5:1, 0.3:1 to 3:1, 0.4:1 to 2.5:1, 0.5:1 to 2:1, 0.7:1 to 1.5:1, 0.8:1 to 1.2:1, or 0.9:1 to 1.1:1. In the embodiments, the ratio of AAV virus particles containing the first AAV vector genome to AAV virus particles containing the second AAV vector genome is less than 0.1:1, less than 0.2:1, less than 0.3:1, less than 0.4:1, less than 0.5:1, less than 0.6:1, less than 0.7:1, less than 0.8:1, less than 0.9:1, or less than 1:1. In the embodiment, the ratio of AAV virus particles containing the first AAV vector genome to AAV virus particles containing the second AAV vector genome is approximately 1:1. In the embodiment, this ratio is calculated by comparing the total number of AAV virus particles containing the first AAV vector genome with the total number of AAV virus particles containing the second AAV vector genome during the administration process (e.g., concurrent or sequential administration).

[0235] kit In embodiments, the drugs, vectors, or compositions described herein may be constructed into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic, or research applications. In embodiments, the kits of the disclosure include one of the modified AAV capsid proteins, AAV vectors, AAV vector genomes, AAV particles, host cells, isolated tissues, compositions, or pharmaceutical compositions described herein.

[0236] The kit may further include instructions for use. Specifically, such a kit may include one or more of the drugs described herein, along with instructions describing the intended use and proper use of these drugs. In embodiments, the kit may include instructions for mixing one or more components of the kit and / or instructions for isolating and mixing samples and applying them to a subject. In embodiments, the drugs in the kit are present in pharmaceutical formulations and dosages suitable for the specific use and method of administration of the drugs. Kits for research purposes may contain these components in concentrations or amounts suitable for performing various experiments.

[0237] The kit may be designed to facilitate the use of the methods described herein and may take many forms. Each of the components of the kit may be provided in liquid form (e.g., solution) or in solid form (e.g., dry powder), where applicable. In certain cases, some of the components may be configurable or otherwise treatable (e.g., into an active form) by adding a suitable solvent or other type (e.g., water or cell culture medium), which may or may not be provided with the kit.

[0238] In embodiments, a kit contains one or more of the components described herein in one or more containers. Thus, in embodiments, a kit may include containers for containing the drugs described herein. The drugs may be in liquid, gel, or solid (powder) form. The drugs may be prepared under sterile conditions, packaged in syringes, and shipped under refrigeration. Alternatively, they may be contained in vials or other containers for storage. A second container may contain other drugs prepared under sterile conditions. Alternatively, a kit may contain active drugs that are pre-mixed and shipped in syringes, vials, tubes, or other containers. A kit may include one or more or all of the components required to administer the drug to a target, such as syringes, topical application devices, or IV needle tubes and bags.

[0239] While the present invention has been described in conjunction with the embodiments described above, it should be understood that the foregoing description and examples are intended to illustrate, and not limit, the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0240] Further numbered embodiments Further numbered embodiments of this disclosure are provided below.

[0241] Embodiment 1. An AAV vector genome, wherein in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Photoreceptor-retinoid-binding protein (IRBP) enhancer, (c) Ocular specific promoter, (d) 5' portion of the introduced gene, (e) Splice donor (SD) site, (f) Recombination site, (g) Polynucleotide encoding a recombinase, (h) Poly A portion, and (i) The AAV vector genome comprising a 3'AAV inverted terminal repeat.

[0242] Embodiment 2. The AAV vector genome according to Embodiment 1, wherein the IRBP enhancer comprises or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 208.

[0243] Embodiment 3. The AAV vector genome according to Embodiment 1 or 2, wherein the eye-specific promoter is a human rod cGMP phosphodiesterase β-subunit promoter (βPDE) promoter comprising or consisting of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 200.

[0244] Embodiment 4. The AAV vector genome according to Embodiment 1 or 2, wherein the eye-specific promoter is a human rhodopsin (hRho) promoter comprising or consisting of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 209.

[0245] Embodiment 5. The AAV vector genome according to Embodiment 1 or 2, wherein the eye-specific promoter is a human rhodopsin kinase (hGRK1) promoter comprising or consisting of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 188.

[0246] Embodiment 6. The eye-specific promoter is a human rhodopsin kinase (hGRK1) promoter, a human rhodopsin (hRho) promoter, a human rod cGMP phosphodiesterase β subunit promoter (βPDE) promoter, a human interphotoreceptor retinoid-binding protein (IRBP) promoter, a human red / green opsin promoter, a human blue opsin (HB) promoter, human vitelliform macular dystrophy 2 (VMD2), or a human RPE65 promoter, and the AAV vector genome according to Embodiment 1 or 2.

[0247] Embodiment 7. An AAV vector genome, in the 5' to 3' direction, (a) 5' AAV inverted terminal repeat, (b) human rhodopsin (hRho) promoter, (c) 5' portion of the transgene, (d) splice donor (SD) site, (e) recombination site, (f) polynucleotide encoding a recombinase, (g) polyA site, and (h) 3' AAV inverted terminal repeat, and the AAV vector genome.

[0248] Embodiment 8. The human rhodopsin (hRho) promoter comprises, or consists of, a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 209, and the AAV vector genome according to Embodiment 7.

[0249] Embodiment 9. An enhancer is included between the 5' AAV inverted terminal repeat and the human rhodopsin (hRho) promoter, and the AAV vector genome according to Embodiment 7 or 8. <00,00921>

[0250] Embodiment 10. A CBA-MVM hybrid sequence is included between the eye-specific promoter and the 5' portion of the transgene, and the AAV vector genome according to any one of Embodiments 1 to 9. <00009,24>

[0251] Embodiment 11. The AAV vector genome according to Embodiment 10, wherein the CBA-MVM hybrid sequence includes or comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 210.

[0252] Embodiment 12. An AAV vector genome, wherein in the 5' to 3' direction, (a) 5'AAV inverted terminal repeat, (b) CMV enhancer, (c) Chicken beta-actin (CBA) promoter, (d) 5' portion of the introduced gene, (e) Splice donor (SD) site, (f) Recombination site, (g) Polynucleotide encoding a recombinase, (h) Poly A portion, and (i) The AAV vector genome comprising a 3'AAV inverted terminal repeat.

[0253] Embodiment 13. The AAV vector genome according to Embodiment 12, wherein the CMV enhancer comprises or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 218.

[0254] Embodiment 14. The AAV vector genome according to Embodiment 12 or 13, wherein the CBA promoter contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 219.

[0255] Embodiment 15. An AAV vector genome according to any one of Embodiments 12 to 14, wherein the sequence between the 5' AAV inverted terminal repeat and the 5' portion of the transgene has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 211.

[0256] Embodiment 16. An AAV vector genome according to any one of Embodiments 1 to 15, wherein the 5' portion of the transgene is at least 0.5 kb, at least 1 kb, at least 1.5 kb, at least 2 kb, or at least 2.5 kb in length.

[0257] Embodiment 17. An AAV vector genome according to any one of Embodiments 1 to 16, wherein the transgene encodes an ATP-binding cassette, subfamily A, member 4 (ABCA4) protein.

[0258] Embodiment 18. The AAV vector genome according to Embodiment 17, wherein the ABCA4 protein contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 190.

[0259] Embodiment 19. The AAV vector genome according to Embodiment 17 or 18, wherein the 5' portion of the transgene contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 212.

