Compositions and methods for treating stargardt disease

Abstract

The disclosure provides dual AAV vector compositions and systems for reconstitution of RNA molecules, including methods for using these molecules. For example, such molecules can be used to deliver a coding sequence for a ABCA4 gene over two AAV vectors, resulting in reconstitution of the full-length ABCA4 protein in a cell. Such methods can be used to deliver therapeutic ABCA4 protein to treat Stargardt Disease.

Description

COMPOSITIONS AND METHODS FOR TREATING STARGARDT DISEASECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application No. 63 / 642,478, filed May 3, 2024 and U.S. Provisional Application No. 63 / 705,907, filed October 10, 2024, the disclosure of each of which are incorporated by reference in their entireties.FIELD

[0002] The disclosure generally relates to dual adeno-associated virus (AAV) particle compositions for delivering an Abca4 coding sequence over two AAV vectors, methods of producing the AAV particles, cells producing the AAV particles, and methods of using the AAV particles for the treatment of Stargardt Disease.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (INMD_214_02WO_SeqList_ST26.xml; Size: approximately 50,876 bytes; and Date of Creation: April 18, 2025) are herein incorporated by reference in its entirety.BACKGROUND

[0004] Stargardt Disease is the most prevalent form of inherited childhood blindness, affecting 1 :9000 individuals in the United States. Stargardt disease is caused by mutations in the retinaspecific ATP -binding cassette transporter (Abca4) gene encoding a large (256kDa) transmembrane protein that clears toxic retinyl-derivatives from photoreceptors. Loss of Abca4 leads to accumulation of toxic byproducts that cause degeneration of the photoreceptors and apposing retinal pigment epithelium. Despite a well-characterized disease mechanism, treatment for Stargardt patients is restricted to symptom management, as there is currently no cure or method for stalling progressive vision loss.

[0005] Monogenic inherited retinal diseases (IRDs) such as Stargardt disease are in principle particularly suitable for gene replacement therapy, both because there is loss of function of a single gene and because of the relative accessibility and “immune privilege” of the affected retinal tissue. In vivo mouse studies of Stargardt disease have established that Abca4 gene replacement can be an effective treatment, however a major challenge in gene therapy has been the ~5-kilobase cargo size limitation of adeno-associated viruses (AAV) and thus an inability to replace large genes such as Abca4 efficiently and safely.

[0006] Thus, there is an urgent need in the art for compositions and methods for the treatment of Stargardt disease.SUMMARY

[0007] The present application utilizes RNA-end joining (REJ) technology to address the challenges related to AAV-based gene therapy by delivering a gene of interest as fragments in two or more individual AAVs and subsequently reconstituting the full-length coding sequence on the mRNA level in the target cell. Thus, the present application addresses the challenges of conventional gene therapy approaches, and in particular advances the use of AAV strategies in large genes implicated in ocular diseases such as Stargardt disease.

[0008] In one aspect of the disclosure, provided herein are dual adeno-associated virus (AAV) particle compositions for expressing a target protein. In some embodiments, the composition comprises a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises from 5’ to 3’ : (i) a first 5 ’-inverted terminal repeat (5’- ITR) sequence; (ii) a first promoter sequence; (iii) a Kozak sequence; (iv) an N-terminal coding sequence encoding an N-terminal portion of a target protein, said N-terminal coding sequence being operably linked to and under control of said first promoter; (v) a nucleic acid sequence of a splicing donor; (vi) a nucleic acid sequence of a first dimerization domain; (vii) a first poly(A) signal sequence; and (viii) a first 3’-ITR sequence; and (b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises: (i) a second 5’-ITR sequence; (ii) a second promoter sequence; (iii) a nucleic acid sequence of a second dimerization domain, said second dimerization domain being operably linked to and under control of said second promoter; (iv) a nucleic acid sequence of a splicing acceptor; (v) a C-terminal coding sequence encoding a C-terminal portion of the target protein; vi) a second poly(A) signal sequence; and (vii) a second 3’-ITR sequence; wherein the first dimerization domain comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 9; and wherein the second dimerization domain comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 16.

[0009] In some embodiments, the composition comprises a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises from 5’to 3’ : (i) a first 5 ’-inverted terminal repeat (5’-ITR) sequence; (ii) a first promoter sequence; (iii) a Kozak sequence; (iv) an N-terminal coding sequence encoding an N-terminal portion of a target protein, said N-terminal coding sequence being operably linked to and under control of said first promoter; (v) a nucleic acid sequence of a splicing donor; (vi) a nucleic acid sequence of a first dimerization domain; (vii) a first poly(A) signal sequence; and viii) a first 3’-ITR sequence; and (b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises: (i) a second 5’-ITR sequence; (ii) a second promoter sequence; (iii) a nucleic acid sequence of a second dimerization domain, said second dimerization domain being operably linked to and under control of said second promoter; (iv) a nucleic acid sequence of a splicing acceptor; (v) a C-terminal coding sequence encoding a C-terminal portion of the target protein; (vi) a second poly(A) signal sequence; and (vii) a second 3’-ITR sequence; wherein the N-terminal coding sequence comprises a first intervening intron sequence within the N-terminal coding sequence; and wherein the C- terminal coding sequence comprises a second intervening intron sequence within the C- terminal coding sequence.

[0010] In some embodiments, the composition comprises (a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises from 5’ to 3’ : (i) a first 5 ’-inverted terminal repeat (5’-ITR) sequence; (ii) a first promoter sequence; (iii) a Kozak sequence; (iv) an N-terminal coding sequence encoding an N-terminal portion of an Abca4 protein, said N-terminal coding sequence being operably linked to and under control of said first promoter; (v) a nucleic acid sequence of a splicing donor; (vi) a nucleic acid sequence of a first dimerization domain; (vii) a first poly(A) signal sequence; and (viii) a first 3’-ITR sequence; and (b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises: (i) a second 5’-ITR sequence; (ii) a second promoter sequence; (iii) a nucleic acid sequence of a second dimerization domain, said second dimerization domain being operably linked to and under control of said second promoter; (iv) a nucleic acid sequence of a splicing acceptor; (v) a C-terminal coding sequence encoding a C-terminal portion of the Abca4 protein; (vi) a second poly(A) signal sequence; and (vii) a second 3’-ITR sequence; wherein the N-terminal coding sequence comprises a nucleic acid sequence that is at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 7; and wherein the C-terminal coding sequence comprises a nucleic acid sequence that is at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 20.

[0011] In some embodiments, the composition comprises (a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises from 5’ to 3’ : (i) a first 5 ’-inverted terminal repeat (5’-ITR) sequence; (ii) a first promoter sequence; (iii) a Kozak sequence; (iv) an N-terminal coding sequence encoding an N-terminal portion of an Abca4 protein, said N-terminal coding sequence being operably linked to and under control of said first promoter; (v) a nucleic acid sequence of a splicing donor; (vi) a nucleic acid sequence of a first dimerization domain; (vii) a first poly(A) signal sequence; and viii) a first 3’-ITR sequence; and (b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises: (i) a second 5’-ITR sequence; (ii) a second promoter sequence; (iii) a nucleic acid sequence of a second dimerization domain, said second dimerization domain being operably linked to and under control of said second promoter; (iv) a nucleic acid sequence of a splicing acceptor; (v) a C-terminal coding sequence encoding a C-terminal portion of the Abca4 protein; (vi) a second poly(A) signal sequence; and vii) a second 3’-ITR sequence; wherein a first transcript of the first transgene and a second transcript of the second transgene can be spliced and joined to form a full-length coding sequence that encodes the Abca4 protein; and wherein the full-length Abca4 coding sequence comprises a nucleic acid sequence that is at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 22.

[0012] In some embodiments, the target protein is an Abca4 protein. In some embodiments, the N-terminal coding sequence is codon optimized. In some embodiments, the N-terminal coding sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:7. In some embodiments, the C-terminal coding sequence is codon optimized. In some embodiments, the C-terminal coding sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 20.

[0013] In some embodiments, a first transcript of the first transgene and a second transcript of the second transgene can be spliced and joined to form a full-length coding sequence that encodes an Abca4 protein. In some embodiments, the full-length Abca4 coding sequence is codon optimized. In some embodiments, the full-length Abca4 coding sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 22.

[0014] In some embodiments, the N-terminal coding sequence comprises a first intervening intron sequence within the N-terminal coding sequence. In some embodiments, the first intervening intron sequence is a modified mouse beta-actin (Actb) intron 2 sequence. In some embodiments, the first intervening intron sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the C-terminal coding sequence comprises a second intervening intron sequence within the C-terminal coding sequence. In some embodiments, the second intervening intron sequence is a modified mouse beta-actin (Actb) intron 2 sequence. In some embodiments, the second intervening intron sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 5

[0015] In some embodiments, the splicing donor comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 8. In some embodiments, the splicing acceptor comprises a nucleic acid nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 17

[0016] In some embodiments, the first dimerization domain and the second dimerization domain comprise sequences that are complementary to each other. In some embodiments, the first dimerization domain and the second dimerization domain contain complementary RNA stem loops. In some embodiments, the complementary RNA stem loops form central kissing loop interaction with four or more loop base pairs for intermolecular pairing. In some embodiments, the positioning of the complementary RNA stem loops is offset by at least 1 nt, at least 2nt, at least 3 nt, at least 4nt, at least 5nt, at least 6 nt, at least 7nt, at least 8 nt, at least 9nt, or at least lOnt so that the respective stem regions of the complementary RNA stem loops base pair in trans through strand invasion. In some embodiments, the stem regions of the complementary RNA stem loops contain about 1% to about 30% mismatches, but match to the other dimerization domain. In some embodiments, the first dimerization domain and the seconddimerization domain form an extended duplex through strand invasion. In some embodiments, the first dimerization domain comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 9. In some embodiments, the second dimerization domain comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 16.

