Recombinant adeno-associated virus vectors for multi-part gene delivery

Abstract

Provided herein are recombinant AAV vectors, AAV viral vectors, and capsid proteins for improved gene therapy, as well as methods for their production and use in multipart (e.g., bipartite) delivery systems. The vectors provide separate parts of a transgene and direct their recombination in cells to provide the complete transgene.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Application No. 63 / 051,721, filed on 14 July 2020, and U.S. Provisional Application No. 63 / 179,612, filed on 26 April 2021, all of which are incorporated herein by reference in their entirety.

[0002] Incorporating sequence lists by reference The contents of the text file submitted electronically with respect to this specification are incorporated herein by reference in their entirety: a computer-readable copy of the sequence listing (filename: ABEO_007_02WO_SeqList_ST25.txt, creation date: July 12, 2021, file size: approximately 721 kilobytes). [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 in length, 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 larger genes. [Overview of the project] [Means for solving the problem]

[0004] This disclosure relates, in general, to the field of gene therapy, and more specifically to recombinant adeno-associated virus (AAV) vector particles (also known as AAV viral vectors), and their use for delivering transgenes in a multi-part (e.g., bipart) approach to treat or prevent disease or disorder.

[0005] In one embodiment, the present disclosure relates to an AAV vector genome, which is oriented from 5' to 3'. (a) 5'AAV inverted terminal repeat, (b) promoter, (c) 5' portion of the introduced gene, (d) Splice donor (SD) site, (e) Recombination parts, (f) Polynucleotide encoding a recombinase, (g) PolyA portion, and (h)3'AAV inverted terminal repeat, including, This invention provides an AAV vector genome in which the expression of a recombinase is operably linked to a promoter.

[0006] In the embodiment, the recombinase is Cre recombinase. In the embodiment, the recombinase includes a nuclear localization sequence (NLS). In the embodiment, the recombination site includes a LoxP71 sequence. In the embodiment, the AAV vector genome includes a polynucleotide encoding an internal cleavage polypeptide located between the recombination site and the polynucleotide encoding the recombinase, and the 5' portion of the transgene, the polynucleotide encoding the internal cleavage polypeptide, and the polynucleotide encoding the recombinase are in the same reading frame. In the embodiment, the internal cleavage polypeptide is a self-cleaving peptide selected from the group consisting of T2A, P2A, E2A, and F2A. 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, and the IRES is operably linked to the polynucleotide encoding the recombinase.

[0007] In the embodiment, the transgene encodes a polypeptide. In the embodiment, the polypeptide is the ABCA4 protein.

[0008] In the embodiments, the promoter is the Roussarcoma virus (RSV) LTR promoter (optionally having an RSV enhancer), cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, beta-actin promoter, phosphoglycerol kinase (PGK) promoter, U6 promoter, H1 promoter, CAG promoter, hybrid chicken beta-actin promoter, MeCP2 promoter, EF1 promoter, ubiquitous chicken β-actin hybrid (CBh) promoter, U1a promoter, U1b promoter, MeCP2 promoter, MeP418 promoter, MeP426 promoter, minimal MeCP2 promoter, VMD2 promoter, mRho promoter, EFla promoter, Ubc promoter, human β-actin promoter, TRE promoter, Ac5 promoter, polyhedrin promoter, CaMKIIa promoter, Gal1 promoter, TEF1 promoter, GDS promoter, ADH1 promoter, Ubi promoter, or α-1-antitrypsin (hAAT) promoter. In the embodiments, the promoter is a human rhodopsin kinase (RK) promoter, a human photoreceptor-binding protein promoter (IRBP), a human red / green opsin promoter (pR2.1), a human blue opsin promoter (HB), a mouse opsin promoter (mOP), a mouse short-wavelength opsin promoter (mBP), or a human rod cGMP phosphodiesterase β-subunit promoter (βPDE).

[0009] In one embodiment, the present disclosure relates to an AAV vector genome, which is oriented from 5' to 3'. (a) 5'AAV inverted terminal repeat, (b) Recombination parts, (c) Splice acceptor (SA) site, (d) The 3' portion of the introduced gene, (e) a polyA site, and (f) a 3’ AAV inverted terminal repeat, and provides an AAV vector genome.

[0010] In one aspect, the present disclosure provides an AAV vector comprising the AAV vector genome of the present disclosure. In one aspect, the present disclosure provides a polynucleotide comprising the AAV vector genome of the present disclosure.

[0011] In one aspect, the present disclosure provides an AAV virus particle 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 is at least 70%, 80%, 90%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 1-3, 67, 71, 196, 205, and 206.

[0012] In one aspect, the present disclosure provides a pharmaceutical composition comprising the AAV vector genome of the present disclosure or the AAV virus particle of the present disclosure. In embodiments, the pharmaceutical composition (i) a first AAV virus particle comprising the AAV vector genome of the present disclosure, and (ii) a second AAV virus particle comprising a second AAV vector genome, 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, and includes a second AAV virus particle, Retrograde recombination is prevented by 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.

[0013] In one aspect, the present disclosure is a method of treating a subject having a disease or disorder caused by a gene deficiency, (1) Administer to a subject a first AAV viral particle comprising: (i) an AAV capsid comprising a first AAV capsid protein, and (ii) an AAV vector genome of the present disclosure; and (2) Administer to the subject a second AAV viral particle comprising: (i) an AAV capsid comprising a second AAV capsid protein, and (ii) a second 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 polyA site, and (f) a 3' AAV inverted terminal repeat. In embodiments, administration of the first AAV viral particle and the second AAV viral 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 viral particle and the recombination site in the second AAV vector genome of the second AAV viral particle. In embodiments, administration of the first AAV viral particle and the second AAV viral particle results in expression of a polypeptide. In embodiments, the first AAV viral particle and the second AAV viral particle are administered simultaneously or sequentially. In embodiments, the first AAV viral particle and the second AAV viral particle are administered simultaneously. In embodiments, the viral particles are administered by subretinal injection. In embodiments, the genetic defect is an ABCA4 gene defect. In embodiments, the ABCA4 gene defect results in one or more conditions selected from the group consisting of reduced expression of the ABCA4 protein, elimination of expression of the ABCA4 protein, expression of a mutant ABCA4 protein, and reduced function of the ABCA4 protein.

[0014] In one embodiment, the present disclosure relates to a method for expressing a polypeptide in cells, (1) Transduction of the cells using a first AAV virus particle containing the AAV vector genome of the present disclosure, wherein the transgene encodes a polypeptide, (2) Transducing cells using a second AAV virus particle containing a second AAV vector genome, wherein the second AAV vector genome is oriented from 5' to 3'. (a) 5'AAV inverted terminal repeat, (b) Recombination parts, (c) Splice acceptor (SA) site, (d) The 3' portion of the introduced gene, (e) Poly A portion, and (f) A method is provided which includes transduction, including a 3'AAV inverted terminal repeat.

[0015] In the embodiment, stable expression of the recombinase is not detected in transduced cells. In the embodiment, the polypeptide is the ABCA4 protein.

[0016] In one embodiment, the present disclosure relates to transduced cells, (i) Gene-deficient genome copies, (ii) A first recombinant nucleic acid, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) promoter, (c) 5' portion of the introduced gene, (d) Splice donor (SD) site, (e) Recombination parts, (f) Splice acceptor (SA) site, (g) 3' portion of the introduced gene, (h) Poly A portion, and (i) Includes 3'AAV inverted terminal repeat, The introduced gene is a first recombinant nucleic acid having a length of 4.6-7.7 kb, (iii) A transduced cell comprising a second recombinant nucleic acid comprising a 5'AAV inverted terminal repeat, a polynucleotide encoding a recombinase, and a 3'AAV inverted terminal repeat in the 5' to 3' direction, wherein the second recombinant nucleic acid lacks a promoter.