[0260] Embodiment 20. The 5' portion of the transgene contains one or more of the following nucleotides: (i) nucleotide "G" at a position corresponding to nucleotide 813 of SEQ ID NO: 212, (ii) nucleotide "A" at a position corresponding to nucleotide 1311 of SEQ ID NO: 212, (iii) nucleotide "A" at a position corresponding to nucleotide 2274 of SEQ ID NO: 212, (iv) nucleotide "C" at a position corresponding to nucleotide 2277 of SEQ ID NO: 212, (v) nucleotide "T" at a position corresponding to nucleotide 2278 of SEQ ID NO: 212, and (vi) nucleotide "T" at a position corresponding to nucleotide 2568 of SEQ ID NO: 212, and is the AAV vector genome according to Embodiment 19.

[0261] Embodiment 21. The 5' portion of the transgene contains all of the nucleotides (i) to (vi), and is the AAV vector genome according to Embodiment 19 or 20.

[0262] Embodiment 22. The 5' portion of the transgene encodes a polypeptide sequence containing amino acids 1 to 884 of human ABCA4 protein (SEQ ID NO: 190), and is the AAV vector genome according to any one of Embodiments 19 to 21.

[0263] Embodiment 23. The splice donor (SD) site contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 186, and is the AAV vector genome according to any one of Embodiments 1 to 22.

[0264] Embodiment 24. The recombination site contains or consists of a Lox71 site having a sequence of SEQ ID NO: 178 or having a maximum of one, two, or three nucleotide mutations (deletions, insertions, and / or substitutions) relative thereto, and is the AAV vector genome according to any one of Embodiments 1 to 23.

[0265] Embodiment 25. An AAV vector genome according to any one of Embodiments 1 to 24, wherein the expression of the recombinase is operably linked to the promoter.

[0266] Embodiment 26. The AAV vector genome according to any one of Embodiments 1 to 25, wherein the recombinase is Cre recombinase.

[0267] Embodiment 27. The AAV vector genome according to Embodiment 26, wherein the Cre recombinase comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 164.

[0268] Embodiment 28. The AAV vector genome according to Embodiment 26 or 27, wherein the polynucleotide encoding the recombinase contains a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 214, or contains an intron consisting thereof.

[0269] Embodiment 29. The AAV vector genome according to any one of Embodiments 26 to 28, wherein the polynucleotide encoding the recombinase includes a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 213.

[0270] Embodiment 30. An AAV vector genome according to any one of Embodiments 1 to 29, wherein the recombinase comprises SEQ ID NO: 165 or an N-terminal nuclear localization sequence (NLS) comprising SEQ ID NO: 165.

[0271] Embodiment 31. The AAV vector genome according to any one of Embodiments 1 to 30, wherein the AAV vector genome comprises a polynucleotide encoding an internally cleaved polypeptide located between the recombination site and the polynucleotide encoding the recombinase, and the 5' portion of the transgene, the polynucleotide encoding the internally cleaved polypeptide, and the polynucleotide encoding the recombinase are all within the same reading frame.

[0272] Embodiment 32. The AAV vector genome according to Embodiment 31, wherein the internally cleaved polypeptide is a self-cleaving peptide selected from the group consisting of T2A, P2A, E2A, and F2A, and optionally the self-cleaving peptide is a P2A peptide containing or consisting of SEQ ID NO: 181.

[0273] Embodiment 33. An AAV vector genome according to any one of Embodiments 1 to 32, wherein the polyA region includes, or consists of, a sequence having up to one, up to two, up to three, up to four, up to five, or up to six nucleotide mutations (deletions, insertions, and / or substitutions) therewith, SEQ ID NO: 189.

[0274] Embodiment 34. The AAV vector genome according to any one of Embodiments 1 to 33, wherein the AAV vector genome includes an internal ribosome entry site (IRES) located between the recombination site and the polynucleotide encoding the recombinase, and the IRES is operably linked to the polynucleotide encoding the recombinase.

[0275] Embodiment 35. An AAV vector genome, wherein in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b)(b-1) A Lox JTZ17 recombinant site comprising or consisting of a sequence containing up to one, up to two, or up to three nucleotide mutations (deletions, insertions, and / or substitutions) of sequence number 217, or (b-2) A Lox 66 recombinant site comprising or consisting of a sequence containing up to one, up to two, or up to three nucleotide mutations (deletions, insertions, and / or substitutions) of sequence number 179, (c) Splice acceptor (SA) site, (d) The 3' portion of the introduced gene, (e) Poly A portion, and (f) The AAV vector genome comprising a 3'AAV inverted terminal repeat.

[0276] Embodiment 36. The AAV vector genome according to Embodiment 35, wherein the splice acceptor (SA) site contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 187.

[0277] Embodiment 37. The AAV vector genome according to Embodiment 35 or 36, wherein the introduced gene encodes the ABCA4 protein.

[0278] Embodiment 38. The AAV vector genome according to Embodiment 37, wherein the 3' portion of the transgene contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 193.

[0279] Embodiment 39. An AAV vector genome according to any one of Embodiments 35 to 38, wherein the polyA region includes, or consists of, a sequence having up to one, up to two, up to three, up to four, up to five, or up to six nucleotide mutations (deletions, insertions, and / or substitutions) therewith, SEQ ID NO: 189.

[0280] Embodiment 40. The AAV vector genome according to any one of Embodiments 1 to 39, wherein the 5' AAV inverted terminal repeat contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 215.

[0281] Embodiment 41. The AAV vector genome according to any one of Embodiments 1 to 40, wherein the 3' AAV inverted terminal repeat contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 216.

[0282] Embodiment 42. AAV virus particle, (i) an AAV capsid comprising an AAV capsid protein, the AAV capsid and (ii) The AAV virus particle comprising the AAV vector genome described in any one of Embodiments 1 to 41.

[0283] Embodiment 43. An AAV virus particle according to Embodiment 42, wherein the AAV capsid protein contains or consists of a sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a sequence selected from SEQ ID NOs: 1-3, 67, 71, 196, 205, and 206.

[0284] Embodiment 44. The AAV virus particle according to Embodiment 42, wherein the AAV capsid protein contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3.

[0285] Embodiment 45. The AAV virus particle according to Embodiment 42, wherein the AAV capsid protein contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 67.

[0286] Embodiment 46. A pharmaceutical composition comprising an AAV vector genome described in any one of Embodiments 1 to 41 or an AAV virus particle described in any one of Embodiments 42 to 45.

[0287] Embodiment 47. A pharmaceutical composition, (i) A first AAV virus particle comprising the AAV vector genome described in any one of Embodiments 1 to 34 and 40 to 41, (ii) The pharmaceutical composition comprising a second AAV virus particle containing an AAV vector genome as described in any one of Embodiments 35 to 41.

[0288] Embodiment 48. A pharmaceutical composition, (i) A first AAV virus particle comprising the AAV vector genome described in any one of Embodiments 1 to 34 and 40 to 41, (ii) An AAV vector genome, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Recombination site, (c) Splice acceptor (SA) site, (d) The 3' portion of the introduced gene, (e) Poly A portion, and (f) The pharmaceutical composition comprising a second AAV virus particle containing the AAV vector genome, which includes a 3'AAV inverted terminal repeat.