[0017] In some embodiments, the first promoter is an ocular tissue-specific promoter. In some embodiments, the ocular tissue-specific promoter is a photoreceptor-specific promotor. In some embodiments, the first promoter is a human rhodopsin kinase (hGRKl) promoter. In some embodiments, the first promoter comprises the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the second promoter is an ocular tissue-specific promoter. In some embodiments, the ocular tissue-specific promoter is a photoreceptor-specific promotor. In some embodiments, the second promoter is a human rhodopsin kinase (hGRKl) promoter. In some embodiments, the second promoter comprises the nucleic acid sequence of SEQ ID NO: 2.

[0018] In some embodiments, the first 5’-ITR is a 5’ AAV2 ITR. In some embodiments, the first 5'-ITR comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 1. In some embodiments, the first 3 ’-ITR is a 3’ AAV2 ITR. In some embodiments, the first 3 ’-ITR comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 11. In some embodiments, the second 5’-ITR is a 5’ AAV2 ITR. In some embodiments, the second 5’-ITR comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 1. In some embodiments, the second 3 ’-ITR is a 3’ AAV2 ITR. In some embodiments, the second 3 ’-ITR comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical toSEQ ID NO: 11

[0019] In some embodiments, the first poly(A) signal sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 10. In some embodiments, the second poly(A) signal sequence comprises a late SV40 poly(A) signal sequence. In some embodiments, the second poly(A) signal sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO:21.

[0020] In some embodiments, the Kozak sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to a sequence of SEQ ID NO:3, 23, 24, or 25.

[0021] In embodiments described herein, the first or second AAV particle comprises an AAV capsid encapsidating a transgene. In some embodiments, the AAV capsid of the first AAV particle and / or the second AAV particle comprises one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.8, AAVrh.10, AAVrh.39, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 capsid proteins. In some embodiments, the first AAV particle and / or the second AAV particle is an AAV8 particle and the AAV capsid of the first AAV particle and / or the second AAV particle comprises one or more AAV8 capsid proteins. In some embodiments, the one or more AAV8 capsid proteins comprises AAV8 capsid protein VP1. In some embodiments, the one or more AAV8 capsid proteins comprise AAV8 capsid protein VP2. In some embodiments, the one or more AAV8 capsid proteins comprise AAV8 capsid protein VP3. In some embodiments, the AAV capsid the first AAV particle and / or the second AAV particle comprises one or more engineered variants of an AAV8 capsid protein. In some embodiments, the one or more engineered variants of the AAV8 capsid protein comprises an engineered variant of AAV8 capsid protein VP1. In some embodiments, the one or more engineered variants of the AAV8 capsid protein comprises an engineered variant of AAV8 capsid protein VP2. In some embodiments, the one or more engineered variants of the AAV8capsid protein comprises an engineered variant of AAV8 capsid protein VP3. In some embodiments, the one or more engineered variants of the AAV8 capsid protein are one or more deimmunized variants of an AAV8 capsid protein.

[0022] Provided herein are pharmaceutical compositions comprising an effective amount of the first AAV particle and the second AAV particle described herein and a pharmaceutically acceptable carrier, excipient, diluent, or buffer. In some embodiments, the pharmaceutical composition comprises an effective amount of the first AAV particle and the second AAV particle at a ratio of about 1 to about 1. In some embodiments, the ratio of about 1 to about 1 is a ratio of the number of first encapsidated transgenes to the number of second encapsidated transgenes (i.e., about 1 to about 1 ratio of vector genomes). In some embodiments, the ratio of about 1 to about 1 is a ratio of the number of first AAV particles to the number of second AAV particles. In some embodiments, the pharmaceutical composition is suspended in a buffer at a concentration of from about | / I O10total vg / mL to about | / I O14total vg / mL. In some embodiments, the pharmaceutical composition is suspended in a buffer at a concentration of from about I x lO10total vg / mL to about 2 / I 013total vg / mL. In some embodiments, the pharmaceutical composition is suspended in a buffer at a concentration of about I x lO10total vg / mL, about 1.25x lO10total vg / mL, about 2x lO10total vg / mL, about 5x lO10total vg / mL, about I x lO11total vg / mL, about 1.25xlOntotal vg / mL, about 2x lOntotal vg / mL, about 5x l0ntotal vg / mL, about I x lO12total vg / mL, about 1 ,25x 1012total vg / mL, about 2x 1012total vg / mL, about 5 x lO12total vg / mL, about I x lO13total vg / mL, about 1.25x l013total vg / mL, about 2x l013total vg / mL, about 5x 1013total vg / mL, or about I x lO14total vg / mL.

[0023] In another aspect of the disclosure, methods for treating Stargardt Disease (STGD) in a subject in need thereof are provided. In some embodiments, the method comprises administering to the subject the pharmaceutical composition described herein. In some embodiments, the administering comprises subretinal administration. In some embodiments, the subretinal administration comprises subretinal administration with foveal placement. In some embodiments, the administering comprises subfoveal administration. In some embodiments, the administering comprises intravitreal administration.

[0024] In some embodiments, the method comprises administering to the subject, by subretinal injection, an effective amount of a pharmaceutical composition comprising first and second AAV particles, wherein the first AAV particle comprises an AAV capsid encapsidating a first transgene, the first transgene comprising from 5’ to 3’: a 5’-ITR sequence of SEQ ID NO: 1; a first promoter sequence comprising an ocular tissue-specific promoter of SEQ ID NO: 2; aKozak sequence of SEQ ID NO: 3; anN-terminal coding sequence of SEQ ID NO: 7 encoding an N-terminal portion of an Abca4 protein, the N-terminal coding sequence being operably linked to and under control of the first promoter; a nucleic acid sequence of a splicing donor of SEQ ID NO: 8; a nucleic acid sequence of a first dimerization domain of SEQ ID NO: 9; a poly(A) signal sequence of SEQ ID NO: 10; and a 3’-ITR sequence of SEQ ID NO: 11; wherein the second AAV particle comprises an AAV capsid encapsidating a second transgene, the second transgene comprising from 5’ to 3’: the 5’-ITR sequence of SEQ ID NO: 1; a second promoter sequence comprising the ocular tissue-specific promoter of SEQ ID NO: 2; a nucleic acid sequence of a second dimerization domain comprising a sequence of SEQ ID NO: 16, said second dimerization domain being operably linked to and under control of said second promoter; a nucleic acid sequence of a splicing acceptor comprising a sequence of SEQ ID NO: 17; a C-terminal coding sequence of SEQ ID NO: 20 encoding a C-terminal portion of the Abca4 protein; a poly(A) signal sequence of SEQ ID NO: 21; and the 3’-ITR sequence of SEQ ID NO: 11, and wherein a first transcript of the first transgene and a second transcript of the second transgene is spliced and joined to form a full-length coding sequence of SEQ ID NO: 22 that encodes the Abca4 protein.

[0025] In some embodiments, wherein the composition is administered to one eye of the subject. In some embodiments, the composition is administered to both eyes of the subject. In some embodiments, the subject is a neonatal human subject. In some embodiments, the subject is an adolescent human subject. In some embodiments, the subject is an adult human subject.

[0026] In some embodiments, the effective amount of the AAV particles in the composition is from about 1 x 109vector genomes (vg) to about 1 x 1014vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is from about 1 x 109vg to about 1 x 1013vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is from about 1 x 109vg to about 1 x 1012vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is from about 1 x 109vg to about 1 x 1011vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is from about 1 x 109vg to about 1 x 1010vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is from about 1 x 109vg to about 5x 1012vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, theeffective amount of the AAV particles in the composition is from about l * 109vg to about I x lO12vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about 1 * 109vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about 5* 109vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about I x lO10vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about 1.5x lO10vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about 5x lO10vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about I x lO11vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about 1.5xl0nvg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about 5x l0nvg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about I x lO12vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about 1.5 x lO12vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about 2x 1012vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about 2.5x 1012vg of each the first AAV vector and the second AAV vector, per eye. In even another embodiment, the effective amount of the AAV particles in the composition is about 5x 1012vg of each the first AAV vector and the second AAV vector, per eye.

[0027] In some embodiments, the effective amount of the AAV particles in the composition administered per eye is from about I x lO10total vg to about 5x l012total vg of the first and second AAV vectors combined. In some embodiments, the about I x lO10total vg comprises about 5x 109vg of the first AAV vector and 5x 109vg of the second AAV vector; and the about 5x l012total vg comprises about 2.5x l012vg of the first AAV vector and 2.5x l012vg of the second AAV vector.

[0028] In some embodiments, the effective amount of the AAV particles is administered as a single dose per eye. In some embodiments, the effective amount of the AAV particles is administered as multiple doses per eye.