[0017] In the embodiment, stable expression of the recombinase is not detected in transduced cells. In the embodiment, the recombinase is Cre recombinase. In the embodiment, the transgene encodes the ABCA4 gene. In the embodiment, the cells are ex vivo cells. The present invention provides, for example, the following items: (Item 1) AAV vector genome, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) promoter, (c) 5' portion of the introduced gene, (d) Splice donor (SD) site, (e) Recombination parts, (f) Polynucleotide encoding a recombinase, (g) PolyA portion, and (h)3'AAV inverted terminal repeat, including, An AAV vector genome in which the expression of the recombinase is operably linked to the promoter. (Item 2) The AAV vector genome described in item 1, wherein the aforementioned recombinase is Cre recombinase. (Item 3) The AAV vector genome described in item 1 or 2, wherein the recombinase contains a nuclear localization sequence (NLS). (Item 4) The AAV vector genome described in item 2 or 3, wherein the aforementioned recombination site includes the LoxP71 sequence. (Item 5) The AAV vector genome according to any one of items 1 to 4, 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. (Item 6) The AAV vector genome described in item 5, wherein the internally cleaved polypeptide is a self-cleaved peptide selected from the group consisting of T2A, P2A, E2A, and F2A. (Item 7) The AAV vector genome according to any one of items 1 to 4, 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. (Item 8) An AAV vector genome according to any one of items 1 to 7, wherein the transgene encodes a polypeptide. (Item 9) The AAV vector genome described in item 8, wherein the polypeptide is the ABCA4 protein. (Item 10) The promoters include the Roussarcoma virus (RSV) LTR promoter (optionally containing an RSV enhancer), cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, beta-actin promoter, phosphoglycerol kinase (PGK) promoter, U6 promoter, H1 promoter, CAG promoter, hybrid chicken beta-actin promoter, MeCP2 promoter, EF1 promoter, ubiquitous chicken β-actin hybrid (CBh) promoter, U1a promoter, and U1b promoter. - An AAV vector genome as described in any one of items 1-9, which is a MeCP2 promoter, MeP418 promoter, MeP426 promoter, minimal MeCP2 promoter, VMD2 promoter, mRho promoter, EFla promoter, Ubc promoter, human β-actin promoter, TRE promoter, Ac5 promoter, polyhedrin promoter, CaMKIIa promoter, Gal1 promoter, TEF1 promoter, GDS promoter, ADH1 promoter, Ubi promoter, or α-1-anti-trypsin (hAAT) promoter. (Item 11) An AAV vector genome according to any one of items 1 to 9, wherein the promoter is a human rhodopsin kinase (RK) promoter, a human photoreceptor-binding protein promoter (IRBP), a human red / green opsin promoter (pR2.1), a human blue opsin promoter (HB), a mouse opsin promoter (mOP), a mouse short-wavelength opsin promoter (mBP), or a human rod cGMP phosphodiesterase β-subunit promoter (βPDE). (Item 12) AAV vector genome, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Recombination parts, (c) Splice acceptor (SA) site, (d) The 3' portion of the introduced gene, (e) Poly A portion, and (f) AAV vector genome containing 3'AAV inverted terminal repeats. (Item 13) AAV virus particles, (i) AAV capsid containing AAV capsid protein, and (ii) AAV virus particles comprising the AAV vector genome described in any one of items 1 to 12. (Item 14) The AAV virus particle described in item 13, wherein the AAV capsid protein is at least 70%, 80%, 90%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 1-3, 67, 71, 196, 205, and 206. (Item 15) A pharmaceutical composition comprising an AAV vector genome as described in any one of items 1 to 12, or an AAV virus particle as described in item 13 or 14. (Item 16) A pharmaceutical composition, (i) A first AAV virus particle containing the AAV vector genome described in any one of items 1 to 11, (ii) A second AAV virus particle containing a second AAV vector genome, (a) 5'AAV inverted terminal repeat, (b) Recombination parts, (c) Splice acceptor (SA) site, (d) The 3' portion of the introduced gene, (e) Poly A portion, and (f) A second AAV virus particle containing a 3'AAV inverted terminal repeat, A pharmaceutical composition in which retrograde recombination is prevented by the recombination sites in the AAV vector genome of the first AAV virus particle and the recombination sites in the second AAV vector genome of the second AAV virus particle. (Item 17) A method for treating a subject having a disease or disorder caused by a gene defect, (1) The subject is a first AAV virus particle, (i) an AAV capsid containing the first AAV capsid protein, and (ii) Administering a first AAV virus particle containing the AAV vector genome described in any one of items 1 to 11, (2) The subject is a second AAV virus particle, (i) an AAV capsid containing a second AAV capsid protein, and (ii) A second AAV vector genome, which is oriented from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Recombination parts, (c) Splice acceptor (SA) site, (d) The 3' portion of the introduced gene, (e) Poly A portion, and A method comprising administering a second AAV virus particle, comprising a second AAV vector genome, comprising a 3'AAV inverted terminal repeat. (Item 18) The method according to item 17, wherein administration of the first AAV virus particle and the second AAV virus particle causes recombination of the first AAV vector genome and the second AAV vector genome via the recombination sites in the AAV vector genome of the first AAV virus particle and the recombination sites in the second AAV vector genome of the second AAV virus particle. (Item 19) The method according to item 17 or 18, wherein the expression of the polypeptide occurs upon administration of the first AAV virus particle and the second AAV virus particle. (Item 20) The method according to any one of items 17 to 19, wherein the first AAV virus particle and the second AAV virus particle are administered simultaneously or sequentially. (Item 21) The method according to item 20, wherein the first AAV virus particle and the second AAV virus particle are administered simultaneously. (Item 22) The method according to any one of items 17 to 21, wherein the virus particles are administered by subretinal injection. (Item 23) The method according to any one of items 17 to 22, wherein the gene deletion is an ABCA4 gene deletion. (Item 24) The method according to item 23, wherein the ABCA4 gene deletion results in one or more conditions selected from the group consisting of decreased expression of ABCA4 protein, removal of ABCA4 protein expression, expression of mutant ABCA4 protein, and decreased function of ABCA4 protein. (Item 25) A method for expressing polypeptides in cells, (1) Transduction of the cells using a first AAV virus particle containing the AAV vector genome described in any one of items 1 to 11, wherein the transgene encodes the polypeptide, (2) Transducing the cells using a second AAV virus particle containing a second AAV vector genome, wherein the second AAV vector genome is oriented from 5' to 3'. (a) 5'AAV inverted terminal repeat, (b) Recombination parts, (c) Splice acceptor (SA) site, (d) The 3' portion of the introduced gene, (e) Poly A portion, and (f) A method comprising introducing a 3'AAV inverted terminal repeat. (Item 26) The method according to item 25, wherein stable expression of the recombinase is not detected in the transduced cells. (Item 27) The method according to item 25 or 26, wherein the polypeptide is the ABCA4 protein. (Item 28) Transduced cells, (i) Gene-deficient genome copies, (ii) A first recombinant nucleic acid, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) promoter, (c) 5' portion of the introduced gene, (d) Splice donor (SD) site, (e) Recombination parts, (f) Splice acceptor (SA) site, (g) The 3' portion of the introduced gene, (h) Poly A portion, and (i) Includes 3'AAV inverted terminal repeat, The introduced gene is a first recombinant nucleic acid having a length of 4.6 to 7.7 kb, (iii) Transduced cells comprising a second recombinant nucleic acid comprising, in the 5'-to-3' direction, a 5'AAV inverted terminal repeat, a polynucleotide encoding a recombinase, and a 3'AAV inverted terminal repeat, wherein the second recombinant nucleic acid lacks a promoter. (Item 29) Transduced cells according to item 28, wherein stable expression of the recombinase is not detected in the transduced cells. (Item 30) Transduced cells according to item 28 or 29, wherein the recombinase is Cre recombinase. (Item 31) Transduced cells as described in any one of items 28-30, wherein the transgene encodes the ABCA4 gene. (Item 32) Transduced cells as described in any one of items 28 to 31, wherein the aforementioned cells are ex vivo cells. [Brief explanation of the drawing]

[0018] [Figure 1A]Figure 1A shows an exemplary 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 "poly-A site". "SA" stands for "splice acceptor". Figure 1B shows an exemplary 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 "poly-A site". "SA" stands for "splice acceptor". [Figure 1B] Same as above. [Figure 2] This shows the ABCA4 construct used in tissue culture research. [Figure 3A] This demonstrates the need for Cre recombinase for full-length ABCA4 protein expression mediated by the recombination of a partially transfected ABCA4 construct in Lec2 cells. [Figure 3B]This shows the expression levels of recombinant full-length ABCA4 protein in Lec2 cells transfected with various ABCA4 constructs. [Figure 4] This shows the lack of stable expression of autocleaved Cre recombinase in Lec2 cells after partial ABCA4 construct recombination. [Figure 5] This shows the expression levels of full-length ABCA4 mRNA in Lec2 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 7A] A representative diagram of the bipartite delivery of the ABCA4 gene in an AAV vector genome is shown. Figure 7A shows the construction of the first AAV vector genome (5'ABCA4+Cre), which includes the human RK promoter, the 5' portion of the ABCA4 gene, the Cre recombinase gene, and other genomic elements. Figure 7B 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 7B] Same as above. [Modes for carrying out the invention]

[0019] Embodiments provided in 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 purpose of describing specific embodiments only and are not intended to limit them.

[0020] 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 it will be further understood that they should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0021] 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. To illustrate, where this specification states that a complex comprises components A, B, and C, it is specifically intended that A, B, or C, or any combination thereof, may be omitted and discarded individually or in any combination.

[0022] Unless otherwise explicitly indicated, all specified embodiments, features, and terms are intended to include both the listed embodiments, features, or terms, and their bioequivalents.

[0023] Embedding by reference All references, articles, publications, patents, patent publications, 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 publication, or patent application cited herein should be construed as an acknowledgment or suggestion in any form that it constitutes valid prior art or forms part of common general knowledge in any country worldwide.

[0024] definition The practical application 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)).

[0025] When used in the description of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context explicitly indicates otherwise.

[0026] Where used herein, the term “contains” is intended to mean that compositions and methods include the enumerated elements but do not exclude others. Where used herein, the transitional phrase “essentially consists of” (and grammatical variations) should be interpreted as encompassing the enumerated materials or steps and those that do not substantially affect the essential and novel features of the enumerated embodiments. Therefore, where used herein, the term “essentially consists of” should not be interpreted as equivalent to “contains.” “Consists of” means excluding other components in amounts greater than trace elements and substantial method steps for administering the compositions disclosed herein. The embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0027] All numerical expressions including ranges (e.g., pH, temperature, time, concentration, and molecular weight) are approximations that change by only + / - 15%, 10%, 5%, or 2% increments of 1.0 or 0.1, as appropriate or alternatively. It should be understood that all numerical expressions are preceded by the term “approximately,” although not necessarily explicitly stated. It should also be understood that the reagents described herein are merely illustrative, and equivalents may be known in the art, although not necessarily explicitly stated. Where used herein to refer to measurable values ​​such as volume or concentration and similar, the term “approximately” means to encompass a 10% variation of the specified volume.

[0028] When used to describe any selection of components, ranges, dosage forms, etc. disclosed herein, the terms “acceptable,” “effective,” or “sufficient” are intended to indicate that such components, ranges, dosage forms, etc. are suitable for the purposes disclosed herein.

[0029] Furthermore, as used herein, “and / or” refers to and encompasses not only every possible combination of one or more of the related enumerated items, but also, when interpreted as an alternative ("or"), the absence of any combination.

[0030] 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.

[0031] As used herein, the term “isolated” means molecules, biological substances, or cellular substances that are substantially free from other materials.

[0032] 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, essentially, 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.

[0033] "Genes" refers to polynucleotides containing at least one open reading frame (ORF) capable of encoding a specific polypeptide or protein. "Genome products," or alternatively, "gene expression products," refer to the amino acid sequence (e.g., peptides or polypeptides) produced when a gene is transcribed and translated.

[0034] As used herein, “expression” refers to the two-step process of transcribing a polynucleotide into mRNA and / or the process by 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.

[0035] "Transcriptionally regulated" is a well-understood term in the art and indicates that the transcription of a polynucleotide sequence, usually a DNA sequence, depends on its operable linkage to elements that contribute to or promote transcription initiation. "Operatable linkage" means that the polynucleotide is arranged in a manner that enables it to function within the cell. For example, a promoter may be operable linkage to a downstream sequence.

[0036] 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.

[0037] 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-exclusive exemplary promoters include the human rhodopsin kinase (RK) promoter, the human photoreceptor-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 promoter or blue opsin promoter), the human rhodopsin cGMP phosphodiesterase β-subunit promoter (βPDE), and the Roussarcoma virus (RSV) LTR promoter ( Examples include promoters containing an RSV enhancer, 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), MeCP2 promoter, MeP418 promoter, MeP426 promoter, minimal MeCP2 promoter, VMD2 promoter, mRho promoter, or EFI promoter.

[0038] Additional, non-exclusive, exemplary promoters provided herein include, but are not limited to, EFla, Ubc, human β-actin, CAG, TRE, Ac5 polyhedrin, Gal1, TEF1, GDS, ADH1, Ubi, and α-1-anti-trypsin (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 (modification of the TATA boxes of the 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 interstitial retinoid-binding protein (IRBP) enhancers, RSV enhancers, or CMV enhancers.

[0039] An enhancer is a regulatory element that increases the expression of a target sequence. A “promoter / enhancer” is a polynucleotide containing a sequence that has the ability to 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 juxtaposed with a gene by 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 act from a distance and regardless of their orientation relative to the location of the endogenous or heterogeneous promoter. Therefore, it is further understood that an enhancer operating at a certain distance from a promoter is "operably coupled" to that promoter, regardless of its location within the vector or its orientation relative to the promoter's location.

[0040] 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, and both D and L optical isomers, amino acid analogs, and peptide mimetic.

[0041] As used herein, the terms “signal peptide” or “signal polypeptide” typically refer to an amino acid sequence located at the N-terminus of a newly synthesized secreted polypeptide or membrane polypeptide or protein. This sequence acts to direct the polypeptide to a specific cellular site, for example, 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. Further non-limiting examples include IDUA signal peptides.