[0289] Embodiment 49. A method for expressing a protein encoded by a transgene in a cell, (1) Transduction of the cells with a first AAV virus particle containing the AAV vector genome described in any one of Embodiments 1 to 34 and 40 to 41, (2) The method comprising transducing the cells with a second AAV virus particle containing the AAV vector genome described in any one of Embodiments 35 to 41.

[0290] Embodiment 50. A method for expressing a protein encoded by a transgene in a cell, (1) Transduction of the cells with a first AAV virus particle containing the AAV vector genome described in any one of Embodiments 1 to 34 and 40 to 41, (2) The cell is given an AAV vector genome, which is in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Recombination site, (c) Splice acceptor (SA) site, (d) The 3' portion of the introduced gene, (e) Poly A portion, and (f) The method comprising transducing a second AAV virus particle containing the AAV vector genome, which contains a 3'AAV inverted terminal repeat.

[0291] Embodiment 51. A method for treating a subject having a disease or disorder, (1) The subject is administered a first AAV virus particle containing the AAV vector genome described in any one of Embodiments 1 to 34 and 40 to 41, (2) The method comprising administering to the subject a second AAV virus particle containing the AAV vector genome described in any one of Embodiments 35 to 41.

[0292] Embodiment 52. A method for treating a subject having a disease or disorder, (1) The subject is administered a first AAV virus particle containing the AAV vector genome described in any one of Embodiments 1 to 34 and 40 to 41, (2) The subject is an AAV vector genome, which is oriented in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Recombination site, (c) Splice acceptor (SA) site, (d) The 3' portion of the introduced gene, (e) Poly A portion, and (f) The method comprising administering a second AAV virus particle comprising the AAV vector genome, which includes a 3'AAV inverted terminal repeat.

[0293] Embodiment 53. The pharmaceutical composition according to Embodiment 47 or 48, or the method according to any one of Embodiments 49 to 52, wherein the recombination sites in the AAV vector genome of the first AAV virus particle and the recombination sites in the AAV vector genome of the second AAV virus particle prevent reverse recombination.

[0294] Embodiment 54. A pharmaceutical composition according to any one of Embodiments 47-48 and 53, or the method according to any one of Embodiments 49-53, wherein the AAV virus particles contain a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a sequence selected from SEQ ID NOs: 1-3, 67, 71, 196, 205, and 206, or an AAV capsid protein comprising such a sequence.

[0295] Embodiment 55. The pharmaceutical composition or method according to Embodiment 54, wherein the AAV capsid protein contains or comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3.

[0296] Embodiment 56. The pharmaceutical composition or method according to Embodiment 54, wherein the AAV capsid protein contains or comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 67.

[0297] Embodiment 57. The method according to any one of Embodiments 49 to 56, wherein administration of the first AAV virus particle and the second AAV virus particle results in recombination of the first AAV vector genome and the second AAV vector genome via the recombination site in the AAV vector genome of the first AAV virus particle and the recombination site in the second AAV vector genome of the second AAV virus particle.

[0298] Embodiment 58. The method according to any one of Embodiments 49 to 57, wherein the administration of the first AAV virus particle and the second AAV virus particle results in complete transgene expression.

[0299] Embodiment 59. The method according to any one of Embodiments 51 to 58, wherein the disease or disorder is Stargardt disease.

[0300] Embodiment 60. The method according to any one of Embodiments 49 to 59, wherein the protein is the ABCA4 protein, or the disease or disorder is caused by a deficiency in the ABCA4 gene.

[0301] Embodiment 61. The method according to Embodiment 60, wherein the ABCA4 gene deficiency results in one or more conditions selected from the group consisting of decreased expression of ABCA4 protein, elimination of ABCA4 protein expression, expression of mutant ABCA4 protein, and impaired function of ABCA4 protein.

[0302] Embodiment 62. The method according to any one of Embodiments 49 to 61, wherein the first AAV virus particle and the second AAV virus particle are administered simultaneously.

[0303] Embodiment 63. The method according to any one of Embodiments 49 to 61, wherein the first AAV virus particle is administered before or after the second AAV virus particle.

[0304] Embodiment 64. The method according to any one of Embodiments 51 to 63, wherein the virus particles are administered by intravitreous, pararetinal, or subretinal injection, optionally by subretinal injection.

[0305] Embodiment 65. The method according to any one of Embodiments 49 to 64, wherein stable expression of the recombinase is not detected in the transduced cells or the subject.

[0306] Embodiment 66. Transduced cells, (i) Endogenous gene deletion genomic copies, (ii) Recombinant nucleic acids, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Photoreceptor-retinoid-binding protein (IRBP) enhancer, (c) Ocular specific promoter, (d) The 5' portion of the transgene, which is a working copy of the endogenous gene in which the transgene is missing, (e) Splice donor (SD) site, (f) Recombination site, (g) Splice acceptor (SA) site, (h) The 3' portion of the introduced gene, (i) PolyA portion, and Transduced cells comprising the recombinant nucleic acid comprising (j)3'AAV inverted terminal repeat.

[0307] Embodiment 67. Transduced cells, (i) Endogenous gene deletion genomic copies, (ii) Recombinant nucleic acids, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Optionally, enhancer, (c) Human rhodopsin (hRho) promoter, (d) The 5' portion of the transgene, which is a working copy of the endogenous gene in which the transgene is missing, (e) Splice donor (SD) site, (f) Recombination site, (g) Splice acceptor (SA) site, (h) The 3' portion of the introduced gene, (i) PolyA portion, and Transduced cells comprising the recombinant nucleic acid comprising (j)3'AAV inverted terminal repeat.

[0308] Embodiment 68. Transduced cells, (i) Endogenous gene deletion genomic copies, (ii) Recombinant nucleic acids, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) CMV enhancer, (c) Chicken beta-actin (CBA) promoter, (d) The 5' portion of the transgene, which is a working copy of the endogenous gene in which the transgene is missing, (e) Splice donor (SD) site, (f) Recombination site, (g) Splice acceptor (SA) site, (h) The 3' portion of the introduced gene, (i) PolyA portion, and Transduced cells comprising the recombinant nucleic acid comprising (j)3'AAV inverted terminal repeat.

[0309] Embodiment 69. A transduced cell according to any one of Embodiments 66 to 68, wherein the deficient endogenous gene is the ABCA4 gene and the transgene encodes the ABCA4 protein.

[0310] Embodiment 70. A transdextrin cell according to any one of Embodiments 66 to 69, wherein the transdextrin cell or the recombinant nucleic acid comprises a nucleic acid sequence comprising a 5'AAV inverted terminal repeat, a recombination site, a polynucleotide encoding a recombinase, and a 3'AAV inverted terminal repeat in the 5' to 3' direction, and the nucleic acid sequence lacks a promoter.

[0311] Embodiment 71. A transduced cell according to Embodiment 70, wherein the nucleic acid sequence is located downstream of the 3' end of the (ii)(j)3'AAV inverted terminal repeat.

[0312] Embodiment 72. Transduced cells according to any one of Embodiments 66 to 71, wherein the cells are ex vivo cells.

[0313] Embodiment 73. Transduced cells according to any one of Embodiments 66 to 72, wherein the cells are photoreceptor cells.