[0029] In some embodiments, the treating comprises restoring the Abca4 protein level in the subject to a normal range orto anon-STGD level. In some embodiments, the treating comprises increasing the Abca4 protein level in the subject to at least about 200%, about 195%, about 190%, about 185%, about 180%, about 175%, about 170%, about 165%, about 160%, about 155%, about 150%, about 145%, about 140%, about 135%, about 130% about 125%, about 120%, about 115%, about 110%, about 105%, about 100%, about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30% about 25%, about 20%, about 15%, about 10%, about 5%, about 2%, or about 1% of the normal or the pre-treatment level. In some embodiments, the treating comprises reducing the severity of one or more STGD symptoms in the subject. In some embodiments, one or more STGD symptoms comprise gray, black or hazy spots in the center of the vision, sensitivity to light, needing more time for eyes to adjust between light and dark places, color blindness, or any combination thereof.

[0030] Provided herein, in another aspect, are DNA plasmids comprising an N-terminal transgene construct, wherein the N-terminal transgene construct comprises from 5’ to 3’ : i) a 5 ’-inverted terminal repeat (5’-ITR) sequence described herein; (ii) a promoter sequence described herein; (iii) a Kozak sequence described herein; (iv) an N-terminal coding sequence encoding an N-terminal portion of a target protein described herein, said N-terminal coding sequence being operably linked to and under control of said promoter; (v) a nucleic acid sequence of a splicing donor described herein; (vi) a nucleic acid sequence of a dimerization domain described herein; (vii) a poly(A) signal sequence described herein; and (viii) a 3’-ITR sequence described herein.

[0031] Provided herein, in another aspect, are DNA plasmids comprising a C-terminal transgene construct, wherein the transgene construct comprises from 5’ to 3’: (i) a 5’-ITR sequence described herein; (ii) a promoter sequence described herein; (iii) a nucleic acid sequence of a dimerization domain described herein, said dimerization domain being operably linked to and under control of said promoter; (iv) a nucleic acid sequence of a splicing acceptor described herein; (v) a C-terminal coding sequence encoding a C-terminal portion of a target protein described herein; (vi) a poly(A) signal sequence described herein; and (vii) a 3’-ITR sequence described herein.

[0032] In yet another aspect of the disclosure, packaging cells are provided, comprising one or more DNA plasmids described herein. The packaging cell, in one embodiment, comprises a DNA plasmid comprising an N-terminal transgene construct, and a DNA plasmid comprising a C-terminal transgene construct. In some embodiments, the packaging cell further comprises a helper plasmid containing adenoviral components necessary for recombinant adeno- associated virus production. In some embodiments, the packaging cell further comprises a plasmid comprising AAV replication (rep) and capsid (cap) genes. In some embodiments, the packaging cell is a HEK293 cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 depicts an exemplary RNA-end joining (REJ) process.

[0034] FIGS. 2A-2C are schematic drawings providing an exemplary dual AAV-REJ-Abca4 transgenes. The first transgene contains an N-terminal Abca4 coding sequence (FIG. 2A). The second transgene comprises a C-terminal Abca4 coding sequence (FIG. 2B). FIG. 2C is a schematic drawing providing transcribed and joined RNA sequence.

[0035] FIG. 3 depicts the design for the doner dimerization domain and acceptor dimerization domain.

[0036] FIGS. 4A-4B are representative immunohistochemistry images showing staining for Abca4 protein in a retinal section. All sections were counterstained with DAPI. FIG. 4B is a higher power image of FIG. 4A showing Abca4 expression localized to photoreceptors in the outer nuclear layer (ONL).

[0037] FIGS. 5A-5F are representative images of in situ hybridization with probes designed to target either the N-terminal fragment (FIG. 5A), C-terminal fragment (FIG. 5B), or the junction of the two fragments (full-length mRNA, FIG. 5C). FIGS. 5D-5F show dual in situ hybridization for N-terminal fragment and C-terminal fragment combined with immunohistochemistry for Abca4 protein.

[0038] FIGS. 6A-6F shows western blot analysis of neural retina tissue lysates from wildtype and Abca4 -I- mice treated dual AAV8-REJ-Abca4 vectors at a 1 : 1 ratio. An N-terminal specific antibody was used in FIGS. 6A-6C, and a C-terminal specific antibody was used in FIGS. 6D-6F

[0039] FIG. 7A is a representative image of retinal sections from the animal VTU1437 with -90% targeting. FIG. 7B is a representative image of retinal sections from the animalVTU1441 with ~5% targeting. FIG. 7C is a representative image of retinal sections from the animal VTU1443 with -50% targeting.

[0040] FIGS. 8A-8D are representative images of retinal sections from the animal VTU1433 with 90% targeting. FIG. 8B is a higher power image of FIG. 8A; and FIG. 8D is a higher power image of FIG. 8C.

[0041] FIGS. 9A-9C are representative images of retinal sections from the Abca4- / - animal treated with a high dose of dual AAV8-REJ-Abca4 vectors. FIG. 9B is a higher power image of FIG. 9A. FIG. 9C shows the staining of Abca4 expression in a wild type mouse.

[0042] FIG. 10 is a graph of INS1203-RD vector genomes (vg) per diploid genome (dg) vs. INS1203-RA vgs per dg in individual eyecups.

[0043] FIG. 11 is a bar graph showing ABCA4 protein expression normalized to a reference murine wild-type lysate for individual eyecups.

[0044] FIG. 12 is a graph of A2E concentration (ng / mL) in wild type (WT) and INS 1203 treated ABCA4- / - mice. Quantification of A2E was by mass spectrometry. Abca-T mice have a statistically significant increase in A2E at 14 weeks of age compared to wild-type mice (p<0.0001). INS1203 treatment at a dose of 2.0E+10 vg significantly reduces A2E in Abca^' mice (p<0.001). *** = p<0.001; **** = p<0.0001; A2E = N-retinylidene-N- retinylethanolamine; vg = vector genomes.

[0045] FIG. 13A is a graph of INS1203-RD vector genomes (normalized to the count of diploid genomes in the same sample) in wild type (WT), INS 1203 -treated Abca-T mice or vehicle-treated Abca.4^ mice, measured by ddPCR. FIG. 13B is a graph of INS1203-RA vector genomes (normalized to the count of diploid genomes in the same sample) in wild type (WT), INS 1203 -treated A bca4^ mice or vehicle-treated A bca4^ mice. ddPCR = droplet digital polymerase chain reaction; vg = vector genomes; dg = diploid genomes.

[0046] FIG. 14 is a graph of INS1203-RD vector genomes (vg) per diploid genome (dg) vs. INS1203-RA vg per dg in individual treated mouse retinas.

[0047] FIG. 15 is a graph of A2E concentration (ng / mL) in wild type (WT) and INS 1203 treated Abca4 / ' mice. Quantification of A2E was by mass spectrometry. ** = p <0.01; **** = p < 0.0001; ng / mL = nanogram per milliliter; vg = vector genomes; A2E = N-retinylidene-N- retinylethanolamine.

[0048] FIGS. 16A and 16B are images from the non-human primate (NHP) retina. FIG. 16A is a pre-treatment fundus image of the NHP macula with the dotted circle indicating the goal treatment area, the arrow indicating fovea, and the pointer indicating the injection site; scale = 200 pm. FIG. 16B is an intraoperative image demonstrating successful bleb placement near the fovea indicated by the arrow, a 0. ImL subretinal bleb.

[0049] FIGS. 17A and 17B depict optical coherence tomography (OCT) evaluations of the NHP retina at the date of the injection (FIG. 17A) and four weeks post-injection (FIG. 17B), with the latter not revealing any test article related abnormalities. ILM = internal limiting membrane; ELM = external limiting membrane. Scale bar = 200 pm.

[0050] FIGS. 18A-18C are representative fluorescent micrographs showing ABCA4 mRNA expression in INS 1203 -treated samples. Using FISH, human ABCA4 mRNA is detected in the high dose-treated macula (D959, OS, FIG. 18A) as well as in the low dose-treated macula (D959, OD, FIG. 18B), with expression in the outer nuclear layer (ONL) where photoreceptor cell bodies are located. ABCA4 mRNA is not detected in the naive untreated non-human primate (NHP) retina with only DAPI stained nuclei shown (A761 OD, FIG. 18C), demonstrating species-specific detection of human codon-optimized ABCA4 mRNA. High magnification, 20X.

[0051] FIGS. 19A and 19B are representative overview fluorescent micrographs of NHP fovea showing the extent of ABCA4 mRNA expression in INS 1203 -treated samples. Using FISH, human ABCA4 mRNA is detected throughout the high dose-treated macula (D959, OS, FIG. 19A) as well as throughout the low dose-treated macula, although to a lesser degree (D959, OD, FIG. 19B). Representative images are shown for sections representing the fovea.DETAILED DESCRIPTION

[0052] It is to be understood that both the foregoing general description and the following detailed description are exemplary, and explanatory only, and are not restrictive of the disclosure.

[0053] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0054] All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.Definitions

[0055] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0056] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the present application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, representative methods and materials are herein described.

[0057] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate. Thus, for example, reference to “a carrier” includes mixtures of one or more carriers, two or more carriers, and the like and reference to “the method” includes reference to equivalent steps and / or methods known to those skilled in the art, and so forth.

[0058] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the present application. Generally, the term “about”, as used herein in references to a measurable value such as an amount of weight, time, dose, etc. is meant to encompass values within an acceptable degree of variability in the art. In some embodiments, degree of variability is based on FDA guidelines.

[0059] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0060] As used herein, the term “wild type” (abbreviated “WT”) refers to a typical form of an organism, strain, gene, protein, or characteristic as it occurs in nature as distinguished from mutant or variant forms. For example, a wild type protein is the typical form of that protein as it occurs in nature.