[0042] The terms “equivalent” or “bioequivalent” are used interchangeably when referring to specific molecules, biological substances, or cellular substances, and are 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).

[0043] "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. "Irrelevant" or "non-homologous" sequences share less than 40% identity and less than 25% identity with one of the sequences of 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 ClustalW (available at https: / / genome.jp / tools-bin / clustalw / ). For example, ClustalW parameters can be generated using the Gonnet (for proteins) weight matrix. In embodiments, the ClustalW parameters used to perform nucleic acid sequence alignment using the nucleic acid sequences found herein are generated using the ClustalW (for DNA) weight matrix.

[0044] As used herein, amino acid modifications may be amino acid substitutions, amino acid deletions, or amino acid 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, the substitution of lysine with arginine, the substitution of aspartic acid with glutamic acid, or the substitution of asparagine with glutamine. Other examples of conservative substitutions include the substitution of alanine to serine, asparagine to glutamine or histidine, aspartate to glutamate, 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.

[0045] 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 the cell, it is understood in the art that the vector may be replicated as an extrachromosomal (episome) element or incorporated into the host cell chromosome. Vectors may include nucleic acids derived from retroviruses, adenoviruses, herpesviruses, baculoviruses, modified baculoviruses, papovaviruses, AAV virus vectors, lentiviral vectors, adenovirus vectors, alphavirus vectors, and the like. Alphavirus vectors, such as Semliki Forest virus vectors and Sindbis virus vectors, have also been developed for use in gene therapy and immunotherapy. See, for example, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med. 5(7):823-827. Exemplary non-viral vectors for nucleic acid delivery 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.

[0046] Regarding common 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.

[0047] As used herein, the terms “recombinant expression system” or “recombinant vector” refer to a gene construct or construct formed by recombination for the expression of a particular genetic material.

[0048] A “gene delivery vehicle” is defined as any molecule capable of delivering an inserted polynucleotide to 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; metal particles; bacteria; viruses such as baculoviruses, adenoviruses, and retroviruses; bacteriophages, cosmids, plasmids, and fungal vectors; and other recombinant vehicles commonly used in the art, which are described for expression in various eukaryotic and prokaryotic hosts and may be used not only for gene therapy but also for simple protein expression. Similarly, liposomes containing, essentially consisting of, or comprising target antibodies or fragments thereof may be used in the methods 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.

[0049] 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 not only vector-mediated gene transfer (e.g., viral infection / transfection, or by various other protein-based or lipid-based gene delivery complexes), but also a variety of 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.

[0050] A plasmid is a DNA molecule that is typically separable 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 microbial populations and typically offer 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 proteins may act as toxins under similar circumstances. Plasmid vectors often exist as extrachromosomal circular DNA molecules, but plasmid vectors 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.

[0051] 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, allowing 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.

[0052] As used herein, the terms “adeno-associated virus” or “AAV” refer to a class of viruses associated with this name and belonging to the genus Dependoparvovirus, family Parvoviridae. Adeno-associated viruses are single-stranded DNA viruses that grow only in cells 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 remarkably similar replication characteristics mediated by homologous rep genes, and all possess three related capsid proteins, including one 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. At least 11 consecutively numbered native AAV serotypes are known in the art.Non-limiting, exemplary serotypes useful in the methods disclosed herein include any of these 11 serotypes, e.g., AAV2, AAV8, AAV9, or variant serotypes, e.g., AAV-DJ and AAV PHP.B. AAV particles contain, essentially consist of, or comprise 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, or AAV13. In this embodiment, the AAV particles contain an AAV capsid protein selected from the group consisting of AAVPHP.B, AAVrh74, AAV 110, AAV 204, AAV 214, AAV 214A, AAV 214e, AAV 214e8, AAV 214e9, AAV 214e10, AAV ITB102_45, and AAV 214AB.

[0053] 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 (ITR) sequences. Such AAV vectors can be replicated in host cells that provide functionality for rep and cap gene products, and can enable the ITRs and inter-ITR nucleic acids to be packaged into infectious viral particles. In embodiments, the 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 into infectious AAV particles. The ITRs and inter-ITR nucleic acids may be capsidated into an AAV cuspid, which may be referred to as the “AAV vector genome.” In addition to the capsid portion, the AAV vector may contain, for example, antibiotic resistance genes or other elements known in the art that are included in a plasmid for manufacturing purposes but are not packaged into AAV particles.

[0054] As used herein, the terms “viral capsid” or “capsid” refer to the proteinaceous shell or covering of a viral particle. The capsid serves to enclose, protect, transport, and / or release the viral genome into a host cell. Capsids generally consist of oligomeric structural subunits of 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 ratio of 1:1:10 (VP1:VP2:VP3) or 1:1:20 (VP1:VP2:VP3) in an icosahedral symmetry with T=1, 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.

[0055] "AAV virion," "AAV virus particle," "AAV virus vector," "AAV vector particle," or "AAV particle" refers to a virus particle comprising at least one AAV capsid protein and a polynucleotide enclosed in a capsid derived from an AAV vector, which is referred to herein as the AAV vector genome.

[0056] As used herein, the term “helper” with respect to a virus or plasmid means 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 an enzyme or other factor necessary for the replication of a viral genome in a host cell. Non-limiting examples of helper viruses and plasmids suitable for use in AAV constructs include pHELP (plasmid), adenovirus (virus), or herpesvirus (virus).

[0057] As used herein, packaging cells (or helper cells) are cells used to produce a viral vector. The production of a recombinant AAV viral vector also requires the AAV Rep and Cap proteins, as well as adenovirus gene sequences that assist in AAV replication, provided in trans. 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.

[0058] As used herein, a reporter protein is a detectable protein operably ligated to a promoter to assay promoter expression (e.g., tissue specificity and / or intensity). In some embodiments, the reporter protein may be operably ligated to another 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-galactosidases; fluorescent proteins such as green fluorescent protein (GFP) or red fluorescent protein (RFP); luciferases; glutathione S-transferase (GST); and maltose-binding proteins (MBPs).

[0059] In embodiments, the compositions herein may include pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” includes any of the following: standard pharmaceutical carriers such as phosphate-buffered saline, water, and emulsions such as oil / water or water / oil emulsions, as well as various types of wetting agents. The compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0060] The “subject” of a diagnosis or treatment is a cell, or an animal such as a mammal or a human. The subject is not limited to a specific species and includes non-human animals that are the subject of a diagnosis or treatment, and non-human animals that are the subject of an infection or animal model, including but not limited to monkeys, mice, rats, dogs, or rabbits, as well as other livestock, sport animals, or pets. In some embodiments, the subject is a human.

[0061] 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, diseased, or have genetic mutations. Biological tissue may include any single tissue (e.g., a collection of interconnected cells) or a group of tissues that make up an organ, part, or region of the body of an organism. Tissues may contain 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, biliary tract, duodenum, abdominal aorta, iliac vein, heart, and intestines, and any combination thereof.

[0062] 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 desirable outcome, including clinical outcomes. Beneficial or desirable outcomes for the purposes of the Art 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 degree of a condition (including disease); stabilization (i.e., no worsening) of a condition (including disease); delay or slowing of a condition (including disease); progression, improvement or mitigation of a condition (including disease); or a condition and remission (whether partial or complete remission).

[0063] 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 may depend on the type and severity of the condition in question, as well as the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. In the context of gene therapy, in embodiments, the effective dose is an amount sufficient to bring about the acquisition of partial or full function of a deficient gene in the subject. In other embodiments, an effective dose of AAV virus particles is an amount sufficient to bring about the expression of the gene in the subject. Those skilled in the art can determine the appropriate dose depending on these and other factors.

[0064] In embodiments, the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the target object, as well as the method of use. Those 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, one or more doses of the composition.

[0065] As used herein, the terms “administer” or “dosage” are intended to mean the delivery of a substance to an animal or human or other subject. Administration can be carried out in a single dose, continuously or intermittently, throughout the course of treatment. The most effective means and methods for determining the dosage of administration are known to those skilled in the art and 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 subject being treated. Single or multiple doses can be administered at dose levels and patterns selected by the treating physician, or, in the case of pets and other animals, by the treating veterinarian.

[0066] Structure and Function of AAV AAV is a replication-deficient parvovirus with a single-stranded DNA genome approximately 4.7 kb long, containing two 145-nucleotide inverted terminal repeats (ITRs). AAV has multiple serotypes. The nucleotide sequences of the AAV serotype genomes 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 complete 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 AAV-10 genome is provided in al., J. Virol., 78:6381-6388 (2004), the AAV-11 genome is provided in Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004). The sequence of the AAV rh.74 genome is provided in U.S. Patent No. 9,434,928, which is incorporated herein by reference in its entirety. U.S. Patent No. 9,434,928 also provides the sequence of the capsid protein and the self-complementary genome. In one embodiment, the genome is a self-complementary genome. Cis-acting sequences that direct viral DNA replication (rep), capsid formation / packaging, and host cell chromosome integration are contained within the AAV ITR. Three AAV promoters (named p5, p19, and p40 based on their relative mapping locations) drive the expression of two AAV internal open reading frames that encode the rep and cap genes.Differential splicing of a single AAV intron (at nucleotides 2107 and 2227) coupled with two rep promoters (p5 and p19) results in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. These rep proteins possess multiple enzymatic properties that ultimately carry out viral genome replication.

[0067] 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 the single mRNA into which each of the VP1, VP2, and VP3 proteins is translated, splicing can occur in two different ways: either a longer or shorter intron can be excised, resulting in the formation of two pools of mRNA (2.3kb and 2.6kb in length). 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, from which VP1 protein synthesis begins, 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 that codon within the same open reading frame is an ACG sequence (encoding threonine) surrounded by the optimal Koszak (translation start) context.This contributes to the low-level synthesis of the VP2 protein, which is actually the VP3 protein 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" As 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 polyA site is located at map position 95 of the AAV genome.The life cycle and genetics of AAVs have been reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992). Non-exclusive and exemplary capsid proteins include, as shown in Tables 1-3 of this disclosure, AAV 110 capsid protein, AAV 204 capsid protein, AAV 214 capsid protein, AAV 214A capsid protein, AAV 214e capsid protein, AAV 214e8 capsid protein, AAV 214e9 capsid protein, AAV 214e10 capsid protein, AAV ITB102_45 capsid protein, AAV 214AB capsid protein, AAV6 capsid protein, AAV8 capsid protein, AAV9 capsid protein, and AAV2 capsid protein. Additional, non-limiting, and exemplary capsid proteins include those disclosed in WO2016 / 081811, which is incorporated herein by reference, particularly with respect to AAV capsid protein sequences.

[0068] 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 an amino acid sequence present in the VP1 protein, also found within 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.

[0069] The VP3 protein can be further divided into individual variable surface regions I-IX (VR-I-IX). Each variable surface region (VR) contains, or may contain, a specific amino acid sequence that can give a distinctive infectious phenotype (e.g., reduced antigenicity, improved transduction, and / or tissue-specific targeting) to a particular serotype, either alone or in combination with the specific amino acid sequence of each of the other VRs, as described in DiMatta et al., "Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9," J. Virol., Vol. 86(12):6947-6958, June 2012, the entire content of which is incorporated herein by reference.