[0314] Embodiment 74. A nucleic acid comprising the 5' portion of the ABCA4 gene, wherein the 5' portion of ABCA4 comprises one or more of the following: (i) nucleotide "G" at the position corresponding to nucleotide 813 of sequence number 212, (ii) nucleotide "A" at the position corresponding to nucleotide 1311 of sequence number 212, (iii) nucleotide "A" at the position corresponding to nucleotide 2274 of sequence number 212, (iv) nucleotide "C" at the position corresponding to nucleotide 2277 of sequence number 212, (v) nucleotide "T" at the position corresponding to nucleotide 2278 of sequence number 212, and (vi) nucleotide "T" at the position corresponding to nucleotide 2568 of sequence number 212.

[0315] Embodiment 75. The nucleic acid according to Embodiment 74, comprising two or more, three or more, four or more, five or more, or all six of the nucleotides (i) to (vi).

[0316] Embodiment 76. The nucleic acid according to Embodiment 74 or 75, wherein the 5' portion of the ABCA4 gene is at least 1 kb, at least 1.5 kb, at least 2 kb, or at least 2.5 kb in length.

[0317] Embodiment 77. The nucleic acid according to any one of Embodiments 74 to 76, wherein the 5' portion of the ABCA4 gene contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 212.

[0318] Embodiment 78. The nucleic acid according to Embodiment 77, wherein the 5' portion of the introduced gene encodes a polypeptide sequence containing amino acids 1 to 884 of the human ABCA4 protein (SEQ ID NO: 190).

[0319] Embodiment 79. An AAV vector genome comprising the nucleic acid described in any one of Embodiments 74 to 78.

[0320] Embodiment 80. AAV virus particle, (i) an AAV capsid comprising an AAV capsid comprising an AAV capsid protein, and (ii) an AAV vector genome as described in Embodiment 79, comprising the AAV virus particle.

[0321] Embodiment 81. A pharmaceutical composition comprising the AAV vector genome described in Embodiment 79 or the AAV virus particles described in Embodiment 80.

[0322] Embodiment 82. A method for treating a subject having a disease or disorder, comprising administering to the subject the AAV virus particles described in Embodiment 80, wherein the disease or disorder is optionally Stargardt disease.

[0323] Embodiment 83. Transduced cells comprising the nucleic acid described in any one of Embodiments 74 to 78. [Examples]

[0324] Example 1: Plasmid construction using bipartite design Several plasmids were generated, and the expression of the full-length ABCA4 gene in cells was tested using a bipartite vector system (Figure 2).

[0325] A plasmid called "5'ABCA4 -FLAG +Cre" was constructed, containing a 5'AAV inverted terminal repeat, a U1a promoter, the 5' portion of the ABCA4 gene ORF, a splice donor (SD) site, a Lox71 site, a nucleic acid sequence encoding the T2A peptide, a nucleic acid sequence encoding Cre recombinase with a nuclear localization sequence (NLS), a polyA site, and a 3'AAV inverted terminal repeat, from 5' to 3'.

[0326] The "5'ABCA4 -FLAG +Cre" plasmid was constructed by incorporating a 3xFLAG tag code sequence into the 5' portion of the ABCA4 gene in the "5'ABCA4 -FLAG +Cre" plasmid.

[0327] The "5'ABCA4 +FLAG +Cre" plasmid was generated by removing the Cre recombinase coding sequence from the "5'ABCA4 +FLAG +Cre" plasmid.

[0328] A "3'ABCA4 +FLAG" plasmid was constructed containing the 3' portion of the ABCA4 gene ORF, a polyA region, and a 3'AAV inverted terminal repeat, along with a 5'AAV inverted terminal repeat, a Lox66 site, a splice acceptor (SA) site, a 3xFLAG tag coding sequence, and the 3'AAV inverted terminal repeat, from 5' to 3'.

[0329] Two positive control plasmid constructs containing the full-length ABCA4 gene were also generated (Figure 2). One positive control construct lacked an intron region, while the other positive control contained an intron region including a splice donor (SD) site, a Lox71 site, and a splice acceptor (SA) site, mimicking the construct produced by recombination between the "5'ABCA4 +FLAG +Cre" plasmid and the "3'ABCA4 +FLAG" plasmid. Both positive control constructs contained the 3xFLAG tag coding sequence in both the 5' and 3' portions of the ABCA4 gene.

[0330] Example 2: Expression of full-length protein using a bipartite vector system Cell studies were performed using HEK293 cells transfected with the plasmids indicated in Example 1 and Figure 2, and the expression of full-length ABCA4 protein was examined by Western blotting. The full-length ABCA4 protein (with the FLAG tag attached) has an estimated molecular weight of approximately 261 kD. As shown in Figure 3A, expression of full-length ABCA4 protein was observed only when both the "5'ABCA4 + FLAG + Cre" plasmid and the "3'ABCA4 + FLAG" plasmid were applied to cells transfected with plasmids other than the positive control. On the other hand, expression of full-length ABCA4 protein was not observed in cells transfected with the Cre recombinase-free dual vector system (i.e., the "5'ABCA4 + FLAG - Cre" and "3'ABCA4 + FLAG" pair). Similarly, in repeated experiments (Figure 3B), only two experimental groups showed expression of full-length ABCA4 among the cell populations transfected with the indicated plasmids other than the positive control. One group was transfected with the "5'ABCA4 +FLAG +Cre" plasmid and the "3'ABCA4 +FLAG" plasmid, while the other group was transfected with the "5'ABCA4 -FLAG +Cre" plasmid and the "3'ABCA4 +FLAG" plasmid. These results demonstrate that the dual vector system enables efficient Cre-lox-mediated recombination between these two plasmids, ultimately leading to the expression of full-length ABCA4 protein.

[0331] Example 3: Cre recombinase expression is significantly reduced after recombination of the AAV vector genome. The expression level of Cre recombinase in plasmid-containing cells was analyzed (Figure 4). HEK293 cells were transfected with various plasmids and controls described in Example 2. 48 hours after transfection, cell samples were treated with sample buffer containing 4M urea to quantify stable Cre recombinase expression by Western blotting using 3% milk as a blocker, rabbit anti-Cre mAb (Cell signaling technology, no. 15036) as the primary antibody at a 1:10,000 dilution, and goat anti-rabbit-HRP (azur) as the secondary antibody at a 1:10,000 dilution. Autocleaved Cre recombinase has a molecular weight of approximately 37 kD. As expected, transfection with either the "5'ABCA4-FLAG + Cre" plasmid or the "5'ABCA4 + FLAG + Cre" plasmid resulted in clear expression of Cre recombinase in HEK293 cells. Notably, in cells co-transfected with the second plasmid "3'ABCA4 +FLAG", Cre recombinase expression was significantly lower. This suggests that the dual-vector system enables efficient recombination between the first and second plasmids, leading to the generation of recombinant nucleic acids containing Cre recombinase ORF without a promoter, and thus resulting in a significant decrease in Cre recombinase expression.

[0332] Example 4: Cre-mediated recombination of a bipartite vector system achieves expression of full-length ABCA4 mRNA. We quantified the expression levels of full-length ABCA4 mRNA in cells transfected with various plasmids. As shown in Figure 5, cells transfected with dual vector systems encoding Cre recombinase ("5'ABCA4 + FLAG + Cre" and "3'ABCA4 + FLAG") showed approximately 55-fold higher full-length ABCA4 mRNA expression levels compared to cells transfected with the corresponding dual vector systems that do not encode Cre recombinase ("5'ABCA4 + FLAG - Cre" and "3'ABCA4 + FLAG"). These results indicate that Cre recombinase effectively mediates recombination between these two initiation vectors immediately after transfecting cells with these two initiation vectors, driving the expression of full-length mRNA.