[0061] The term “nucleic acid,” “nucleotide,” “polynucleotide” or “oligonucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleicacids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al. (1991). Nucleic Acid Res. 19:5081; Ohtsuka et al. (1985). J. Biol. Chem. 260:2605-2608; and Rossolini et al. (1994). Mol. Cell. Probes 8:91-98).

[0062] The term “gene” can refer to the segment of DNA involved in producing or encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). Alternatively, the term “gene” can refer to the segment of DNA involved in producing or encoding a non-translated RNA, such as an rRNA, tRNA, guide RNA (gRNA), shortinterfering RNA (siRNA), or micro-RNA (miRNA).

[0063] As used herein, the term “transgene” refers to an exogenous gene, for example supplied by a vector, such as AAV. In one embodiment, a transgene comprises a coding region that encodes a portion of a target protein, such as about a third, half, or two-thirds of a target protein. Transgenes in embodiments described herein include regions preceding and following the coding region, regulatory elements, as well as elements necessary for RNA-end joining (REJ) (e.g., a splicing domain and a dimerization domain). The transgene is flanked on each end by an inverted terminal repeat (ITR), and each ITR is a component of the transgene. The transgene according to embodiments described herein, comprises a fragment of the full Abca4 coding sequence (e.g., N-terminal or C-terminal Abca4 coding sequence). In embodiments described herein, a transgene includes, a 5’ inverted terminal repeat (ITR); a promoter; an N-terminal Abca4 coding sequence; a splicing donor; a dimerization domain; a poly(A) signal; and a 3’ ITR. In another embodiment, a transgene includes, a 5’ inverted terminal repeat (ITR); a promoter; a dimerization domain; a splicing acceptor; a C-terminal Abca4 coding sequence; a poly(A) signal; and a 3’ ITR.

[0064] “Vector genome” or “vg”, as used herein, refers to a transgene that is encapsidated by a viral capsid, e.g., an AAV capsid. In embodiments described herein, a subject is administered an effective amount of a composition of the disclosure. The effective amount, in someembodiments, is a certain vector genome dose (“vg dose”), and refers to the number of vector genomes administered to a subject. A “vg dose” may refer to the dose of a single vector or the dose of multiple vectors (e.g., a first vector and a second vector). In some embodiments, compositions of the disclosure are described by the number of vector genomes present.

[0065] As used herein, the term “endogenous” with reference to a nucleic acid, for example, a gene, or a protein in a cell, is a nucleic acid or protein that occurs in that particular cell as it is found in nature, for example, at its natural genomic location or locus. Moreover, a cell “endogenously expressing” a nucleic acid or protein expresses that nucleic acid or protein as it is found in nature.

[0066] A “promoter” refers to one or more nucleic acid control sequence(s) that direct transcription of a nucleic acid, e.g., a fragment of the Abca4 coding sequence and REJ elements (e.g., a splicing domain and a dimerization domain), and is present within a transgene or when the transgene is encapsidated, a vector genome. As used herein, a promoter includes nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0067] A “regulatory element”, as used herein, refers to a nucleic acid sequence capable of regulating transcription of a gene (e.g., comprising a fragment of Abca4 coding sequence and REJ elements), and / or regulate the stability or translation of a transcribed mRNA product, and can be present within a vector genome. In some embodiments, regulatory elements can regulate tissue-specific transcription of a gene, for example, eye. Regulatory elements can comprise at least one transcription factor binding site. Regulatory elements as used herein increase or enhance promoter-driven gene expression when compared to the transcription of the gene from the promoter alone in the absence of the regulatory element. Regulatory elements as used herein may occur at any distance (i.e., proximal or distal) to the coding sequence. Regulatory elements as used herein may comprise part of a larger sequence involved in transcriptional control, e.g., part of a promoter sequence. However, regulatory elements alone are typically not sufficient to initiate transcription on its own and require the presence of a promoter.

[0068] A first nucleic acid is “operably linked” to a second nucleic acid when the first nucleic acid is placed into a functional relationship with the second nucleic acid. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation of the coding sequence. A first nucleic acid that is “operably linked” to a second nucleic acid need not be directly linked. In other words, there may be intervening sequences between two operably linked nucleic acids.

[0069] “Polypeptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, and functional fragments thereof, wherein the amino acid residues are linked by covalent peptide bonds.

[0070] A nucleic acid sequence that is “flanked” by two nucleic acid elements indicates that one element is located 5’ to the sequence and the other element is located 3’ to the sequence. The term “flanked” is not intended to indicate that the respective sequences are necessarily contiguous. For example, there may be intervening sequences between one nucleotide sequence, e.g., a coding sequence, and an ITR.

[0071] As used herein, a first nucleic acid sequence is “upstream” of a second nucleic acid sequence, if the first nucleic acid sequence is located 5’ of the second nucleic acid. As used herein, a first nucleic acid sequence is “downstream” of a second nucleic acid sequence, if the first nucleic acid sequence is located 3’ of the second nucleic acid.

[0072] As used herein, the term “complementary” refers to specific base pairing between nucleotides or nucleic acids. Complementary nucleotides are, generally, A and T (or A and U), and G and C. The polynucleotides and nucleic acids described herein can comprise sequences that are perfectly complementary or substantially complementary (e.g., having a small fraction of mismatched bases) to a genomic sequence. A single stranded nucleic acid for the purposes described herein, also refers to the complementary nucleic acid and a double stranded nucleic acid comprises both strands.

[0073] As used herein, the terms “introducing” or “delivering” in the context of nucleic acids, for example, AAV vectors, refers to the translocation of the nucleic acid from outside a cell to inside the cell. In some cases, introducing refers to translocation of the nucleic acid from outside the cell to inside the nucleus of the cell. Various methods of such translocation are contemplated, including but not limited to, electroporation, contact with nanowires or nanotubes, receptor mediated internalization, translocation via cell penetrating peptides, liposome-mediated translocation, and the like.

[0074] As used herein, the terms “packaged” or “encap si dated” refers to the inclusion of a transgene in a capsid comprising viral capsid proteins to form an AAV particle encapsidatingthe transgene. Once the transgene is encapsidated, in some embodiments, the transgene is referred to a vector genome. The capsid, in one embodiment of the disclosure, is an AAV9 capsid. In one preferred embodiment of the disclosure, the capsid is an AAV8 capsid.

[0075] The terms “ITR” or “inverted terminal repeat” refer to the stretch of nucleic acid sequences that exist in adeno-associated viruses (AAV) and / or recombinant adeno-associated viral vectors (rAAV) that can form a T-shaped palindromic structure, which is required for completing AAV lytic and latent life cycles. The term “non-resolvable ITR” refers to a modified ITR such that the resolution by the Rep protein is reduced. An ITR can be rendered non-resolvable when the terminal resolution site (trs) and / or D sequence in the ITR is deleted or mutated.

[0076] The term “binding” refers to an association between two substances or molecules, such as the hybridization of one nucleic acid molecule to another (or itself), such as between two dimerization domains. An oligonucleotide molecule binds or stably binds to another nucleic acid molecule if there are a sufficient number of complementary base pairs between the oligonucleotide molecule and the target nucleic acid to permit detection of that binding. In some examples, binding between nucleic acid molecules may occur directly. In some examples, binding between nucleic acid molecules may occur indirectly, e.g., through an intermediate molecule. Either direct binding or indirect binding may occur by standard base pairing, by non- canonical base pair interactions, by non-base pair interactions, or a combination thereof. Non- canonical base pair interactions may occur by any means of stabilization known to those of skill in the art, including but not limited to Hoogsteen base pairs and wobble base pairs. Nonbase pair interactions can include binding through an intermediate molecule. In some examples, direct binding is between kissing loop dimerization domains.

[0077] The term “C-terminal portion” refers to a region of a protein sequence that includes a contiguous stretch of amino acids that begins at or near the C-terminal residue of the protein. A C-terminal portion of the protein can be defined by a contiguous stretch of amino acids (e.g., a number of amino acid residues).

[0078] The term “N-terminal portion” refers to a region of a protein sequence that includes a contiguous stretch of amino acids that begins at the N-terminal residue of the protein. An N- terminal portion of the protein can be defined by a contiguous stretch of amino acids (e.g., a number of amino acid residues).

[0079] The term “kissing loop / kissing stem loop” refers to an RNA structure that forms when bases between two hairpin loops form pair interactions. These intermolecular “kissing interactions” occur when the unpaired nucleotides in one hairpin loop, base pair with the unpaired nucleotides in another hairpin loop to form a stable interaction complex.

[0080] Hybridization of a nucleic acid occurs when two nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acids used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993 ). The Tm is the temperature at which 50% of a given strand of nucleic acid is hybridized to its complementary strand.

[0081] The term “substantial identity” or “substantially identical,” as used in the context of polynucleotide or polypeptide sequences, refers to a sequence that has at least 60% sequence identity to a reference sequence. Alternatively, percent identity can be any integer from 60% to 100%. Exemplary embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

[0082] As used herein, the term “strand invasion” refers to the displacement of one strand of a first double stranded nucleic acid molecule by a single stranded portion of a second nucleic acid molecule, wherein the single strand has nucleotide sequence that is substantially identical to the displaced strand and can selectively hybridize to the strand complementary to the displaced strand.

[0083] As used herein, the terms “AAV vector,” “AAV particle,” “recombinant AAV particle,” and “rAAV” are used interchangeably and refer to the AAV capsid with a transgene or vector genome comprising ITRs as produced by a packaging cell.