[0070] AAV possesses unique characteristics that make it attractive, for example, as a vector for delivering foreign DNA to cells 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 a wide range of mammalian cells, allowing for the potential to target numerous different tissues in vivo. Additionally, AAV transducers both slow-dividing and non-dividing cells and can persist essentially throughout the cell's life as a transcriptionally active nuclear episome (extrachromosomal element). Moreover, since the signals directing AAV replication and genomic capsid formation are contained within the ITR of the AAV genome, some or all of approximately 4.3 kb inside the genome (encoding the replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA to generate the AAV vector genome. The rep and cap proteins may be supplied trans. Another important characteristic of AAV is that it is an extremely stable and potent virus. This allows AAV to easily withstand the conditions used to inactivate adenoviruses (56°C to 65°C for several hours), making low-temperature storage of AAV less risky. AAV can even be freeze-dried. Finally, AAV-infected cells are not resistant to co-infection.

[0071] Multiple studies have demonstrated long-term (>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). See also Chao et al., Mol Ther, 2:619-623 (2000), and Chao et al., Mol Ther, 4:217-222 (2001). Furthermore, because muscles are highly angiogenic, recombinant AAV transduction results in the transgene product appearing in the systemic circulation after intramuscular injection, 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). In addition, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the cellular factors necessary for the correct glycosylation, folding, and secretion of antibodies, showing that muscles have the ability to 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 be derived from any AAV serotype capable of inducing recombinant viruses, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV PHP.B, and AAV rh74. The production of pseudotyped rAAVs is disclosed, for example, in WO2001 / 083692. Other types of rAAV variants, such as exemplary rAAVs with capsid mutations, are also being considered.For example, see Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). In this field, the nucleotide sequences of various AAV serotype genomes are known.

[0072] AAV vector particles, capsid proteins, and AAV vectors This specification provides AAV vector particles, AAV vectors, and capsid proteins that find use in the delivery of various 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 of approximately 4.7 kb. Delivery of larger transgenes may be achieved by delivering portions of the transgene into two or more AAV vector particles and recombining those portions in cells to provide the completed transgene (e.g., as illustrated in Figures 1A and 1B). In the novel preferred approach described herein, the 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 recombinated transgene. The recombination site may also direct the direction of the recombination event (e.g., to reduce or eliminate reverse recombination). In a further advantage, the recombination event may also remove the promoter operably linked to the Cre recombinase protein, resulting in a promoter-free vector genome containing Cre recombinase, thereby preventing further undesirable expression of Cre recombinase in host cells.

[0073] AAV Capsid Protein This disclosure provides AAV particles and AAV vector genomes for multi-part (e.g., bipartite) 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.

[0074] Capsid proteins include sequences having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that are mutated, deleted, or added compared to any one of the amino acid sequences listed in Table 1. Optionally, up to 20, 30, or 40 amino acids may be mutated, deleted, or added compared to these sequences. Capsid proteins are encoded by sequences having up to 5, 10, 30, or 60 nucleotide changes compared to any one of the nucleic acid sequences listed in Table 1. [Table 1]

[0075] In embodiments, the AAV capsid protein includes, essentially consists of, or consists of, 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. The polynucleotides encoding the VP1 proteins include, essentially consist of, or consist of, the sequences of SEQ ID NOs: 15, 18-23, 47, 66, 70, 82, 98, and 197, or sequences having up to 5, up to 10, or up to 30 nucleotide changes from the sequences of SEQ ID NOs: 15, 18-23, 47, 66, 70, 82, 98, and 197.

[0076] In the embodiment, 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 the embodiment, the AAV capsid protein is a variant of the AAV214 capsid protein. In the embodiment, 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 the embodiment, the AAV capsid protein is AAV9 capsid protein (SEQ ID NO: 71).

[0077] Exemplary sequences of the VP2 and VP3 proteins are provided in Tables 2 and 3. Given the VP2 and VP3 sequences, the VP1 portion may be determined by alignment with the complete VP1 protein sequence. [Table 2]

[0078] An exemplary nucleic acid for ITB102_45 is Sequence ID No. 47. Exemplary nucleic acids for other capsid VP2 moieties may be derived from the corresponding moieties of VP1 capsid protein nucleic acids. [Table 3]

[0079] 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.

[0080] In embodiments, the AAV VP2 protein comprises, essentially comprises, or comprises one amino acid sequence from any one of sequence numbers 35-40, 50, or 85, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids different from sequence numbers 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 sequence number 47, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes from sequence number 47.

[0081] 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 amino acids different from SEQ ID NOs: 17, 41-46, 51, or 86. Polynucleotides encoding these VP3 proteins are also provided. In embodiments, the polynucleotides encoding the protein comprises, essentially comprises, or comprises the sequences 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 SEQ ID NOs: 16, 24-29, 48, and 83.

[0082] 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.

[0083] In embodiments, an AAV capsid protein is provided herein that includes 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 of the AAV capsid protein are numbered relative to the amino acid positions of the amino acid sequence of SEQ ID NO: 2. In some cases, the protein contains the amino acid sequence of SEQ ID NO: 2. In other cases, these amino acids may be introduced into other capsid proteins.

[0084] In embodiments, an AAV VP1 capsid protein comprising a VP1 portion, a VP2 portion, and a VP3 portion is provided herein, wherein the VP1 portion contains a leucine (L) residue at amino acid 129, the VP2 portion contains a threonine (T) residue or an asparagine (N) residue at amino acid 157, a lysine (K) residue or a serine (S) residue at amino acid 162, and the VP3 portion contains an asparagine (N) residue at amino acid 223, an alanine (A) residue at amino acid 224, a histidine (H) residue at amino acid 272, a threonine (T) residue at amino acid 410, a histidine (H) residue at amino acid 724, and a proline (P) residue at amino acid 734, and the amino acid positions within 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).

[0085] In the embodiment, the VP1 portion further includes 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 includes one or more of the following: (i) a proline (P) residue at amino acid 148, (ii) an arginine (R) residue inserted at amino acid 152, (iii) an arginine (R) residue at amino acid 168, (iv) an isoleucine (I) residue at amino acid 189, and (v) a serine (S) residue at amino acid 200, 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.

[0086] 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 specified in Table 4 below. Their locations and the identity of additional alternatives can be determined 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. [Table 4]

[0087] 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 serotype, AAV8 serotype, or AAV9 serotype.

[0088] AAV Vector An AAV vector supplies not only nucleic acids that are capsid-formed into the AAV vector particle containing elements involved in regulating nucleic acid expression in the target, but also ITRs to facilitate capsid formation. 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 the target cell and, when recombined with the rest of the HNA sequence, encodes a transgene that treats a disease or disorder. Thus, the HNA sequence comprises a 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.

[0089] This disclosure provides a method for expressing transgenes encoded by genes exceeding a size limit. The size limit is determined by the packaging ability of the viral particle carrying the heterologous nucleic acid sequence, and for most AAV viral vectors, the size limit of the capsid-formed vector genome is typically about 4.7 kb. This disclosure provides an approach using AAV viral particles to deliver transgenes to host cells and express transgenes that are up to about 8 kb, up to about 7.9 kb, up to about 7.8 kb, up to about 7.7 kb, up to about 7.6 kb, up to about 7.5 kb, or up to about 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.

[0090] Previous approaches to delivering transgenes that exceed the size limitations of the AAV system have included using homologous recombination mediated by the host cell's own cellular mechanisms. However, the efficiency of homologous recombination varies greatly between different organisms and cell types, and the endogenous homologous recombination process is prone to errors.

[0091] The novel method described herein utilizes an exogenous recombinase to mediate efficient and precise recombination between AAV vector genomes encoding different portions of a transgene, thereby overcoming the 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. A 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. Using the Cre recombination approach disclosed herein, highly efficient recombination was obtained regardless of the cellular environment and in the absence of additional cofactors. Other exogenous recombinases, such as yeast flippase (Flp), may also be used.

[0092] The multipart viral delivery system of this disclosure uses an exogenous recombinase (e.g., Cre recombinase) to mediate the recombination and fusion of different portions of a target transgene via specific recombination sites within the AAV vector genome that are delivered to a host cell inside the viral vector. In embodiments, three or more portions of a target transgene may be delivered using the multipart viral delivery system to form a complete transgene in a host cell using the same strategy.

[0093] Figure 1A shows 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 split into two parts: a 5' portion and a 3' portion. The 5' portion of the transgene 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), such as Cre recombinase. When expressed in cells, the recombinase mediates recombination between the first and second AAV vector genomes through recombination sites (RSs) in the first and second AAV vector genomes, resulting in the formation of two recombinant nucleic acids, as shown in Figure 1A. The first recombinant nucleic acid contains both the 5' and 3' portions of the transgene, which, upon transcription, join together during RNA splicing due to the presence of splicing donor (SD) and splicing acceptor (SA) sites, thereby enabling the expression of the full-length transgene. The second recombinant nucleic acid does not contain the promoter region, and therefore the expression of recombinase from the second recombinant nucleic acid is suppressed.

[0094] 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. Recombinant sites (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' Includes AAV inverted terminal repetition.

[0095] Typically, there is no stop codon between the 5' portion of the transgene and the recombinase nucleic acid, meaning that the HNA region (which optionally has a nuclear localization sequence) containing the 5' portion of the transgene and the gene encoding the recombinase forms a continuous ORF (i.e., it lies within a reading frame).

[0096] The second AAV vector genome is oriented from 5' to 3'. a. 5'AAV inverted terminal repetition, b. Recombination parts, c. Splice acceptor (SA) site, d. The 3' portion of the transgene ORF, e. Poly A area, It may also include f. and 3'AAV inverted terminal repeat.

[0097] Elements of the first and second AAV vector genomes promote the production of the full-length transgene. 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 the recombination of the first and second AAV vector genomes via recombination sites in the first and second AAV vector genomes, resulting in the formation of two recombinant nucleic acids. The first 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, a full-size mRNA is produced from the first recombinant nucleic acid. The second recombinant nucleic acid contains the residual nucleic acid portion, including the recombinase. In particular, the recombinase is hardly expressed from the second recombinant nucleic acid because it is no longer operably linked to the promoter due to the absence of the promoter in the nucleic acid. See Figure 1A.

[0098] Three or more portions of a transgene can be delivered to host cells using a multipart viral delivery system, where they can be 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 more than three 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. Next, the AAV vector genomes are encapsulated in a capsid to produce AAV vector particles, which are delivered to host cells to introduce the AAV vector genomes. 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, a nucleic acid sequence encoding a recombinase (Rec) having an optional internal cleavage (IC) polypeptide or internal ribosome entry site (IRES), an optional 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.

[0099] When three AAV vector genomes are delivered into 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., there is minimal or no recombination between RS1 / RS1' and RS2 / RS2'). In 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 an integrated transgene that does not have any of the recombination sites. In this embodiment, the SD1 / SA1 splicing pair and the SD2 / SA2 splicing pair are orthogonal to each other (i.e., no splicing occurs between the SD1 / SA2 pair or the SD2 / SA1 pair), thereby ensuring that all parts of the transgene are retained in their correct positions within the mature mRNA transcript.