[0333] Example 5: Delivery of a bipartite vector system via dual AAV virus particles results in the expression of full-length ABCA4 mRNA. We tested whether a dipartite vector system could be delivered by AAV virus particles to express full-length ABCA4 mRNA in cells. Two types of AAV9 virus particles were prepared using the expression cassette described in Example 1. One type of AAV9 virus particle contained an AAV vector genome encoding "5'ABCA4 +FLAG +Cre", and the other type of AAV9 virus particle contained an AAV vector genome encoding "3'ABCA4 +FLAG". As shown in Figure 6, ABCA4-negative cells were transduced with the instructed AAV9 virus particles, and the expression level of full-length ABCA4 mRNA was quantified in each group. Only cells transduced with both types of AAV9 virus particles exhibited high expression levels of full-length ABCA4 mRNA, and the expression level continued to increase over 120 hours after transduction. This result demonstrates that AAV virus particles can effectively deliver a dipartite vector system within the same cell and be recombined so that the mRNA payload is properly reconstituted by the dipartite vector system.

[0334] Example 6: The hABCA4 dual AAV vector system achieved robust in vivo expression of full-length recombinant RNA encoding hABCA4 in wild-type mice. We evaluated an AAV dual-vector system designed for full-size human ABCA4 (hABCA4) protein expression in photoreceptors, delivered by subretinal injection to 4-week-old B6129SF1 / J mice. Two AAV virus particles, AAV8 and AAV214, were tested according to the study design in Table 8 below. Animals received one dose on day 1 of the study.

[0335] [Table 8]

[0336] On day 28 (±1 day), all animals were euthanized by CO2 asphyxiation followed by cervical vertebral dislocation. Both eyes were collected from each animal, and the lenses were dissected. Each eye sample was placed in an RNase / DNase-free tube and flash-frozen in liquid nitrogen.

[0337] Total mouse RNA was isolated using the RNeasy Mini Kit (QIAGEN). RNA quantification was performed using a Nanodrop spectrophotometer (THERMOFISHER). Cre-dependent ABCA4 gene expression was evaluated by qPCR assay. Seven mouse eyes from each test group (untreated, AAV8 ABCA4 N13 / C17 vector administered, and AAV214 ABCA4 N13 / C17 vector administered) were used for protein extraction. The mouse eyes were suspended in RIPA lysis buffer supplemented with protease inhibitors and disrupted using a FastPrep Bead Beating Grinder and Lysis System (MP BIOMEDICALS) as recommended by the manufacturer.

[0338] ABCA4 and Cre gene expression analysis: As shown in Figure 8, mouse ABCA4 gene expression was successfully detected in all prepared test samples, ranging from 5.5E+4 copies to 6.2E+5 copies per qPCR reaction. Human ABCA4 gene expression was successfully detected in all administered mice, ranging from 117 copies (0.18% of mABCA4 RNA) to 6.5E+5 copies (245% of mABCA) per 10 ng of total RNA input. Fewer than 100 copies of hABCA4 were detected in 2 out of 14 administered animals (214 OS and 214 OD).

[0339] These results confirmed Cre-dependent hABCA4 recombination in administered mice. No statistically significant difference (p=0.29) was observed in ABCA4 gene expression between AAV8 capsid and AAV214 capsid.

[0340] Human Cre gene expression was not detected in untreated animals. Cre gene expression in treated mice ranged from 593 copies to 1.4E+5 copies per 10 ng of total RNA input. Fewer than 100 Cre copies were detected in 2 of the 14 treated animals tested (214 OS and 214 OD). In this study, Cre gene expression correlated well with hABCA4 gene expression.

[0341] ABCA4 and Cre protein expression analysis: Protein lysates were probed on an automated Western blotting platform, and Figure 9 shows the signals for endogenous mouse ABCA4, recombinant human ABCA4 (FLAG-tagged), and Cre recombinase. Endogenous mouse ABCA4 protein was detected in all mouse eye protein lysates. The molecular weight of the ABCA4 protein band was an average of 226 kDa. The amount of mouse ABCA4 protein ranged from 2.8E+4 chemiluminescent units to 1.9E+5 chemiluminescent units (measured as protein peaks).

[0342] Human recombinant ABCA4 signaling was detected using NOVUS BIOLOGICALS' rabbit monoclonal anti-FLAG antibody MAB8529. The corresponding human ABCA4 protein signal was observed only in 216 OS mouse eye lysates. This signal was confirmed by manual Western blotting assay.

[0343] Cre recombinase protein was not detected in untreated mouse eyes. Transgenic Cre recombinase protein was detected in all treated mouse eyes and also in positive control samples. The molecular weight of the detected Cre protein band was an average of 38 kDa. 216 OS mouse eye lysates (which produced detectable transgenic human ABCA4 protein) also had the highest levels of detectable transgenic Cre recombinase.

[0344] Conclusion: Overall, the results of this proof-of-concept in vivo trial demonstrated that Cre recombinase can induce full-size human ABCA4 gene (mRNA) expression after subretinal administration of a partially hABCA4 expression cassette capsidized to AAV8 or AAV214 virions. Further optimization of regulatory sequences (e.g., within the promoter region) would be desirable to achieve higher hABCA4 protein expression.

[0345] Example 7: Optimized regulatory sequences in the bipartite AAV system resulted in increased hABCA4 protein expression. We evaluated different regulatory sequences for hABCA4 protein expression using bipartite AAV systems. As shown in Figure 10, four novel bipartite AAV systems with novel regulatory sequences were tested in ABCA4(- / -) mice. The bipartite AAV viral vectors for each group were capsidized onto AAV8, and 1 μL of AAV viral particles were subretinally administered to deliver 3.0E+9vg to the eyes of eight mice. For each bipartite AAV system, the ratio of the first AAV vector genome to the second AAV vector genome was maintained at 1:1. Four weeks after administration, all 16 eye samples were collected from each group. Of these, six eyes were subjected to RTqPCR, six eyes to Western blotting, and the remaining four eyes to IHC.

[0346] For Western blotting analysis, mouse retinas were suspended in RIPA lysis buffer supplemented with protease inhibitors and disrupted using a FASTPREP bead beating grinder and lysis system. The protein lysates were processed and then probed on an automated Western blotting platform using an anti-HA tag antibody to confirm the recombination event and estimate the amount of transgenic human ABCA4 protein accumulated in mouse eyes one month after injection.

[0347] Design of test groups and two-part AAV systems Group 1 mice served as controls and underwent simulated treatment.

[0348] In group 2 (novel hGRK1 promoter), the first AAV vector genome pA-ABCA4.N101 is oriented from 5' to 3'. - 5'AAV ITR according to sequence number 215, - IRBP enhancer according to sequence number 208, - hGRK1 promoter according to Sequence ID No. 188, - CBA-MVM hybrid sequence according to sequence number 210, - The 5' portion of the hABCA4 gene according to sequence number 212, - Splice donor (SD) site according to sequence number 186, - Lox71 recombinant site according to sequence number 178, - P2A (Sequence ID 181) code sequence, - A polynucleotide encoding Cre recombinase (SEQ ID NO: 164) having a nuclear localization signal (SEQ ID NO: 165) and an intron (SEQ ID NO: 214), - SPA49 polyA moiety according to Sequence ID No. 189, and - Includes 3'AAV ITR, following sequence number 216. pA-ABCA4.N101 was encoded by a vector containing sequence number 220.