[0084] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0085] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215, pp. 403-410 and Altschul et al. (1977). Nucleic Acids Res. 25, pp. 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1977). Nucleic Acids Res. 25, pp. 3389-3402). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=l, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff (1989). Proc. Natl. Acad. Sci. USA 89, p. 10915).

[0086] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993). Proc. Nat’l. Acad. Sci. USA 90, pp. 5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10-5, and most preferably less than about 10-20.

[0087] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps.

[0088] Aspects of the disclosure relate to target gene replacement systems / compositions that express a target protein from two or more vectors. The two or more vectors can be any viral or non-viral vectors including, but not limited to, plasmid vectors, adenoviral vectors, retroviral vectors and adeno-associated vectors (AAV). Each vector drives expression of a split pre- mRNA molecule comprising a portion of the coding sequence of the target gene (e.g., N- or C- terminal fragment of the full coding sequence). The two or more split pre-mRNA molecules (e.g., N-terminal split pre-mRNA and C-terminal split pre-mRNA) are spliced through RNA- end joining (REJ) domains comprising RNA dimerization domains and splicing domains (also referred to as a splicing donor or a splicing acceptor). In some embodiments, the systems / compositions comprise elements that are designed to inhibit fragment expression and function to block translation of truncated protein from either single vector. Descriptions of REJ technology can be found, for example, in international patent application publication Nos. WO 2020 / 205604 and WO 2021 / 096605, the contents of which are incorporated by reference in their entirety.

[0089] In some embodiments, provided herein are system that express a target protein from two or more AAV vectors, such as at least two, at least three, at least four, or at least five different AAV vectors (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 different AAV vectors). Each AAV vector drives expression of a split pre-mRNA molecule comprising a portion of the coding sequence of the target gene. Each split pre-mRNA molecule can be spliced sequentially through RNA-end joining (REJ) domains.

[0090] In some embodiments, provided herein are target gene replacement systems / compositions that express a target protein from two AAV vectors (also referred to as “dual AAV-REJ”). In some embodiments, provided herein is a ATP -binding cassette, subfamily A, member 4 (Abca4) gene replacement systems / compositions that express Abca4 protein from two AAV vectors (also referred to as “dual AAV-REJ-Abca4”). Each vector drives expression of a split pre-mRNA molecule comprising a portion of the Abca4 coding sequence (e.g., N- or C-terminal fragment of the full Abca4 coding sequence). The dual AAV vectors, in one embodiment, are dual AAV8 vectors.

[0091] The REJ technology involves in part: (1) splitting the full-length coding sequence of the target gene (e.g., a.Abca4 gene) into an N-terminal fragment and a C-terminal fragment and constructing two different transgenes, each of which comprises the N or C-terminal fragment of the coding sequence; (2) packaging each transgene individually into two independent AAV particles; (3) coinfecting two independent AAV particles into the same target cell; (4) forming split pre-mRNAs from transcription of the two different transgenes; (5) non-covalent binding between the two RNA dimerization domains; (6) recruiting spliceosome by the splicing domains; (7) spliceosome mediated RNA-end joining; (8) suppressing protein expression from un-joined RNA fragments; and (9) translating a full-length protein. See e.g., FIG.l for an overview of the process.

[0092] The present disclosure provides distinct and improved sequences for dimerization domains that promote strong / ra / rs-in teractions with a complementary RNA strand, while reducing the likelihood / minimizing cis-binding interactions. These uniquely structured dimerization domains are combined with novel and improved elements including, among others, a splicing donor and acceptor, to enhance RNA splicing processes. By improving the trans-dimerization of the RNA strands in the context of the appropriate elements that mediate efficient splicing, it is demonstrated herein that two different RNA fragments of a coding sequence can be joined precisely and efficiently in the same cell producing physiological or even supraphy si ologi cal levels of functional proteins in vivo with no or minimal risk of accumulating unjoined RNAs that encode non-functional and / or deleterious proteins, or truncated protein from either single AAV.Transgene and Vector Genome Architectures

[0093] In one aspect of the disclosure, provided herein are dual-AAV particle compositions comprising two different AAV particles, each comprising an encapsidated transgene. Thetransgene of each AAV particle comprises nucleic acid elements required for transcript expression and subsequent mRNA trans-splicing. The transgene encapsidated by an AAV particle can be referred to as a “vector genome,”. In the majority of instances discussed herein, nucleic acid elements are discussed as being part of a transgene. However, one of ordinary skill in the art will appreciate that vector genomes of the disclosure will also include such elements, once the transgene is encapsidated by an AAV capsid.

[0094] In some embodiments, the AAV particle containing the N-terminal coding sequence comprises an N-terminal transgene comprising one or more of the following sequences from 5’ to 3’: (i) a 5’ ITR; (ii) a promoter; (iii) a Kozak sequence; (iv) an N-terminal coding sequence of a target protein; (v) a dimerization domain; (vi) a splicing donor; (vii) a poly(A) signal; and (viii) a 3’ ITR. In one embodiment, the N-terminal transgene comprises one or more of the following sequences from 5’ to 3’ : (i) a 5’ ITR; (ii) a promoter; (iii) a Kozak sequence; (iv) an N-terminal Abca4 coding sequence; (v) a dimerization domain; (vi) a splicing donor; (vii) a poly(A) signal; and (viii) a 3’ ITR. See, e.g., FIG. 2A. In a further embodiment, the N-terminal transgene comprises one or more additional regulatory elements operably linked to the coding sequence.

[0095] In some embodiments, the AAV particle containing the C-terminal coding sequence comprises a C-terminal transgene comprising one or more of the following sequences from 5’ to 3’: (i) a 5’ ITR; (ii) a promoter; (iii) a dimerization domain; (iv) a splicing acceptor; (v) a C- terminal coding sequence of a target protein; (vi) a poly(A) signal; and (vii) a 3’ ITR. In one embodiment, the C-terminal transgene comprises one or more of the following sequences from 5’ to 3’: (i) a 5’ ITR; (ii) a promoter; (iii) a dimerization domain; (iv) a splicing acceptor; (v) a C-terminal Abca4 coding sequence; (vi) a poly(A) signal; and (vii) a 3’ ITR. See, e.g., FIG. 2B. In a further embodiment, a vector genome construct comprises one or more additional regulatory elements operably linked to the dimerization domain. In even a further embodiment, the C-terminal transgene of an AAV vector comprises one or more of the following sequences from 5’ to 3’, (i) a 5’ ITR; (ii) a promoter; (iii) a dimerization domain; (iv) a splicing acceptor; (v) a C-terminal Abca4 coding sequence; (vi) a poly(A) signal; and (vii) a 3’ ITR.

[0096] Where one or more additional regulatory elements is employed, in one embodiment, the one or more additional regulatory elements is an expression control element such as an enhancer. In one embodiment, the one or more regulatory elements is upstream of a coding sequence or a REJ element (e.g., a dimerization domain), and operably linked thereto. In a further embodiment, the element is an enhancer, and the enhancer is upstream of the promoterand downstream of the 5 ’ ITR. The enhancer and promoter are operably linked to the coding sequence or REJ element.N-terminal or C-terminal coding sequences

[0097] Coding sequences for use with the present disclosure are nucleic acid sequences encoding a target protein, or fragment thereof, and are delivered via the vectors (e.g., AAV particles) described herein. One application of this system is expression of large diseasecausing genes using viral vectors with restricted packaging capacity. Disease and genes include but are not limited to, Stargardt disease 1 (Abca4 and Leber congenital amaurosis (CEP290 and RPE65).

[0098] In some embodiments, the coding sequences of the present disclosure are nucleic acid sequences encoding ATP -binding cassette, sub-family A, member 4 (Abca4) protein, or fragment thereof.

[0099] In some embodiments, the coding sequence of a target protein is divided into two portions, such as about two equal halves (or other proportions, such as portion A expressing about 1 / 3 and portion B expressing about 2 / 3, or portion A expressing about 1 / 4 and portion B expressing about 3 / 4, etc.). However, it is not required that each portion be the same number of nucleotides (or encode the same number of amino acids). In one example, the target gene is an Abca4 gene. In one embodiment, the coding sequence of the Abca4 protein is split into an N-terminal coding sequence encoding an N-terminal portion of the Abca4 protein, and a C- terminal coding sequence encoding a C-terminal portion of the Abca4 protein.

[0100] In some embodiments, the N-terminal and / or C-terminal coding sequence of a target protein is a wild type coding sequence. For example, the coding sequence is one that is found in the cell or organism into which the disclosed system is introduced (e.g., a human coding sequence when introduced into a human cell or subject). In some embodiments, the N-terminal and / or C-terminal coding sequence of a target protein is codon optimized relative to a wild type coding sequence, for example to maximize tRNA availability, or to de-enrich for cryptic splice sites (e.g., to reduce or avoid incorrect splicing and promote the correct junction formation). The coding sequence is the portion of the mRNA sequence that encodes the amino acids for translation. During translation, each of 61 trinucleotide codons are translated to one of 20 amino acids, leading to a degeneracy, or redundancy, in the genetic code. However, different cell types, and different animal species, utilize tRNAs (each bearing an anticodon) coding for the same amino acids at different frequencies. When a gene sequence contains codons that areinfrequently represented by the corresponding tRNA, the ribosome translation machinery may slow, impeding efficient translation. Expression can be improved via codon optimization for a particular species, where the coding sequence is altered to encode the same protein sequence, but utilizing codons that are highly represented, and / or utilized by highly expressed human proteins (Cid-Arregui et al., 2003, J. Virol. 77: 4928). In some embodiments, the coding sequence of a target protein (e.g., an Abca4 protein) is modified to replace codons infrequently expressed in mammal or in primates with codons frequently expressed in primates. In some embodiments, the coding sequence of a target protein (e.g., an Abca4 protein) is a codon- optimized version of human gene to increase joined mRNA stability upon transcription. For the optimization, GeneArt® software may be used, increasing the GC content and removing cryptic splice sites in order to avoid transcriptional silencing and therefore increase transgene expression. Alternatively, any optimization method known in the art may be used.