[0100] In the embodiment, the recombinase is cleaved from the N-terminal region of the protein via an internal cleavage polypeptide. Recombinase expression may be driven by an internal ribosome entry site (IRES).

[0101] The first 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 efficient transcription of the HNA sequence containing the transgene, and the 5' and 3' portions of the transgene are joined together after splicing of the RNA transcript via the splice donor and acceptor sites, thereby resulting in the formation of a full-length transgene mRNA transcript.

[0102] The second recombinant nucleic acid may include, in the 5'-3' direction, a 5' AAV inverted terminal repeat (ITR), a recombinant site (RS) formed by the recombination process, optionally an HNA sequence encoding an internal cleavage (IC) polypeptide or internal ribosome entry site (IRES), optionally a nuclear localization sequence (NLS), a nucleic acid sequence encoding a recombinase, a polyA site (pA), and a 3' AAV inverted terminal repeat. In the embodiment, the gene encoding the recombinase located within the second 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 using both the first and second AAV vector genomes.

[0103] The recombinase may be a Cre recombinase. The Cre recombinase may contain, essentially consist of, or 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. Optionally, the Cre recombinase may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acids that are different from SEQ ID NO: 164.

[0104] Cre recombinase may be fused to a nuclear localization sequence (NLS). The location of the NLS may be at the N-terminus, C-terminus, or mid-terminus of Cre recombinase. The NLS may have the amino acid sequence of PKKKRKV (SEQ ID NO: 165), or have up to one, two, or three amino acid mutations from SEQ ID NO: 165. Cre recombinase having an NLS may contain, essentially consist of, or consist of the amino acid sequence of SEQ ID NO: 166, or 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. Cre recombinase having an NLS may be encoded by 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 100% identity with SEQ ID NO: 167.

[0105] The recombination site recognized by Cre recombinase may contain one or more LoxP (crossover 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. Pairing of 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 retrograde recombination.

[0106] The recombination sites recognized by Cre recombinase may, in essence, consist of one or more variant Lox sites containing spacer region sequences different from the standard sequence (ATGTATGC). In embodiments, the variant Lox sites are orthogonal to the LoxP sites (i.e., there is no cross-recombination between variant Lox sites and LoxP sites). 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, Lox sites undergo 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, both of which are incorporated herein by reference in their entirety. Exemplary variant Lox site sequences are provided in Table 5 below. [Table 5]

[0107] The recombination site in the first AAV vector genome may include, or be essentially, a Lox 71 sequence, e.g., sequence number 178, and the recombination site in the second AAV vector genome may include, or be essentially, a Lox 66 sequence, e.g., sequence number 179.

[0108] The first AAV vector genome may encode an internal cleavage polypeptide to facilitate the cleavage of the recombinase from the remaining polypeptide. The nucleic acid encoding the internal cleavage 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, approximately 1 to 300 bp, approximately 1 to 200 bp, approximately 1 to 150 bp, approximately 1 to 100 bp, approximately 1 to 50 bp, approximately 1 to 30 bp, approximately 1 to 20 bp, or approximately 1 to 10 bp upstream of the 5' end of the nucleic acid portion encoding the recombinase.

[0109] The internally cleaved polypeptide may also be a 2A self-cleaving peptide. Preferred examples of 2A self-cleaving peptides are listed in Table 6 below. 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. [Table 6]

[0110] In the embodiments, stable expression of recombinase is little to no detectable in cells transduced using both the first and second AAV vector genomes. In the embodiments, recombinase expression cannot be detected 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 with both AAV vector genomes. In the embodiments, recombinase expression cannot be detected by Western blotting in cells approximately 48 hours after transduction with both AAV vector genomes. Western blotting analysis of recombinase can be performed using commercially available antibodies by those skilled in the art. For example, the expression level of Cre recombinase can be analyzed by Western blotting using a rabbit anti-Cre mAb (Cell signaling technology #15036) at a 1:10,000 dilution according to the protocol described in Example 3.

[0111] In the embodiments, stable expression of recombinase-encoding mRNA is little to no detectable in cells transduced by both the first and second AAV vector genomes. In the embodiments, expression of recombinase-encoding mRNA cannot be detected 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 with both AAV vector genomes. In the embodiments, expression of recombinase-encoding mRNA cannot be detected by qPCR in cells approximately 1 week after transduction with both AAV vector genomes.

[0112] The first AAV vector genome may include an internal ribosome entry site (IRES) to promote the expression of downstream recombinases.

[0113] 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.

[0114] 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.

[0115] The splice donor (SD) and splice acceptor (SA) sites may be located within different AAV vector genomes. As a result, 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.

[0116] The splice donor (SD) site may contain, essentially consist of, the nucleotide sequence of SEQ ID NO: 186, or a sequence having up to 1, 3, 5, or 10 nucleotide changes relative to SEQ ID NO: 186. The splice acceptor (SA) site may contain, essentially consist of, the nucleotide sequence of SEQ ID NO: 187, or a sequence having up to 1, 3, 5, or 10 nucleotide changes relative to SEQ ID NO: 187.

[0117] The regulation of transgene expression in host cells may be regulated by regulatory elements contained within the AAV vector genome, including the promoter sequence, and within the poly-A region. The AAV vector may also encode a signal peptide. In embodiments, the AAV vector has 5' and 3' inverted terminal repeats (ITRs). The 5'ITR is located upstream of the promoter, which is also upstream of the transgene. In embodiments, the 5' and 3'ITRs have the same sequence. In embodiments, they have different sequences. In embodiments, the AAV vector 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.

[0118] An HNA (e.g., an HNA containing a transgene or a portion thereof) is operably ligated 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 includes, essentially consists of, or consists of, the Roussarcoma (RSV) LTR promoter (optionally having 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. See, for example, Gao et al. (2018) Mol.Ther.:Nucleic Acids 12:135-145 (modification of the TATA boxes of the 7SK, U6, and H1 promoters to abolish RNA polymerase III transcription and stimulate RNA polymerase II-dependent mRNA transcription). In embodiments, the HNA sequence is operably ligated to a tissue-specific control promoter or an inducible promoter. In embodiments, the tissue-specific control promoter is a central nervous system (CNS) cell-specific promoter, a lung-specific promoter, a skin-specific promoter, a muscle-specific promoter, a liver-specific promoter, or an eye-specific promoter. In embodiments, the tissue-specific control promoter includes, essentially consists of, or consists of an eye-specific promoter.In embodiments, the tissue-specific control promoter includes, essentially consists of, or consists of, the human rhodopsin kinase (RK) promoter, the human photoreceptor-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 promoter or the blue opsin promoter), the human rod cGMP phosphodiesterase β-subunit promoter (βPDE), the VMD2 promoter, or the mRho promoter.

[0119] The promoters used herein may 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). The promoter may be a U1a or U1b promoter, an EF1 promoter, or CBA (chicken beta-actin). The promoter may be a human rhodopsin kinase (RK) promoter (SEQ ID NO: 188). The promoter may include, essentially consist of, or consist of, any one of the nucleic acid sequences listed in Table 7, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes to any one of the nucleic acid sequences listed in Table 7. [Table 7]

[0120] In embodiments, 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 embodiments, 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.

[0121] In embodiments, the HNA sequence is operably linked to a polyadenylation (poly-A) site. The poly-A site includes, essentially consists of, or consists of, the MeCP2 poly-A site, the retinol dehydrogenase 1 (RDH1) poly-A site, the bovine growth hormone (BGH) poly-A site, the SV40 poly-A site, the SPA49 poly-A site (SEQ ID NO: 189), the sNRP-TK65 poly-A site, the sNRP poly-A site, or the TK65 poly-A site. An exemplary SPA49 poly-A sequence is described by reference in Ostedgaard et al., Proc. Nat'l Acad. Sci. USA (Feb. 22, 2005) 102:2952-2957, which is incorporated herein by reference.

[0122] 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 and optionally translated in the cell of the target tissue. In some embodiments, the new recombinant HNA sequence includes a transgene encoding a protein.

[0123] For protein-coding transgenes, a full-length protein-coding transgene is divided into two parts: a 5' portion and a 3' portion. The length of each portion is shorter than the length of the entire 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 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 division point of the transgene does not need to be between two codons). This is because, 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. Therefore, the transgene and portions of the corresponding amino acid sequences it codes for may be a perfect match, or they may have a small mismatch of one or two nucleotides at the division 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. The 3' portion of the transgene may have a size limitation of approximately 4.6kb, 4.5kb, 4.4kb, or 4.3kb in the second AAV vector genome.

[0124] A typical 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 that follows Uniprot accession number P78363 (sequence number 190). An exemplary nucleic acid sequence encoding the ABCA4 protein is sequence number 191.

[0125] A suitable ABCA4 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: 190. The ABCA4 protein may be identical to SEQ ID NO: 190, or it may have up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that are different from SEQ ID NO: 190.

[0126] The nucleic acid encoding the ABCA4 protein is divided into a 5' and a 3' portion, which are placed into separate AAV vector genomes. The 5' portion of the ABCA4 transgene may be a nucleotide sequence having at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, or at least approximately 99% identity with SEQ ID NO: 192, and the 3' portion of the ABCA4 transgene may be a nucleotide sequence having at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, or at least approximately 99% identity with SEQ ID NO: 193.

[0127] Examples of additional transgenes suitable for bipartite gene delivery include MYO7A and CEP290.

[0128] 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 preferred 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 amino acids that are different from SEQ ID NO: 201.

[0129] The CEP290 gene encodes a centrosome protein localized to photoreceptor-binding cilia and involved in both ciliary body formation and ciliary body transport. 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 preferred 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 amino acids that are different from SEQ ID NO: 203.

[0130] 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.

[0131] In the embodiments, the cells are packaging or helper cell lines. 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.

[0132] The helper plasmid may, for example, include at least one viral helper DNA sequence derived from a non-replicating viral genome, without producing a replicating AAV, for transcoding all the virion proteins required to package a non-replicating AAV, and for producing a virion protein capable of packaging a high-titer, non-replicating AAV.

[0133] Helper plasmids for packaging AAV are known in the art; see, for example, U.S. Patent Publication 2004 / 0235174 A1, incorporated herein by reference. As described therein, the AAV helper plasmid may, as a non-limiting example, contain the Ad5 genes E2A, E4, and VA as helper viral DNA sequences, controlled by their respective original promoters or by 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.

[0134] This disclosure provides a method for generating AAV particles, comprising transfecting a packaging cell line with any one of the AAV helper plasmids and any of the AAV vectors disclosed herein. In embodiments, the AAV helper plasmid and the AAV vector are co-transfected into a 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 any of the AAV vectors, AAV vector genomes, and / or AAV particles disclosed herein.

[0135] Pharmaceutical composition This disclosure provides pharmaceutical compositions comprising any of the AAV vectors, AAV vector genomes, AAV capsids, and / or AAV particles described herein. Typically, AAV particles are administered for therapeutic purposes.