[0349] In group 3 (hRho promoter), the first AAV vector genome pA-ABCA4.N103 contains the same sequence as pA-ABCA4.N101 except for the hRho promoter (sequence number 209), in which the hGRK1 promoter sequence is used instead. pA-ABCA4.N103 was encoded by a vector containing sequence number 221.

[0350] In group 4 (βPDE promoter), the first AAV vector genome pA-ABCA4.N105 contains the same sequence as pA-ABCA4.N101 except for the βPDE promoter (sequence number 200), in which the hGRK1 promoter sequence is used instead. pA-ABCA4.N105 was encoded by a vector containing sequence number 222.

[0351] In group 5 (CBh promoter), the first AAV vector genome pA-ABCA4.N107 contains the same sequence as pA-ABCA4.N101 except for the CBh promoter (which includes SEQ ID NO: 211, CMV enhancer, chicken β-actin promoter, and CVA exon), in which the latter uses an IRBP enhancer instead for the CBA-MVM hybrid sequence region. pA-ABCA4.N107 was encoded by a vector containing SEQ ID NO: 223.

[0352] For each of groups 2 to 5, the second AAV vector genome pA-ABCA4.C77 is oriented from 5' to 3'. - 5'AAV ITR according to sequence number 215, - Lox JTZ17 recombinant site according to Sequence ID No. 217, - Splice acceptor (SA) site according to sequence number 187, - The 3' portion of the hABCA4 gene following sequence number 193, which has a C-terminal HA tag coding sequence, - SPA49 polyA moiety according to Sequence ID No. 189, and - Includes 3'AAV ITR, following sequence number 216. pA-ABCA4.C77 was encoded by a vector containing sequence number 224.

[0353] Schematic diagrams of all these vectors are shown in Figure 11A. Cre-mediated recombination of the first AAV vector genome and the second AAV vector genome is shown in Figure 11B.

[0354] Test results As shown in Figure 12A, robust expression of the full-length hABCA4 protein was detected in most mice in groups 3 and 4 (with photoreceptor-specific hRho or βPDE promoters) and group 5 (with a universal CBh promoter) that were administered the novel bipartite AAV system. In contrast, protein expression was not detected much with the previous promoter designs in Example 6 (shown in Figure 12B) and group 2 (both with hGRK1 promoters).

[0355] Subsequently, Cre recombinase expression was evaluated in groups 1 and 3-5. Protein lysate samples were probed with anti-Cre antibody and anti-Brn3a antibody, and retested with anti-HA antibody. As shown in Figure 13A, Cre recombinase expression was detected in many eye samples that showed hABCA4 protein expression. However, in contrast to the strong correlation detected in the previous in vitro study using HEK293 cells, there was no strong correlation between Cre protein expression and hABCA4 protein expression in this animal study (Figure 13B).

[0356] In summary, the bipartite AAV system with a novel promoter resulted in more robust in vivo expression of the hABCA4 protein without a strong correlation with Cre recombinase expression. Therefore, these bipartite AAV systems have the potential to achieve selectively high expression of the target protein while simultaneously reducing recombinase expression.

[0357] While preferred embodiments of the Disclosure are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Those skilled in the art can perform numerous modifications, alterations, and substitutions without departing from the Disclosure. It should be understood that various alternative forms of the embodiments of the Disclosure described herein may be used when performing the Disclosure.

[0358] Embedding by reference All references, articles, publications, patents, patent gazettes, and patent applications cited herein are incorporated in their entirety by reference for all purposes. However, references to references, articles, publications, patents, patent gazettes, and patent applications cited herein should not be construed as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country worldwide.

Claims

1. AAV vector genome, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Photoreceptor-retinoid-binding protein (IRBP) enhancer, (c) Ocular specific promoter, (d) The 5' portion of the introduced gene, (e) Splice donor (SD) site, (f) Recombination site, (g) Polynucleotide encoding a recombinase, (h) Poly A portion, and (i) The AAV vector genome comprising a 3' AAV inverted terminal repeat.

2. The AAV vector genome according to claim 1, wherein the IRBP enhancer comprises or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

208.

3. The AAV vector genome according to claim 1 or 2, wherein the eye-specific promoter is a human rod cGMP phosphodiesterase β-subunit promoter (βPDE) promoter comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

200.

4. The AAV vector genome according to claim 1 or 2, wherein the eye-specific promoter is a human rhodopsin (hRho) promoter comprising or consisting of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

209.

5. The AAV vector genome according to claim 1 or 2, wherein the eye-specific promoter is a human rhodopsin kinase (hGRK1) promoter comprising or consisting of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

188.

6. The AAV vector genome according to claim 1 or 2, wherein the eye-specific promoter is a human rhodopsin kinase (hGRK1) promoter, a human rhodopsin (hRho) promoter, a human rod cGMP phosphodiesterase β-subunit promoter (βPDE) promoter, a human photoreceptor-retinoid-binding protein (IRBP) promoter, a human red / green opsin promoter, a human blue opsin (HB) promoter, a human vitiligo macular dystrophy 2 (VMD2), or a human RPE65 promoter.

7. AAV vector genome, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Human rhodopsin (hRho) promoter, (c) The 5' portion of the introduced gene, (d) Splice donor (SD) site, (e) Recombinant site, (f) Polynucleotide encoding a recombinase, (g) Poly A portion, and (h) The AAV vector genome comprising a 3' AAV inverted terminal repeat.

8. The AAV vector genome according to claim 7, wherein the human rhodopsin (hRho) promoter contains or comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

209.

9. The AAV vector genome according to claim 7 or 8, comprising an enhancer between the 5' AAV inverted terminal repeat and the human rhodopsin (hRho) promoter.

10. The AAV vector genome according to any one of claims 1 to 9, comprising a CBA-MVM hybrid sequence between the eye-specific promoter and the 5' portion of the transgene.

11. The AAV vector genome according to claim 10, wherein the CBA-MVM hybrid sequence includes or comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

210.

12. AAV vector genome, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) CMV enhancer, (c) Chicken beta-actin (CBA) promoter, (d) The 5' portion of the introduced gene, (e) Splice donor (SD) site, (f) Recombination site, (g) Polynucleotide encoding a recombinase, (h) Poly A portion, and (i) The AAV vector genome comprising a 3' AAV inverted terminal repeat.

13. The AAV vector genome according to claim 12, wherein the CMV enhancer comprises or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

218.

14. The AAV vector genome according to claim 12 or 13, wherein the CBA promoter contains or comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

219.

15. The AAV vector genome according to any one of claims 12 to 14, wherein the 5' AAV inverted terminal repeat and the 5' portion of the transgene have a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

211.

16. The AAV vector genome according to any one of claims 1 to 15, wherein the 5' portion of the introduced gene is at least 0.5 kb, at least 1 kb, at least 1.5 kb, at least 2 kb, or at least 2.5 kb in length.

17. The AAV vector genome according to any one of claims 1 to 16, wherein the introduced gene encodes an ATP-binding cassette, subfamily A, member 4 (ABCA4) protein.

18. The AAV vector genome according to claim 17, wherein the ABCA4 protein contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

190.

19. The AAV vector genome according to claim 17 or 18, wherein the 5' portion of the introduced gene contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

212.