[0101] In some embodiments, the N-terminal transgene comprises an N-terminal coding sequence that is a wild type coding sequence. In some embodiments, the N-terminal transgene comprises an N-terminal coding sequence that is codon optimized. In some embodiments, the N-terminal coding sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 7. In a further embodiment, the N-terminal coding sequence comprises a nucleic acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 7.

[0102] In some embodiments, the C-terminal transgene comprises a C-terminal coding sequence that is a wild type coding sequence. In some embodiments, the C-terminal transgene comprises a C-terminal coding sequence that is codon-optimized. In some embodiments, the C-terminal coding sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 20. In a further embodiment, the C-terminal coding sequence comprises a nucleic acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 20.

[0103] In some embodiments, the N-terminal coding sequence and the C-terminal coding sequence can be spliced and joined to form a full-length coding sequence that encodes a full- length target protein. In one embodiment, the N-terminal coding sequence and the C-terminalcoding sequence can be spliced and joined to form a full-length coding sequence that encodes a full-length Abca4 protein, wherein the full-length coding sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 22. In some embodiments, the full-length coding sequence comprises a nucleic acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 22. In some embodiment, the full-length coding sequence is a wild type coding sequence. In some embodiment, the full-length coding sequence is codon-optimized. In one embodiment, the codon-optimized full-length coding sequence shows about 65% to about 95%, about 70% to about 90%, about 75% to 85% identity to the wild type coding sequence, with no changes in the amino acids sequence of the target protein. For example, the codon-optimized full-length Abca4 coding sequence of the present disclosure shows about 74% identity to the wild type human Abca4 mRNA transcript variant 1 (i.e., NM_000350.3) and about 71% identity to the wild type human Abca4 mRNA transcript variant 2 (i.e., NM_001425324.1), with no changes in the amino acids sequence of the Abca4 protein.

[0104] In some embodiments, the N-terminal coding sequence and / or the C-terminal coding sequence comprises an intervening intron sequence within the coding sequence. The intervening intron sequence is either natural or synthetic in nature. Inclusion of such introns can be used to stimulate splicing machinery attachment to the trans-splicing intron donor and acceptor. In some embodiments, the N-terminal coding sequence comprises an intervening intron sequence situated close to the 3’ end of the coding sequence. In one embodiment, the intervening intron sequence is situated about 95 nt, about 100 nt, about 105 nt, about 110 nt, or about 115 nt upstream of the 3 ’ end of the N-terminal coding sequence. In another embodiment, the intervening intron sequence is situated about 105 nt upstream of the 3 ’ end of the N-terminal coding sequence. In one embodiment, the intervening intron sequence within the N-terminal coding sequence is a modified mouse beta-actin (Actb) intron 2 sequence. In one embodiment, the intervening intron sequence within the N-terminal coding sequence comprises a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 5 In some embodiments, the C-terminal coding sequence comprises an intervening intron sequence situated close to the 5’ end of the coding sequence. In one embodiment, the intervening intron sequence is situated about 140 nt, about 145 nt, about 150nt, about 155 nt, or about 160 nt downstream of the 5’ end of the C-terminal coding sequence. In another embodiment, the intervening intron sequence is situated about 153 nt downstream of the 5’ end of the C-terminal coding sequence. In one embodiment, the intervening intron sequence within the C-terminal coding sequence is a modified mouse beta-actin (Actb) intron 2 sequence. In one embodiment, the intervening intron sequence within the C-terminal coding sequence comprises a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 5.Splicing Domain

[0105] RNA splicing depends on the recruitment of spliceosome components to the splicing domain of each split pre-mRNA. In some embodiments, the N-terminal split pre-mRNA comprises a splicing donor at the 3’ end of the N-terminal coding sequence. See e.g., FIG. 2A, a synthetic half intron. In some embodiments, the C-terminal split pre-mRNA comprises a splicing acceptor at the 5’ end of the C-terminal coding sequence. See e.g., FIG. 2B, a synthetic half intron. Different ribonucleoproteins are recruited to the synthetic half intron through base pairing of protein associated small nuclear RNA (snRNA) with intronic sequences.

[0106] In some embodiments, the N-terminal transgene comprises a splicing donor that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 8. In a further embodiment, the N-terminal transgene comprises a splicing donor that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 8. The C-terminal transgene comprises a splicing acceptor that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 17. In a further embodiment, the C-terminal transgene comprises a splicing donor that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 17.Dimerization Domain

[0107] The association of the two split pre-mRNA molecules (the N-terminal fragment and the C-terminal fragment) is mediated by the RNA dimerization domain, allowing the spliceosome components to recombine N-terminal coding sequence and C-terminal coding sequence. Asshown in FIG. 1, interaction and hybridization (base pairing) between the dimerization domain of the N-terminal split pre-mRNA and the dimerization domain of the C-terminal split pre- mRNA allows the spliceosome components to recombine N-terminal coding sequence and C- terminal coding sequence, which results in the joining of the N-terminal and the C-terminal coding sequences, allowing for expression of the full-length protein.

[0108] In some embodiments, the first and second dimerization domains contain complementary RNA stem loops (also called hairpin loops) that can form strong kissing loop interactions with their counter parts. Each stem loop comprises at least two complementary sequences (e.g., form a stem) separated by a region of non-complementary sequence (e.g., form a loop). Complementary sequences between two stem loops result in base pairing, and generation of a kissing loop / kissing stem loop interaction. In some embodiments, the complementary sequences between the two stem loops comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more loop base pairs for intermolecular pairing. In some embodiments, the stems of the kissing loops are chosen to base pair in trans between the two dimerization domains. In such an example, after forming a kissing loop interaction of one stem loop on one dimerization domain with another stem loop on a second dimerization domain, the respective stem (or helix) regions of the initial hairpin loops can base pair in trans between the two dimerization domains through strand replacement / invasion and result in extended duplex formation. In one embodiment, extended duplex formation is favored by inclusion of mismatches in the initial stems that favor pairing in the extended duplex. In some embodiments, the dimerization domains contain helices destabilized by the inclusion of as about 1% to about 30%, about 5% to about 30%, about 10% to about 30%, or about 25% to about 30% mismatches, but match to the other dimerization domain, to favor extended duplex formation after initial kissing / pairing. In some embodiments, these stem loops contain at least 10 nt, such as at least 20 nt, at least 25 nt, at least 50 nt, at least 75 nt, or at least 100 nt in length, such as 10 to 50, 20 to 25, 10 to 100, 10 to 20, or 20 to 40 nt in length. In some embodiments, each dimerization domain can contain at least 1 individual stem loop, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20, such as 1 to 20, 2 to 5 or 1 to 10 individual stem loops. In one embodiment, each dimerization domain can contain a central stem loop.

[0109] In one embodiment, the first and second dimerization domains are designed to contain sequences that are complementary to each other (e.g., the first dimerization domain comprising a nucleic acid sequence of SEQ ID NO:9 and the second dimerization domain comprising anucleic acid sequence of SEQ ID NO: 16). The first and second dimerization domains contain complementary RNA stem loops that can form central kissing loop interaction with four or more loop base pairs for intermolecular pairing. See, e.g., FIG. 3. In one embodiment, the positioning of the stem loops is offset in such a way that the respective stem regions of the initial stem loops can base pair in trans through strand invasion, which leads to an extended area of interaction (a wider synapse) between the first and second dimerization domains. In one embodiment, the positioning of the kissing loops is offset by at least 1 nt, at least 2nt, at least 3 nt, at least 4nt, at least 5nt, at least 6 nt, at least 7nt, at least 8 nt, at least 9nt, or at least lOnt. In one embodiment, extended strand invasion is followed for the full dimerization of the two dimerization domains. The extended area of interaction facilitates more robust molecule interactions. In addition, mismatches are introduced in the two stem loops to disfavor the intramolecular base pairing and favor intermolecular pairing.

[0110] In some embodiments, the N-terminal transgene comprises a dimerization domain comprising a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 9. In a further embodiment, the N-terminal transgene comprises a dimerization domain comprising a nucleic acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 9. The C-terminal transgene comprises a dimerization domain, in one embodiment, comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 16. In a further embodiment, the C-terminal transgene comprises a dimerization domain comprising a nucleic acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 16. In one embodiment, the N-terminal transgene comprises a dimerization domain comprising the nucleic acid sequence of SEQ ID NO: 9 and the C-terminal transgene comprises a dimerization domain comprising the nucleic acid sequence of SEQ ID NO: 16.Inverted Terminal Repeats (ITRs)[OHl] ITRs are the genetic elements responsible for the replication and packaging of the transgene into an AAV capsid during AAV particle production and are the only viral cis elements required to generate a recombinant AAV particle. The minimal sequences required to package the transgene into an AAV viral particle are the AAV 5’ and 3’ ITRs, which flank theother elements of the transgene, and may be of the same AAV origin as the capsid proteins, or of a different AAV origin. The 5’ and 3’ ITRs of an AAV vector genome are necessary for both the integration of the coding sequence into the host cell genome (e.g., chromosome 19 in humans) and for encapsidation of the transgene into the AAV particle.