[0136] The pharmaceutical composition may be formulated by any method known or developed in the art of drug development, as described herein, which includes, but is 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 some embodiments, the pharmaceutical composition is suitable for ocular injection.

[0137] In embodiments, the pharmaceutical composition may further include physiological 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 mimetic bodies, or combinations thereof. In embodiments, the pharmaceutical composition is formulated as nanoparticles. In embodiments, the pharmaceutical composition is formulated as sterile solutions and substantially isotonic solutions.

[0138] The pharmaceutical compositions according to this disclosure may be prepared, packaged, and / or sold in bulk as single unit doses and / or as multiple single unit doses. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject, and / or a favorable proportion of such doses (e.g., 1 / 2 or 1 / 3 of such doses). The formulations of the present invention may contain one or more excipients in amounts that each together increases the stability of the viral vector, increases cell transfection or transduction by the viral vector, increases the expression of proteins encoded by the vector genome, and / or alters the release profile of proteins 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.

[0139] In embodiments, the pharmaceutical composition includes an antifreeze agent. The term "antifreeze agent" refers to an agent that has the ability to 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.

[0140] Treatment method This disclosure provides a method for preventing or treating a genetic disorder, which includes, essentially consists of, or comprises administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to a target.

[0141] In the embodiment, the genetic 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.

[0142] In embodiments, 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's crystalline dystrophy, and retinal schizophrenia (e.g., degenerative, hereditary). (tractional, exudative), 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 MPSIIIA), Sanfilippo syndrome B (mucopolysaccharidosis IIIB or MPSIIIB), 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.

[0143] In addition to the specific transgenes disclosed herein, known active enzymes, structural proteins, or RNA sequences may be used as transgenes for delivering functional activity.

[0144] The disorder may be an eye disease. The eye is an immune-privileged tissue, and low doses of the virus may offer therapeutic benefits. The eye disease may affect photoreceptors and / or RPE cells. For example, the eye disease may be retinitis pigmentosa (e.g., autosomal recessive (SPATA7 gene, LRAT gene, TULP1 gene), autosomal dominant (AIPL1 gene), and X-linked (RPGR gene)), eye disorders related to mutations in the bestrophin-1 (BEST-1) gene (e.g., vitiligo macular dystrophy, age-related macular degeneration, autosomal dominant vitreoretinopathy, glaucoma, cataract), or Leber congenital amaurosis (LCA; aryl-hydrocarbon interacting protein-like 1 (AIPL1) gene). The child may also have cone-rod dystrophy (CRD; ABCA4 gene), Stargardt disease (ABCA4 gene), congenital choroidal absence (CHM gene), Usher syndrome (MYO7A gene, CDH23 gene; USH2A gene; CLRN1 gene), retinoschisis (RS1 gene), Vietti's crystalline dystrophy (CYP4V2 gene), or color blindness (CNGA3 gene, CNGB3 gene, GNAT2 gene, PDE6C gene, or PDE6H gene).

[0145] Suitable conditions for treatment using 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, optionally, vestibular dysfunction. Usher syndrome type I (USH1) is the most severe form, characterized by pre-pubescent onset of retinitis pigmentosa leading to severe to severe congenital sensorineural hearing loss, balance defects, and blindness. Six loci of USH1 (USH1B-USH1G) have been mapped. Mutations in the MYO7A gene have been found to cause USH1B, the most common subtype of USH1. The MYO7A gene encodes the myosin VIIA protein. In the human retina, myosin VIIA exhibits active functions in the migration of RPE melanosomes, phagocytosis of the lateral segment tips of photoreceptor cells, and opsin transport through the cilia of these photoreceptors. The coding sequence for MYO7A is approximately 6.6kb 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 causes of LCA. The CEP290 gene codes for a centrosome protein that is localized to photoreceptor-binding cilia and is involved in both ciliary body formation and ciliary body transport. CEP290 plays a crucial role in early ciliation and ciliary body protein transport. Patients with CEP290-associated LCA have several cone nuclei in the foveal region, which is rich in central cones. However, these cone photoreceptors have abnormal medial and lateral segments, resulting in severe vision loss in most patients. The coding sequence for the CEP290 gene is approximately 7.4kb in size and encodes a protein sequence according to Uniprot accession number O15078-1 (www.uniprot.org / uniprot / O15078#O15078-1).

[0146] In the embodiment, the cancer is a solid tumor, such as bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, lip cancer, oral cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung tumor, or thyroid cancer.

[0147] The subjects may be mammals, such as humans. Humans may be infants, such as those under 3 years old, under 2 years old, or under 1 year old.

[0148] The treatment and prevention methods disclosed herein may be combined with appropriate diagnostic techniques to identify and select patients for treatment or prevention. For example, a method for treating or preventing a disorder disclosed herein, such as Stargardt disease, may further include a step of performing genetic testing to identify gene mutations or deletions associated with the disorder in question. In embodiments, a method for treating or preventing a disorder, such as Stargardt disease, includes administration to a subject previously identified as carrying a mutation associated with the disorder or at high risk of developing the disorder (e.g., based on genetic factors).

[0149] This disclosure provides a method for increasing the level of a protein in a host cell, the method comprising contacting the host cell with two AAV virus particles disclosed herein, the first AAV virus particle comprising a first AAV vector genome, and the second AAV virus particle comprising a second AAV vector genome. Cre-lox-mediated recombination between the first AAV vector genome and the second AAV vector genome results in a protein-coding HNA sequence. 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.

[0150] The present disclosure provides a method for preventing or treating a disorder in a subject by introducing a transgene into the subject. In embodiments, the method includes co-administering an effective amount of two AAV viral particles, wherein the first AAV viral particle includes a first AAV vector genome that includes a 5' portion of the transgene, and the second AAV viral particle includes a second AAV vector genome that includes 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 includes co-administering two AAV viral particles, wherein the first AAV viral particle includes a first AAV vector genome that includes a 5' portion of the ABCA4 gene, and the second AAV viral particle includes a second AAV vector genome that includes a 3' portion of the ABCA4 gene.

[0151] Dosage and Administration Methods for determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, and the subject being treated. Single or multiple administrations can be carried out at dosage levels and patterns selected by the treating physician. It should be noted that this 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 can be from as few as about 10 9 per administration to as many as about 10 17 vector genomes.

[0152] 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 per subject. Specifically, in some embodiments, from about 10 10 to about 10 12 , from about 10 11 to about 10 13 , from about 10 11 to about 10 12 , from about 10 11 to about 10 14 , from about 5×l0 11 to about 5×10 12 , or from about 10 12~about 10 13 A number of virus particles are administered to the target. For administration to the human eye, approximately 1 x 10⁻⁶ particles are administered. 10 A total dose of vg / eye may be used, and for mouse eyes, 5 × 10 9 The total dose of vg / eye may be used. Non-invasive in vivo imaging techniques may be used to monitor efficacy / safety in animals, and these techniques include, but are not limited to, scanning laser ophthalmography (SLO), optical coherence tomography (OCT), multiphoton microscopy, and fluorescein angiography.

[0153] In embodiments, viral particles are administered to a target intravenously, intrathecally, intracerebrally, intraventricularly, intranasally, intratracheally, intraaurally, intraocularly, or periocularly, orally, intrarectally, transmucosally, inhaled, percutaneously, parenterally, subcutaneously, intradermally, intramuscularly, intrapleurally, topically, intralymphatically, or intracapsularly. Such delivery may also be intraarterial, intracardiac, subventricular, epidural, intracerebral, intraventricular, subretinal, intravitreous, intraarticular, intraperitoneal, intrauterine, or any combination thereof. In embodiments, viral particles are delivered to desired target tissues, such as the lungs, eyes, or CNS, in non-limiting examples. In embodiments, delivery of viral particles is systemic. Intracapsular administration routes include 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.

[0154] There are several modes of administration known to those skilled in the art for treating ocular diseases (ocular disorders) intraocularly, including but not limited to lacrimal gland (LG) administration, topical eye drops, intramatriate administration into the cornea, intrachorionic administration, intravitreal administration, subretinal administration, systemic administration, or combinations thereof. 80% of genetic ocular disorders occur within the photoreceptors. Intravitreal delivery of small amounts of gene therapy can be performed in an outpatient clinic.

[0155] Some AAV particles exhibit brain and cervical spine directivity and can cross the blood-brain barrier (BBB). Some AAV particles exhibit high retinal directivity via subretinal and intravitreous injection. AAV particles target multiple ophthalmic cell types, such as cones, rods, and retinal pigment epithelium (RPE). Advantageously, AAV particles evade neutralizing antibodies against native serotypes, thus enabling the possibility of re-administration. In further embodiments, AAV particles and compositions may be administered in combination with other known treatments for the disorder being treated.

[0156] AAV particles may be administered via subretinal injection. Subretinal injection routes include (1) a transcorneal route through the pupil passing through the lens, vitreous humor, and retina; (2) a transscleral route entering the ciliary body squamous or marginal region passing on the opposite side of the vitreous humor and retina into the subretinal space; and (3) a transscleral route passing through the choroid and Bruch's membrane without penetrating the retina. Subretinal injection is as described in Peng et al. Ophthalmic Res. 2017;58(4):217-226, which is incorporated herein by reference in its entirety.

[0157] Each of the AAV vectors, AAV virus particles, or compositions of this disclosure may be administered in a single dose, continuously or intermittently, throughout the entire course of treatment. In embodiments, the AAV vectors, AAV particles, or compositions of this disclosure are administered parenterally by injection, infusion, or implantation.

[0158] The AAV virus particles containing the first AAV vector genome and the AAV virus particles containing the second AAV vector genome may be administered simultaneously or sequentially. "Simultaneous administration" means that the first and second AAV virus particles are administered 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 at time intervals so that they can act together on target cells and achieve a physiological effect (i.e., transduce the same cells in the target, recombining the transgene portion to 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.

[0159] AAV virus particles containing the first AAV vector genome and AAV virus particles containing the 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 the first AAV vector genome to AAV virus particles containing the 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 1.3:1, 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 embodiment, 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 embodiment, 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., simultaneous or sequential administration).

[0160] kit The agents, vectors, or compositions described herein may, in embodiments, be assembled into a medicament or a diagnostic or research kit to facilitate their use in therapeutic, diagnostic, or research applications. In embodiments, the kits of the present disclosure may include any one of a modified AAV capsid protein, an AAV vector, an AAV vector genome, AAV particles, a host cell, an isolated tissue, a composition, or a pharmaceutical composition, as described herein.

[0161] The kit may further include instructions for use. Specifically, such kits may include one or more agents described herein, along with instructions that explain the intended use and proper use of these agents. By way of example, in embodiments, the kit may include handling instructions for mixing one or more components of the kit and / or for isolating and mixing samples and applying them to a subject. In embodiments, the agents in the kit are in a pharmaceutical formulation and dosage suitable for a particular use and method of administration of the agent. Kits for research purposes may contain appropriate concentrations or amounts of components for performing various experiments.