20. The AAV vector genome according to claim 19, wherein the 5' portion of the introduced gene includes one or more of the following nucleotides: (i) nucleotide "G" at the position corresponding to nucleotide 813 of SEQ ID NO: 212, (ii) nucleotide "A" at the position corresponding to nucleotide 1311 of SEQ ID NO: 212, (iii) nucleotide "A" at the position corresponding to nucleotide 2274 of SEQ ID NO: 212, (iv) nucleotide "C" at the position corresponding to nucleotide 2277 of SEQ ID NO: 212, (v) nucleotide "T" at the position corresponding to nucleotide 2278 of SEQ ID NO: 212, and (vi) nucleotide "T" at the position corresponding to nucleotide 2568 of SEQ ID NO:

212.

21. The AAV vector genome according to claim 19 or 20, wherein the 5' portion of the introduced gene comprises all of the nucleotides (i) to (vi).

22. The AAV vector genome according to any one of claims 19 to 21, wherein the 5' portion of the introduced gene encodes a polypeptide sequence containing amino acids 1 to 884 of the human ABCA4 protein (SEQ ID NO: 190).

23. The AAV vector genome according to any one of claims 1 to 22, wherein the splice donor (SD) site includes or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with sequence number 186.

24. The AAV vector genome according to any one of claims 1 to 23, wherein the recombination site is a Lox71 site comprising sequence number 178, or a sequence having up to one, up to two, or up to three nucleotide mutations (deletions, insertions, and / or substitutions) therewith.

25. The AAV vector genome according to any one of claims 1 to 24, wherein the expression of the recombinase is operably linked to the promoter.

26. The AAV vector genome according to any one of claims 1 to 25, wherein the recombinase is Cre recombinase.

27. The AAV vector genome according to claim 26, wherein the Cre recombinase comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

164.

28. The AAV vector genome according to claim 26 or 27, wherein the polynucleotide encoding the recombinase contains a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 214, or contains an intron consisting of such a sequence.

29. The AAV vector genome according to any one of claims 26 to 28, wherein the polynucleotide encoding the recombinase includes a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

213.

30. The AAV vector genome according to any one of claims 1 to 29, wherein the recombinase comprises SEQ ID NO: 165 or an N-terminal nuclear localization sequence (NLS) consisting thereof.

31. The AAV vector genome according to any one of claims 1 to 30, wherein the AAV vector genome comprises a polynucleotide encoding an internally cleaved polypeptide located between the recombination site and the polynucleotide encoding the recombinase, and the 5' portion of the transgene, the polynucleotide encoding the internally cleaved polypeptide, and the polynucleotide encoding the recombinase are all within the same reading frame.

32. The AAV vector genome according to claim 31, wherein the internally cleaved polypeptide is a self-cleaving peptide selected from the group consisting of T2A, P2A, E2A, and F2A, and optionally the self-cleaving peptide is a P2A peptide containing or consisting of SEQ ID NO:

181.

33. The AAV vector genome according to any one of claims 1 to 32, wherein the polyA region includes or consists of a sequence having up to one, up to two, up to three, up to four, up to five, or up to six nucleotide mutations (deletions, insertions, and / or substitutions) therewith, or is a sequence thereof, i.e., SEQ ID NO:

189.

34. The AAV vector genome according to any one of claims 1 to 33, wherein the AAV vector genome includes an internal ribosome entry site (IRES) located between the recombination site and the polynucleotide encoding the recombinase, and the IRES is operably linked to the polynucleotide encoding the recombinase.

35. AAV vector genome, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) (b-1) A Lox JTZ17 recombinant site comprising or consisting of a sequence containing up to one, up to two, or up to three nucleotide mutations (deletions, insertions, and / or substitutions) of sequence number 217, or (b-2) A Lox 66 recombinant site comprising or consisting of a sequence containing up to one, up to two, or up to three nucleotide mutations (deletions, insertions, and / or substitutions) of sequence number 179, (c) Splice acceptor (SA) site, (d) The 3' portion of the introduced gene, (e) Poly A portion, and (f) The AAV vector genome comprising a 3' AAV inverted terminal repeat.

36. The AAV vector genome according to claim 35, wherein the splice acceptor (SA) site includes or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with sequence number 187.

37. The AAV vector genome according to claim 35 or 36, wherein the introduced gene encodes the ABCA4 protein.

38. The AAV vector genome according to claim 37, wherein the 3' portion of the introduced gene contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

193.

39. The AAV vector genome according to any one of claims 35 to 38, wherein the polyA region includes or consists of a sequence having up to one, up to two, up to three, up to four, up to five, or up to six nucleotide mutations (deletions, insertions, and / or substitutions) therewith, or is a sequence having up to one, up to two, up to three, up to four, up to five, or up to six nucleotide mutations therewith.

40. The AAV vector genome according to any one of claims 1 to 39, wherein the 5' AAV inverted terminal repeat includes or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

215.

41. The AAV vector genome according to any one of claims 1 to 40, wherein the 3' AAV inverted terminal repeat includes or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

216.

42. AAV virus particles, (i) AAV capsid, wherein the AAV capsid contains an AAV capsid protein, (ii) The AAV virus particle comprising the AAV vector genome according to any one of claims 1 to 41.

43. The AAV virus particle according to claim 42, wherein the AAV capsid protein contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a sequence selected from SEQ ID NOs: 1-3, 67, 71, 196, 205, and 206.

44. The AAV virus particle according to claim 42, wherein the AAV capsid protein contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

3.

45. The AAV virus particle according to claim 42, wherein the AAV capsid protein contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

67.

46. A pharmaceutical composition comprising an AAV vector genome according to any one of claims 1 to 41 or an AAV virus particle according to any one of claims 42 to 45.

47. A pharmaceutical composition, (i) A first AAV virus particle comprising the AAV vector genome described in any one of claims 1 to 34 and 40 to 41, (ii) The pharmaceutical composition comprising a second AAV virus particle containing the AAV vector genome described in any one of claims 35 to 41.

48. A pharmaceutical composition, (i) A first AAV virus particle comprising the AAV vector genome described in any one of claims 1 to 34 and 40 to 41, (ii) An AAV vector genome, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Recombinant site, (c) Splice acceptor (SA) site, (d) The 3' portion of the introduced gene, (e) Poly A portion, and (f) The pharmaceutical composition comprising a second AAV virus particle containing the AAV vector genome, which includes a 3'AAV inverted terminal repeat.

49. A method for expressing a protein encoded by an introduced gene within a cell, (1) Transduction of the cells with a first AAV virus particle containing the AAV vector genome described in any one of claims 1 to 34 and 40 to 41, (2) The method comprising transducing the cells with a second AAV virus particle containing the AAV vector genome described in any one of claims 35 to 41.

50. A method for expressing a protein encoded by an introduced gene within a cell, (1) Transduction of the cells with a first AAV virus particle containing the AAV vector genome described in any one of claims 1 to 34 and 40 to 41, (2) The cell is given an AAV vector genome, which is oriented from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Recombinant site, (c) Splice acceptor (SA) site, (d) The 3' portion of the introduced gene, (e) Poly A portion, and (f) The method comprising transducing a second AAV virus particle containing the AAV vector genome, which includes a 3'AAV inverted terminal repeat.

51. A method for treating a subject with a disease or disability, (1) Administering to the subject a first AAV virus particle containing the AAV vector genome described in any one of claims 1 to 34 and 40 to 41, (2) The method comprising administering to the subject a second AAV virus particle containing the AAV vector genome described in any one of claims 35 to 41.