[0112] ITRs described herein may be used to facilitate the packaging of either a self- complementary or single stranded vector genome. In some embodiments, one of the ITRs is a non-resolvable ITR that is used to provide a self-complementary vector genome in a host cell. The non-resolvable ITR may be produced by any method known in the art. For example, insertion into the ITR will displace the nicking site and result in a non-resolvable ITR. The designation of the various regions or elements within the ITR are known in the art. In one embodiment, the insertion is made into the sequence of the terminal resolution site (trs). Alternatively, the insertion may be made at a site between the Rep Binding Element (RBE) within the A element and the trs, which is adjacent to the D sequence. In another embodiment, the ITR may be rendered non-resolvable by deletion of the trs site. In addition to the trs, some or all of the D sequence may be deleted. In some embodiments, the 5’ ITR comprises an AAV trs. In some embodiments, the 5’ ITR does not comprise an AAV trs.

[0113] In some embodiments, transgenes of the disclosure comprise ITR sequences from any one AAV serotype, e.g., AAVrh.8, AAVrh.10, AAVrh.39, AAVrh.74, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13. The ITR sequence, as provided above, can facilitate the formation of either a single stranded or self- complementary vector genome.

[0114] In some embodiments, the transgene disclosed herein comprise a 5’ AAV2 ITR and a 3’ AAV2 ITR sequence.

[0115] In one embodiment, the N-terminal transgene comprises a 5’ ITR that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 1. In some embodiments, the 5’ AAV ITR comprises the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the 5’ AAV ITR consists of the nucleic acid sequence of SEQ ID NO: 1. In a further embodiment, the N-terminal transgene comprises a 3’ AAV ITR that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99%, or 100% sequence identical to the nucleic acid sequence of SEQ ID NO: 11. In a further embodiment, the 3’ AAV ITR comprises SEQ ID NO: 11. In a further embodiment, the 3’ AAV ITR consists of the nucleic acid sequence of SEQ ID NO: 11

[0116] In one embodiment, the C-terminal transgene comprises a 5’ ITR that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 1. In some embodiments, the 5’ AAV ITR comprises the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the 5’ AAV ITR consists of the nucleic acid sequence of SEQ ID NO: 1. In a further embodiment, the C-terminal transgene comprises a 3’ AAV ITR that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to the nucleic acid sequence of SEQ ID NO: 11. In a further embodiment, the 3’ AAV ITR comprises SEQ ID NO: 11. In a further embodiment, the 3’ AAV ITR consists of the nucleic acid sequence of SEQ ID NO: 11Promoter

[0117] In some N-terminal transgene embodiments, a promoter is operably linked to the N- terminal coding sequence. In embodiments described herein, the promoter is located upstream (5’) of the N-terminal coding sequence and downstream (3’) of the 5’ ITR.

[0118] In some embodiments C-terminal transgene embodiments, a promoter is operably linked to the REJ dimerization domain. In embodiments described herein, the promoter is located upstream (5’) of the REJ dimerization domain and downstream (3’) of the 5’ ITR.

[0119] In one embodiment, the N-terminal transgene comprises the same promoter as the C- terminal transgene. In another embodiment, the N-terminal transgene comprises a promoter that is different than the promoter present in the C-terminal transgene.

[0120] In one embodiment, the promoter is an ocular tissue-specific promoter. In some embodiments, the ocular tissue-specific promoter is a photoreceptor-specific promoter.

[0121] In some embodiments, transgenes and AAV vector genomes of the present disclosure comprise a mammalian promoter, for example, human, non-human primate (e.g., cynomolgus macaque), mouse, horse, cow, pig, cat, and dog promoters. In some embodiments, vectorgenomes disclosed herein comprise strong, constitutively active promoters to drive high-level expression of the coding sequence. For example, in some embodiments, the promoter is a cytomegalovirus (CMV) promoter / enhancer, an elongation factor la (EFla) promoter, a simian virus 40 (SV40) promoter, a chicken P-actin hybrid promoter, or a CAG promoter. In a further embodiment, the promoter has been engineered to be depleted of CpG (5’-C-phosphate- G-3’) dinucleotides. In even a further embodiment, the promoter has been engineered to be free of CpG (5’-C-phosphate-G-3’) dinucleotides.

[0122] In some embodiments, a promoter provided herein is an ocular tissue-specific promoter. For example, in some embodiments, the ocular tissue-specific promoter is a CHX10 promoter, a human RHO promoter, a human interphotoreceptor retinoid binding protein (hIRBP) promoter, a CAR / ARR3 promoter, a Synpl61 promoter, a Chxl0-SV40 promoter, a ProBl promoter, a Synl promoter, a short human GRM6-based promoter (e.g., short 200-bp Grm6 enhancer in combination with a basal viral simian virus 40 (SV40) promoter (200En-SV40), 770En_454P(hG 6) promoter, a 407En_566P(hG 6) promoter, a 770En_454P(hGRM6) promoter), or a shortened RPE65 promoter.

[0123] In one embodiment, the promoter is a human rhodopsin kinase (hGRKl) promoter. The hGRKl promoter is a photoreceptor-specific promoter. In one embodiment, the hGRKl promoter comprises the nucleic acid sequence of SEQ ID NO: 2. In one embodiment, the N- terminal transgene described herein comprises a promoter comprising a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 2. In a further embodiment, the N-terminal transgene comprises a promoter comprising a nucleic acid sequence that is at least about 90%, at least about 95%, at least about 99%, or 100% identical to SEQ ID NO: 2 In one embodiment, the C-terminal transgene described herein comprises a promoter comprising a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 2. In a further embodiment, the C-terminal transgene comprises a promoter comprising a nucleic acid sequence that is at least about 90%, at least about 95%, at least about 99%, or 100% identical to SEQ ID NO: 2.Poly(A) Signal

[0124] As discussed throughout, the transgenes and AAV vector genomes of the present disclosure comprise a poly(A) signal nucleic acid sequence.

[0125] In one embodiment, a suitable poly(A) signal sequence is derived from bovine growth hormone (bGH), human growth hormone (hGH), simian virus 40 (SV40), P-globin, rabbit P- globin (RGB), modified RGB (mRGB) or thymidine kinase (TK). The poly(A) signal sequence in one embodiment is a SV40-poly(A) signal sequence. The poly(A) signal sequence, in another embodiment, is a bovine growth hormone (bGH)-poly(A) signal sequence. In even another embodiment, the poly(A) signal sequence is a synthetic poly(A) signal sequence or from bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit P-globin (RGB), or modified RGB (mRGB).

[0126] In some embodiments, the N-terminal transgene comprise a poly(A) signal sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 10. In some embodiments, the poly(A) signal sequence comprises the nucleic sequence of SEQ ID NO: 10. In some embodiments, the poly(A) signal sequence consists of the nucleic sequence of SEQ ID NO: 10.

[0127] In some embodiments, the C-terminal transgene comprise a late SV40 poly(A) signal sequence. In some embodiments, the C-terminal AAV vector comprise a poly(A) signal sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO:21 In some embodiments, the poly(A) signal sequence comprises the nucleic sequence of SEQ ID NO:21. In some embodiments, the poly(A) signal sequence consists of the nucleic sequence of SEQ ID NO:21.Enhancer

[0128] In one embodiment, a transgene provided herein comprises one or more enhancer elements. In one embodiment, the one or more enhancers are operably linked to the coding sequence in the N-terminal transgene. In one embodiment, the one or more enhancers are operably linked to the dimerization domain sequence in the C-terminal transgene. An enhancer sequence, in one embodiment, can increase the level of transcription of the coding sequence,for example, by serving as a binding site for transcription factors and co-regulators that assist in DNA looping and recruitment of the transcriptional machinery to promoters.

[0129] In one embodiment of an N-terminal transgene, the enhancer is downstream (i.e., 3’) of the 5’ ITR and upstream (i.e., 5’) of the promoter. In some embodiments, the enhancer is downstream (i.e., 3’) of the promoter and upstream (i.e., 5’) of the coding sequence. In some embodiments, the enhancer is downstream (i.e., 3’) of the coding sequence and upstream (i.e., 5’) of the 3’ ITR. In one embodiment of a C-terminal transgene, the enhancer is downstream (i.e., 3’) of the 5’ ITR and upstream (i.e., 5’) of the promoter. In some embodiments, the enhancer is downstream (i.e., 3’) of the promoter and upstream (i.e., 5’) of the dimerization domain. In some embodiments, the enhancer is downstream (i.e., 3’) of the coding sequence and upstream (i.e., 5’) of the 3’ ITR.

[0130] In some embodiments, a transgene of the present disclosure comprises an enhancer that significantly promotes the transcription of a coding sequence in ocular cells.

[0131] In one embodiment, the enhancer used in a vector genome of the disclosure is the En34 enhancer (34 bp core enhancer from the human apolipoprotein hepatic control region).

[0132] In another embodiment, the EnTTR (100 bp enhancer sequence from transthyretin) is included in a vector genome of the disclosure. See, Wu et al, Molecular Therapy, 16(2):280- 289, February 2008, which is incorporated herein by reference.