[0162] The kits may be designed to facilitate the use of the methods described herein and may take many forms. Each of the compositions of the kit may, where applicable, be provided in liquid form (e.g., a solution) or in solid form (e.g., a dry powder). In certain instances, some of the compositions may be configurable or otherwise treatable (e.g., to an active form) by the addition of a suitable solvent or other species (e.g., water or cell culture medium), which may or may not be provided with the kit.

[0163] In embodiments, a kit contains one or more of the components described herein in one or more containers. Therefore, in embodiments, a kit may include a container for containing the drugs described herein. The drugs may be in liquid, gel, or solid (powder) form. The drugs may be sterile-prepared, packaged in syringes, and shipped refrigerated. Alternatively, they may be contained in vials or other containers for storage. A second container may contain other sterile-prepared drugs. Alternatively, a kit may contain pre-mixed and shipped active drugs in syringes, vials, tubes, or other containers. A kit may include one or more or all of the components necessary for administering the drug to a target, such as syringes, topical application devices, or IV needles and bags.

[0164] Although the present invention is 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 who relate to the invention.

[0165] Further numbered embodiments Further numbered embodiments of this disclosure are provided below.

[0166] Embodiment 1. An AAV vector genome, wherein in the 5' to 3' direction, (a) 5'AAV inverted terminal repeat, (b) promoter, (c) 5' portion of the introduced gene, (d) Splice donor (SD) site, (e) Recombination parts, (f) Polynucleotide encoding a recombinase, (g) PolyA portion, and (h)3'AAV inverted terminal repeat, including, AAV vector genome in which recombinase expression is operably linked to a promoter.

[0167] Embodiment 2. The AAV vector genome according to Embodiment 1, wherein the recombinase is Cre recombinase.

[0168] Embodiment 3. The AAV vector genome according to Embodiment 1 or 2, wherein the recombinase comprises a nuclear localization sequence (NLS).

[0169] Embodiment 4. An AAV vector genome according to Embodiment 2 or 3, wherein the recombination site includes the LoxP71 sequence.

[0170] Embodiment 5. The AAV vector genome according to any one of Embodiments 1 to 4, wherein the AAV vector genome comprises a polynucleotide encoding an internally cleaved polypeptide located between a recombination site and a polynucleotide encoding a recombinase, and 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.

[0171] Embodiment 6. The AAV vector genome according to Embodiment 5, wherein the internally cleaved polypeptide is a self-cleaving peptide selected from the group consisting of T2A, P2A, E2A, and F2A.

[0172] Embodiment 7. The AAV vector genome according to any one of Embodiments 1 to 4, wherein the AAV vector genome includes an internal ribosome entry site (IRES) located between a recombination site and a polynucleotide encoding the recombinase, and the IRES is operably ligated to the polynucleotide encoding the recombinase.

[0173] Embodiment 8. An AAV vector genome according to any one of Embodiments 1 to 7, wherein the transgene encodes a polypeptide.

[0174] Embodiment 9. The AAV vector genome according to claim 8, wherein the polypeptide is the ABCA4 protein.

[0175] Embodiment 10. The promoter is a Roussarcoma virus (RSV) LTR promoter (optionally having an RSV enhancer), a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a beta-actin promoter, a phosphoglycerol kinase (PGK) promoter, a U6 promoter, an H1 promoter, a CAG promoter, a hybrid chicken beta-actin promoter, a MeCP2 promoter, an EF1 promoter, a ubiquitous chicken β-actin hybrid (CBh) promoter, a U1a promoter, a U1b promoter, An AAV vector genome according to any one of Embodiments 1 to 9, wherein the promoter is MeCP2 promoter, MeP418 promoter, MeP426 promoter, minimal MeCP2 promoter, VMD2 promoter, mRho promoter, EFla promoter, Ubc promoter, human β-actin promoter, TRE promoter, Ac5 promoter, polyhedrin promoter, CaMKIIa promoter, Gal1 promoter, TEF1 promoter, GDS promoter, ADH1 promoter, Ubi promoter, or α-1-anti-trypsin (hAAT) promoter.

[0176] Embodiment 11. An AAV vector genome according to any one of Embodiments 1 to 9, wherein the promoter is a human rhodopsin kinase (RK) promoter, a human photoreceptor-binding protein promoter (IRBP), a human red / green opsin promoter (pR2.1), a human blue opsin promoter (HB), a mouse opsin promoter (mOP), a mouse short-wavelength opsin promoter (mBP), or a human rod cGMP phosphodiesterase β-subunit promoter (βPDE).

[0177] Embodiment 12. An AAV vector genome, wherein in the 5' to 3' direction, (a) 5'AAV inverted terminal repeat, (b) Recombination parts, (c) Splice acceptor (SA) site, (d) The 3' portion of the introduced gene, (e) a polyA site, and (f) a 3’ AAV inverted terminal repeat, an AAV vector genome.

[0178] Embodiment 13. An AAV virus particle comprising: (i) an AAV capsid comprising an AAV capsid protein, and (ii) the AAV vector genome according to any one of Embodiments 1 to 12, an AAV virus particle.

[0179] Embodiment 14. The AAV virus particle according to Embodiment 13, wherein the AAV capsid protein is at least 70%, 80%, 90%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 1 to 3, 67, 71, 196, 205, and 206.

[0180] Embodiment 15. A pharmaceutical composition comprising the AAV vector genome according to any one of Embodiments 1 to 12, or the AAV virus particle according to Embodiment 13 or 14.

[0181] Embodiment 16. A pharmaceutical composition comprising: (i) a first AAV virus particle comprising the AAV vector genome according to any one of Embodiments 1 to 11, and (ii) a second AAV virus particle comprising a second AAV vector genome, 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, a second AAV virus particle, and wherein retrograde recombination is prevented by 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.

[0182] Embodiment 17. A method for treating a subject having a disease or disorder caused by a gene deficiency, (1) The target is the first AAV virus particle, (i) an AAV capsid containing the first AAV capsid protein, and (ii) Administering a first AAV virus particle containing the AAV vector genome described in any one of Embodiments 1 to 11, (2) The target is a second AAV virus particle, (i) an AAV capsid containing a second AAV capsid protein, and (ii) A second AAV vector genome, which is oriented from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) Recombination parts, (c) Splice acceptor (SA) site, (d) The 3' portion of the introduced gene, (e) Poly A portion, and A method comprising administering a second AAV virus particle, comprising a second AAV vector genome, comprising a 3'AAV inverted terminal repeat.

[0183] Embodiment 18. The method according to Embodiment 17, wherein administration of a first AAV virus particle and a 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.

[0184] Embodiment 19. The method according to Embodiment 17 or 18, wherein polypeptide expression occurs upon administration of a first AAV virus particle and a second AAV virus particle.

[0185] Embodiment 20. The method according to any one of Embodiments 17 to 19, wherein the first AAV virus particle and the second AAV virus particle are administered simultaneously or sequentially.

[0186] Embodiment 21. The method according to Embodiment 20, wherein a first AAV virus particle and a second AAV virus particle are administered simultaneously.

[0187] Embodiment 22. The method according to any one of Embodiments 17 to 21, wherein the virus particles are administered by subretinal injection.

[0188] Embodiment 23. The method according to any one of Embodiments 17 to 22, wherein the gene deletion is an ABCA4 gene deletion.

[0189] Embodiment 24. The method according to Embodiment 23, wherein ABCA4 gene deletion results in one or more conditions selected from the group consisting of decreased expression of ABCA4 protein, removal of ABCA4 protein expression, expression of mutant ABCA4 protein, and decreased function of ABCA4 protein.

[0190] Embodiment 25. A method for expressing a polypeptide in cells, (1) Transduction of cells using a first AAV virus particle containing the AAV vector genome described in any one of Embodiments 1 to 11, wherein the transgene encodes a polypeptide, (2) Transducing cells using a second AAV virus particle containing a second AAV vector genome, wherein the second AAV vector genome is oriented from 5' to 3'. (a) 5'AAV inverted terminal repeat, (b) Recombination parts, (c) Splice acceptor (SA) site, (d) The 3' portion of the introduced gene, (e) Poly A portion, and (f) A method comprising introducing a 3'AAV inverted terminal repeat.

[0191] Embodiment 26. The method according to Embodiment 25, wherein stable expression of recombinase is not detected in transduced cells.

[0192] Embodiment 27. The method according to Embodiment 25 or 26, wherein the polypeptide is the ABCA4 protein.

[0193] Embodiment 28. Transduced cells, (i) Gene-deficient genome copies, (ii) A first recombinant nucleic acid, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) promoter, (c) 5' portion of the introduced gene, (d) Splice donor (SD) site, (e) Recombination parts, (f) Splice acceptor (SA) site, (g) 3' portion of the introduced gene, (h) Poly A portion, and (i) Includes 3'AAV inverted terminal repeat, The introduced gene is a first recombinant nucleic acid having a length of 4.6-7.7 kb, (iii) Transduced cells comprising a second recombinant nucleic acid comprising a 5'AAV inverted terminal repeat, a polynucleotide encoding a recombinase, and a 3'AAV inverted terminal repeat in the 5'-to-3' direction, wherein the second recombinant nucleic acid lacks a promoter.

[0194] Embodiment 29. Transduced cells according to Embodiment 28, wherein stable expression of recombinase is not detected in the transduced cells.

[0195] Embodiment 30. Transduced cells according to Embodiment 28 or 29, wherein the recombinase is Cre recombinase.

[0196] Embodiment 31. A transduced cell according to any one of Embodiments 28 to 30, wherein the transgene encodes the ABCA4 gene.

[0197] Embodiment 32. Transduced cells according to any one of Embodiments 28 to 31, wherein the cells are ex vivo cells. [Examples]

[0198] Example 1: Plasmid construction using bipartite design Using a bipartite vector system, several plasmids were generated to test the expression of the full-length ABCA4 gene in cells (Figure 2).

[0199] A plasmid called "5'ABCA4-FLAG+Cre" was constructed, containing, from 5' to 3', an 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.

[0200] The "5'ABCA4+FLAG+Cre" plasmid was generated by incorporating the 3xFLAG tag code sequence into the 5' portion of the ABCA4 gene in the "5'ABCA4-FLAG+Cre" plasmid.

[0201] The "5'ABCA4+FLAG-Cre" plasmid was generated by removing the Cre recombinase coding sequence from the "5'ABCA4+FLAG+Cre" plasmid.

[0202] A "3'ABCA4+FLAG" plasmid was constructed, containing a 5'AAV inverted terminal repeat, a Lox66 site, a splice acceptor (SA) site, the 3' portion of the ABCA4 gene ORF incorporated into the 3xFLAG tag coding sequence, a polyA site, and a 3'AAV inverted terminal repeat from 5' to 3'.

[0203] 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 a construct generated by recombination between the "5'ABCA4+FLAG+Cre" and "3'ABCA4+FLAG" plasmids. Both positive control constructs contained the 3xFLAG tag coding sequence in both the 5' and 3' portions of the ABCA4 gene.