52. A method for treating a subject with a disease or disability, (1) Administering to the subject a first AAV virus particle containing the AAV vector genome described in any one of claims 1 to 34 and 40 to 41, (2) The subject is an AAV vector genome, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Recombinant site, (c) Splice acceptor (SA) site, (d) The 3' portion of the introduced gene, (e) Poly A portion, and (f) The method comprising administering a second AAV virus particle comprising the AAV vector genome, which includes a 3'AAV inverted terminal repeat.

53. The pharmaceutical composition according to claim 47 or 48, or the method according to any one of claims 49 to 52, wherein the recombination sites in the AAV vector genome of the first AAV virus particle and the recombination sites in the AAV vector genome of the second AAV virus particle prevent reverse recombination.

54. A pharmaceutical composition according to any one of claims 47 to 48 and 53, or the method according to any one of claims 49 to 53, wherein the AAV virus particles include a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a sequence selected from SEQ ID NOs: 1 to 3, 67, 71, 196, 205, and 206, or an AAV capsid protein comprising such a sequence.

55. The pharmaceutical composition or method according to claim 54, wherein the AAV capsid protein comprises or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

3.

56. The pharmaceutical composition or method according to claim 54, wherein the AAV capsid protein comprises or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with Sequence ID No.

67.

57. The method according to any one of claims 49 to 56, wherein the administration of the first AAV virus particle and the second AAV virus particle results in recombination of the first AAV vector genome and the second AAV vector genome via the recombination site in the AAV vector genome of the first AAV virus particle and the recombination site in the second AAV vector genome of the second AAV virus particle.

58. The method according to any one of claims 49 to 57, wherein the administration of the first AAV virus particle and the second AAV virus particle results in complete expression of the transgene.

59. The method according to any one of claims 51 to 58, wherein the disease or disorder is Stargardt disease.

60. The method according to any one of claims 49 to 59, wherein the protein is an ABCA4 protein, or the disease or disorder is caused by an ABCA4 gene deficiency.

61. The method according to claim 60, wherein the ABCA4 gene deficiency results in one or more conditions selected from the group consisting of decreased expression of ABCA4 protein, elimination of ABCA4 protein expression, expression of mutant ABCA4 protein, and impaired function of ABCA4 protein.

62. The method according to any one of claims 49 to 61, wherein the first AAV virus particle and the second AAV virus particle are administered simultaneously.

63. The method according to any one of claims 49 to 61, wherein the first AAV virus particle is administered before or after the second AAV virus particle.

64. The method according to any one of claims 51 to 63, wherein the virus particles are administered by intravitreous, pararetinal, or subretinal injection, optionally by subretinal injection.

65. The method according to any one of claims 49 to 64, wherein stable expression of the recombinase is not detected in the transduced cells or the subject.

66. Transduced cells, (i) Endogenous gene deletion genome copies, (ii) Recombinant nucleic acid, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Photoreceptor-retinoid-binding protein (IRBP) enhancer, (c) Ocular specific promoter, (d) The 5' portion of the transgene, which is a working copy of the endogenous gene in which the transgene is missing, (e) Splice donor (SD) site, (f) Recombination site, (g) Splice acceptor (SA) site, (h) The 3' portion of the introduced gene, (i) Poly A portion, and (j) The transduced cell comprising the recombinant nucleic acid comprising a 3'AAV inverted terminal repeat.

67. Transduced cells, (i) Endogenous gene deletion genome copies, (ii) Recombinant nucleic acid, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Optionally, enhancer, (c) Human rhodopsin (hRho) promoter, (d) The 5' portion of the transgene, which is a working copy of the endogenous gene in which the transgene is missing, (e) Splice donor (SD) site, (f) Recombination site, (g) Splice acceptor (SA) site, (h) The 3' portion of the introduced gene, (i) Poly A portion, and (j) The transduced cell comprising the recombinant nucleic acid comprising a 3'AAV inverted terminal repeat.

68. Transduced cells, (i) Endogenous gene deletion genome copies, (ii) Recombinant nucleic acid, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) CMV enhancer, (c) Chicken beta-actin (CBA) promoter, (d) The 5' portion of the transgene, which is a working copy of the endogenous gene in which the transgene is missing, (e) Splice donor (SD) site, (f) Recombination site, (g) Splice acceptor (SA) site, (h) The 3' portion of the introduced gene, (i) Poly A portion, and (j) The transduced cell comprising the recombinant nucleic acid comprising a 3'AAV inverted terminal repeat.

69. The transduced cell according to any one of claims 66 to 68, wherein the deficient endogenous gene is the ABCA4 gene, and the transgene encodes the ABCA4 protein.

70. The transdextrinsed cell or recombinant nucleic acid comprises a nucleic acid sequence comprising a 5'AAV inverted terminal repeat, a recombination site, a polynucleotide encoding a recombinase, and a 3'AAV inverted terminal repeat in the 5' to 3' direction, wherein the nucleic acid sequence lacks a promoter, according to any one of claims 66 to 69.

71. The transduced cell according to claim 70, wherein the nucleic acid sequence is located downstream of the 3' end of the (ii)(j)3'AAV inverted terminal repeat.

72. Transduced cells according to any one of claims 66 to 71, wherein the cells are ex vivo cells.

73. The transduction cell according to any one of claims 66 to 72, wherein the cell is a photoreceptor cell.

74. A nucleic acid comprising the 5' portion of the ABCA4 gene, wherein the 5' portion of ABCA4 comprises one or more of the following: (i) nucleotide "G" at the position corresponding to nucleotide 813 of sequence number 212, (ii) nucleotide "A" at the position corresponding to nucleotide 1311 of sequence number 212, (iii) nucleotide "A" at the position corresponding to nucleotide 2274 of sequence number 212, (iv) nucleotide "C" at the position corresponding to nucleotide 2277 of sequence number 212, (v) nucleotide "T" at the position corresponding to nucleotide 2278 of sequence number 212, and (vi) nucleotide "T" at the position corresponding to nucleotide 2568 of sequence number 212.

75. The nucleic acid according to claim 74, comprising two or more, three or more, four or more, five or more, or all six of the nucleotides (i) to (vi).

76. The nucleic acid according to claim 74 or 75, wherein the 5' portion of the ABCA4 gene is at least 1 kb, at least 1.5 kb, at least 2 kb, or at least 2.5 kb in length.

77. The nucleic acid according to any one of claims 74 to 76, wherein the 5' portion of the ABCA4 gene contains or consists of a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with sequence number 212.

78. The nucleic acid according to claim 77, wherein the 5' portion of the introduced gene encodes a polypeptide sequence containing amino acids 1 to 884 of human ABCA4 protein (SEQ ID NO: 190).

79. An AAV vector genome comprising the nucleic acid described in any one of claims 74 to 78.

80. AAV virus particles, (i) AAV capsid, wherein the AAV capsid contains an AAV capsid protein, (ii) The AAV virus particle comprising the AAV vector genome described in claim 79.

81. A pharmaceutical composition comprising the AAV vector genome described in claim 79 or the AAV virus particles described in claim 80.

82. A method for treating a subject having a disease or disorder, comprising administering to the subject the AAV virus particles described in claim 80, wherein the disease or disorder is optionally Stargardt disease.

83. Transduced cells comprising the nucleic acid described in any one of claims 74 to 78.

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