[0204] Example 2: Expression of full-length proteins using a bipartite vector system Cell studies were performed using Lec2 cells transfected with the shown plasmids described in Example 1 and Figure 2, and the expression of full-length ABCA4 protein was examined using Western blotting. The full-length ABCA4 protein (with FLAG tag) has an estimated molecular weight of approximately 261 kD. As shown in Figure 3A, among cells transfected with plasmids other than the positive control, 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 the cells. On the other hand, in cells transfected with a dual-vector system without Cre recombinase, there was no expression of full-length ABCA4 protein (i.e., the "5'ABCA4+FLAG-Cre" and "3'ABCA4+FLAG" pairs). Similarly, in replicate experiments (Figure 3B), among the cell populations transfected with the shown plasmids other than the positive control, only two experimental groups showed expression of full-length ABCA4. 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 can perform efficient Cre-lox-mediated recombination between the two plasmids, which in turn leads to the expression of the full-length ABCA4 protein.

[0205] Example 3: No detectable expression of Cre recombinase from recombinant nucleic acids. The expression level of Cre recombinase in plasmid-containing cells was analyzed (Figure 4). Lec2 cells were transfected with various plasmids and controls as described in Example 2. 48 hours post-transfection, cell samples were treated with sample buffer containing 4M urea, and stable expression of Cre recombinase was quantified by Western blotting using 3% milk as a blocking agent, a 1:10,000 dilution of rabbit anti-Cre mAb (Cell signaling technology #15036) as the primary antibody, and a 1:10,000 dilution of goat anti-rabbit HRP (azure) as the secondary antibody. Self-cleaving Cre recombinase has a molecular weight of approximately 37 kD. As expected, transfection with either the "5'ABCA4-FLAG+Cre" or "5'ABCA4+FLAG+Cre" plasmid resulted in clear expression of Cre recombinase in Lec2 cells. Notably, in cells co-transfected with the second plasmid "3'ABCA4+FLAG", there was no detectable expression of autocleaved Cre recombinase by Western blotting. This suggests that the dual-vector system enables efficient recombination between the first and second plasmids, which in turn leads to the generation of recombinant nucleic acids containing Cre recombinase ORF without a promoter, thus preventing Cre recombinase from being expressed.

[0206] Example 4: Full-length ABCA4 mRNA expression is achieved through Cre-mediated recombination of a bipartite vector system. The expression levels of full-length ABCA4 mRNA were quantified in cells transfected with various plasmids. As shown in Figure 5, cells transfected with a dual-vector system encoding Cre recombinase showed approximately 55-fold higher full-length ABCA4 mRNA expression levels compared to cells transfected with the corresponding dual-vector system encoding Cre recombinase ("5'ABCA4+FLAG-Cre" and "3'ABCA4+FLAG"). These results indicate that Cre recombinase effectively mediates the recombination between the two start vectors immediately after they are transfected into cells, driving the expression of full-length mRNA.

[0207] Example 5: Delivery of a bipartite vector system via dual AAV virus particles results in the expression of full-length ABCA4 mRNA. To express full-length ABCA4 mRNA within cells, we tested whether a bipartite vector system could be delivered by AAV virus particles. 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'ABACA4+FLAG+Cre," and the other type of AAV9 virus particle contained an AAV vector genome encoding "3'ABACA4+FLAG." As shown in Figure 6, ABCA4-negative cells were transduced with the indicated 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 showed high expression levels of full-length ABCA4 mRNA, and the expression level continued to increase for more than 120 hours after transduction. This result indicates that AAV virus particles effectively deliver the bipartite vector system within the same cell, and the mRNA payload is recombined so that it is appropriately reconstituted by the system.

Claims

1. AAV vector genome, in the direction from 5' to 3', (a) 5'AAV inverted terminal repeat, (b) promoter, (c) The 5' portion of the introduced gene, (d) Splice donor (SD) site, (e) Recombination parts, (f) Polynucleotide encoding a recombinase, (g) Poly A portion, and (h) Includes 3'AAV inverted terminal repeat, An AAV vector genome in which the polynucleotide encoding the recombinase is operably ligated to the promoter.

2. The AAV vector genome according to claim 1, wherein the recombinase is Cre recombinase.

3. The AAV vector genome according to claim 1 or 2, wherein the recombinase comprises a nuclear localization sequence (NLS).

4. The AAV vector genome according to claim 2 or 3, wherein the recombination site includes the LoxP71 sequence.

5. The AAV vector genome according to any one of claims 1 to 4, wherein the AAV vector genome includes 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.

6. The AAV vector genome according to claim 5, wherein the internally cleaved polypeptide is a self-cleaved peptide selected from the group consisting of T2A, P2A, E2A, and F2A.

7. The AAV vector genome according to any one of claims 1 to 4, 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.

8. The AAV vector genome according to any one of claims 1 to 7, wherein the introduced gene encodes a polypeptide.

9. The AAV vector genome according to claim 8, wherein the polypeptide is ABCA4 protein.

10. The promoters include: Roussarcoma virus (RSV) LTR promoter (optionally containing an RSV enhancer), cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, beta-actin promoter, phosphoglycerol kinase (PGK) promoter, U6 promoter, H1 promoter, CAG promoter, hybrid chicken beta-actin promoter, MeCP2 promoter, EF1 promoter, ubiquitous chicken β-actin hybrid (CBh) promoter, U1a promoter, and U1b promoter. The AAV vector genome according to any one of claims 1 to 9, wherein the promoter is a MeCP2 promoter, a MeP418 promoter, a MeP426 promoter, a minimal MeCP2 promoter, a VMD2 promoter, an mRho promoter, an EFla promoter, an Ubc promoter, a human β-actin promoter, a TRE promoter, an Ac5 promoter, a polyhedrin promoter, a CaMKIIa promoter, a Gal1 promoter, a TEF1 promoter, a GDS promoter, an ADH1 promoter, an Ubi promoter, or an α-1-anti-trypsin (hAAT) promoter.

11. The AAV vector genome according to any one of claims 1 to 9, wherein the promoter is a human rhodopsin kinase (RK) promoter, a human photoreceptor-binding protein promoter (IRBP), a human red / green opsin promoter (pR2.1), a human blue opsin promoter (HB), a mouse opsin promoter (mOP), a mouse short-wavelength opsin promoter (mBP), or a human rod cGMP phosphodiesterase β-subunit promoter (βPDE).

12. AAV virus particles, (i) AAV capsid containing AAV capsid protein, and (ii) AAV virus particle comprising the AAV vector genome according to any one of claims 1 to 11.

13. The AAV virus particle according to claim 12, wherein the AAV capsid protein is at least 90%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 1-3, 67, 71, 196, 205, and 206.

14. A pharmaceutical composition comprising an AAV vector genome according to any one of claims 1 to 11, or an AAV virus particle according to claim 12 or 13.

15. A pharmaceutical composition, (i) A first AAV virus particle comprising the AAV vector genome described in any one of claims 1 to 11, (ii) A second AAV virus particle containing a second AAV vector genome, oriented in the 5' to 3' direction, (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) A second AAV virus particle comprising a 3'AAV inverted terminal repeat, A pharmaceutical composition in which retrograde recombination is prevented by 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.

16. (1) The first AAV virus particle, (i) an AAV capsid containing a first AAV capsid protein, and (ii) A first AAV virus particle comprising the AAV vector genome according to any one of claims 1 to 11, (2) A second AAV virus particle, (i) an AAV capsid containing a second AAV capsid protein, and (ii) A second 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) A combination of a second AAV virus particle and a second AAV vector genome containing a 3' AAV inverted terminal repeat, A combination of substances used for treating a subject with a disease or disorder caused by a gene defect.

17. (i) an AAV capsid containing a first AAV capsid protein, and (ii) A composition comprising a first AAV virus particle, comprising the AAV vector genome according to any one of claims 1 to 11, A composition for use in treating subjects with diseases or disorders caused by gene defects, The composition is (i) an AAV capsid containing a second AAV capsid protein, and (ii) A second 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) A composition characterized by being administered in combination with a second AAV virus particle, which comprises a second AAV vector genome containing a 3'AAV inverted terminal repeat.

18. (i) an AAV capsid containing a second AAV capsid protein, and (ii) A second 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) A composition comprising a second AAV virus particle comprising a second AAV vector genome comprising a 3' AAV inverted terminal repeat, A composition for use in treating subjects with diseases or disorders caused by gene defects, The composition is (i) an AAV capsid containing a first AAV capsid protein, and (ii) A composition characterized by being administered in combination with a first AAV virus particle, comprising the AAV vector genome according to any one of claims 1 to 11.

19. The combination or composition for use according to any one of claims 16 to 18, wherein administration of the first AAV virus particles and the second AAV virus particles to the subject results in recombination of the first AAV vector genome and the second AAV vector genome via the recombination sites in the AAV vector genome of the first AAV virus particles and the recombination sites in the second AAV vector genome of the second AAV virus particles.

20. The combination or composition for use according to any one of claims 16 to 19, wherein the expression of the transgene occurs upon administration of the first AAV virus particle and the second AAV virus particle to the subject.

21. The combination or composition for use according to any one of claims 16 to 20, wherein the first AAV virus particle and the second AAV virus particle are for simultaneous administration or for sequential administration.

22. The combination or composition for use according to claim 21, wherein the first AAV virus particle and the second AAV virus particle are intended for simultaneous administration.

23. The combination or composition for use according to any one of claims 16 to 22, wherein the virus particles are for subretinal injection.

24. The combination or composition for use according to any one of claims 16 to 23, wherein the gene deficiency is an ABCA4 gene deficiency.

25. The combination or composition for use according to claim 24, wherein the ABCA4 gene deletion results in one or more conditions selected from the group consisting of decreased expression of ABCA4 protein, removal of ABCA4 protein expression, expression of mutant ABCA4 protein, and decreased function of ABCA4 protein.

26. (1) A first AAV virus particle comprising the AAV vector genome according to any one of claims 1 to 11, wherein the introduced gene encodes a polypeptide, (2) A second AAV virus particle comprising a second AAV vector genome, wherein the second AAV vector genome 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) A combination with a second AAV virus particle, which includes a 3'AAV inverted terminal repeat, A combination for use in expressing the polypeptide in cells.

27. A composition comprising a first AAV virus particle comprising an AAV vector genome according to any one of claims 1 to 11, wherein the transgene encodes a polypeptide, the composition is for use in expressing the polypeptide in a cell, the composition is characterized in that it is administered in combination with a second AAV virus particle comprising a second AAV vector genome, the second AAV vector genome is oriented 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) A composition comprising a 3'AAV inverted terminal repeat.

28. A composition comprising a second AAV virus particle comprising a second AAV vector genome, wherein the second AAV vector genome 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) Includes 3'AAV inverted terminal repeat, The composition is for use in expressing a polypeptide in cells, and is characterized in that it is administered in combination with a first AAV virus particle containing an AAV vector genome according to any one of claims 1 to 11, wherein the transgene encodes the polypeptide.

29. The combination or composition for use according to any one of claims 26 to 28, wherein the stable expression of the recombinase is not detected in the cells, and the cells are transduced with the combination or composition and express the polypeptide.

30. The combination or composition for use according to any one of claims 26 to 29, wherein the polypeptide is ABCA4 protein.

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