[0133] In yet another embodiment, the al-microglogulin / bikunin precursor enhancer is included in a vector genome of the disclosure. In yet another embodiment, the ABPS (shortened version of the 100 bp distal enhancer from the al-microglogulin / bikunin precursor [ABP] to 42 bp) enhancer is included. In a further embodiment, the enhancer is upstream of a promoter element.

[0134] In yet another embodiment, the ApoE enhancer is included in a vector genome of the disclosure. In a further embodiment, the enhancer is upstream of a promoter element.

[0135] In another embodiment, two or more enhancers are present in a transgene of the disclosure. Such combination may include more than one copy of any of the enhancers described herein, and / or more than one type of enhancer.Other Regulatory Sequences

[0136] In addition to a promoter and optionally an enhancer, a transgene / vector genome / DNA plasmid of the disclosure may contain other appropriate transcription initiation,termination and / or RNA processing signals. One embodiment of such a signal is a Kozak sequence to increase translation efficiency. In a further embodiment, a Kozak sequence is present in a transgene / vector genome / DNA plasmid, provided herein. The Kozak sequence directs the pre-initiation complex (PIC) and ribosome to the translation initiation site (start codon) and mediates ribosome assembly ensuring the correct protein sequence is translated. In one embodiment, the N-terminal AA vector comprises a Kozak sequence immediately upstream of the ATG start codon in the coding sequence. Embodiments of a Kozak sequence for use herein include, GCCGCCACC (SEQ ID NO:3), GCCACC (SEQ ID NO:23), CTGCCACC (SEQ ID NO:24), GCTGCCACC (SEQ ID NO:25).AAV Vector Backbones

[0137] In another aspect of the disclosure, a DNA plasmid is provided comprising a transgene nucleic acid of the disclosure.

[0138] In some embodiments, a DNA plasmid used to generate the N-terminal transgene components comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to a sequence provided in Table 1.

[0139] In some embodiments, a DNA plasmid used to generate the C-terminal transgene components comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to a sequence provided in Table 2.

[0140] In some embodiments, a transgene of the disclosure, and ultimately, an AAV vector genome of the present disclosure can be assembled by inserting the transgene nucleic acid sequence, or a portion thereof, as described herein, into an appropriate adenovirus plasmid backbone using standard molecular biology techniques (see, for example, Sambrook et al. (1989). “Molecular Cloning: A Laboratory Manual, 2nd Ed.”; Ausubel et al. (1987). “Current Protocols in Molecular Biology”). The adenovirus plasmid backbone, in one embodiment, comprises the 5’ ITR and 3’ ITR sequences described herein. As such, in one embodiment, the transgene components other than the ITR sequences can be inserted into the adenovirus plasmid backbone between the ITR sequences, i.e., downstream of the 5’ ITR sequence and upstream of the 3’ ITR sequence. In another embodiment, a DNA plasmid comprising one of thetransgenes provided herein can be assembled by inserting the ITR sequences into the DNA plasmid backbone, along with the remaining nucleic acid sequences that make up the transgene.AAV Particles

[0141] One aspect of the present disclosure relates to a dual AAV particle composition comprising a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises: i) a first 5 ’-inverted terminal repeat (5 ’-ITR) sequence; ii) a first promoter sequence; iii) a Kozak sequence; iv) an N-terminal coding sequence encoding an N-terminal portion of a target protein, said N-terminal coding sequence being operably linked to and under control of said first promoter; v) a nucleic acid sequence of a splicing donor; vi) a nucleic acid sequence of a first dimerization domain; vii) a first poly(A) signal sequence; and viii) a first 3 ’-ITR sequence; and b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises: i) a second 5 ’-ITR sequence; ii) a second promoter sequence; iii) a nucleic acid sequence of a second dimerization domain, said second dimerization domain being operably linked to and under control of said second promoter; iv) a nucleic acid sequence of a splicing acceptor; v) a C-terminal coding sequence encoding a C-terminal portion of the target protein; vi) a second poly(A) signal sequence; and vii) a second 3 ’-ITR sequence; wherein the N-terminal coding sequence and the C-terminal coding sequence can be spliced and joined to form a recombined nucleic acid that encodes a full-length target protein. In some embodiments, the target protein is an Abca4 protein.

[0142] In one embodiment, the AAV particle is an AAV2 particle comprising an AAV2 capsid. In a preferred embodiment, the AAV particle is an AAV8 particle comprising an AAV8 capsid. In another preferred embodiment, the AAV particle is a deimmunized AAV8 particle.

[0143] Transgenes and vector genomes provided herein may be single stranded or self- complementary. In one embodiment, the transgene or vector genome is single stranded. It should be noted that reference to “single stranded” or “self-complementary” is not intended to limit the structure of the transgene or vector genome when encapsidated by an AAV capsid. Without wishing to be bound by theory, it is believed that DNA packaging in AAV likely precludes a double-stranded structure of the transgene / vector genome while encapsidated. Rather, “single stranded” or “self-complementary” is intended to refer to the transgene or vector genome structure once present within a target cell, subsequent to viral uncoating.

[0144] The transgene and vector genome constructs provided herein are described as including a coding sequence (i.e., the “plus” or “sense” sequence). However, one of ordinary skill will appreciate that the transgenes and vector genomes of the disclosure are described in their broadest sense to indicate the strand of DNA corresponding to the mRNA transcript which is translatable into a polypeptide sequence. As such, it is understood that the transgenes and vector genomes describe herein also encompass non-translated sequences, including the complementary sequence to the transgene, i.e., the “minus” or “antisense” nucleic acid sequences.

[0145] Single stranded (ss) vector genomes provided herein can encompass both the coding (plus or sense) nucleic acid sequence and the minus (antisense) nucleic acid sequence of the transgene. For the ss vector genomes provided herein, without wishing to be bound by theory, packaging of the vector genome into assembled capsids occurs from the 3’ end of both the plus (coding) and minus (antisense) strand of the transgene. Without wishing to be bound by theory, it is thought that the resulting AAV particle population comprises about an equal mix of capsids containing the plus strand of the transgene (coding strand) and the minus strand (antisense) of the transgene. When encapsidated into particles, such transgenes are referred to as vector genomes. Each AAV particle can then deliver a single stranded version of the transgene to the target cell, which needs to become double-stranded before it can express the desired therapeutic protein. This can occur either by annealing of the plus strand of a transgene to a minus strand delivered to the same cell or from native nuclear mechanisms initiating second-strand synthesis from a single-stranded transgene.

[0146] The assembly of self-complementary AAV vector genomes is known in the art, and is described, for example, in U.S. Patent No. 8,361,457, the contents of which are incorporated by reference in their entirety. One of ordinary skill in the art will understand, for example, that by employing a non-resolvable ITR in a transgene, together with a resolvable ITR, that the transgene will ultimately comprise resolvable ITRs at both ends, with a centrally-located non- resolvable ITR. Additionally, one of skill in the art will appreciate that each half of the transgene on either side of the non-resolvable ITR is approximately the same length and substantially complementary to the other half. One portion will include a coding sequence, a REJ domain, a poly(A) signal and one or more regulatory elements, and the other portion will be complementary thereto.

[0147] As used herein, an “adeno-associated virus (AAV) particle”, refers to an AAV virion comprising an AAV capsid encapsidating a transgene or an AAV capsid encapsidating a vectorgenome. The vector genome comprises a transgene comprising a promoter, a coding sequence, and a REJ domain that are flanked by a 5’ and 3’ AAV ITR sequences. An AAV capsid is typically composed of 60 capsid (cap) protein subunits, VP1, VP2, and VP3, that are arranged in an icosahedral symmetry in a ratio of approximately 1 :1 : 10 to 1 : 1 :20, depending upon the selected AAV capsid proteins. For example, in embodiments described herein, the AAV capsid shell is serotype 8 (AAV8), T=1 icosahedral capsid consisting of 60 copies of three AAV viral proteins, VP1, VP2, and VP3, at a ratio of 1 : 1 : 10. In a further embodiment, the capsid encapsidates a single-stranded vector genome.

[0148] Various AAVs and combinations thereof may be selected as sources for capsids and capsid proteins. See, e.g., U.S. Patent Application Publication No. 2007 / 0036760; U.S. Patent Application Publication No. 2009 / 0197338; EP 1,310,571; WO 2003 / 042397 (AAV7 and other simian AAV), U.S. Patent Nos. 7,790,449 and 7,282,199 (AAV8); WO 2005 / 033321 and US 7,906,111 (AAV9); and W02006 / 110689, and WO 2003 / 042397 (rh.10), each of which is incorporated by reference in its entirety for all purposes. Unless otherwise specified, the AAV capsid, ITRs, and other selected AAV components described herein, may be readily selected from among any AAV, including, without limitation, the AAVs commonly identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8 and AAVAnc80, engineered variants of any of the known or mentioned AAVs or AAVs yet to be discovered or variants or mixtures thereof. In one embodiment, the AAV capsid is an AAV8 capsid or variant thereof, an AAV9 capsid or variant thereof, an AAVrh.10 capsid or variant thereof, an AAVrh64Rl capsid or variant thereof, an AAVhu.37 capsid or variant thereof, or an AAV3B or variant thereof. In one embodiment, the capsid is an AAV9 capsid. In another embodiment, the capsid is an AAV8 capsid. In even another embodiment, the capsid is an engineered variant of an AAV8 capsid or an engineered variant of an AAV9 capsid.

[0149] “Engineered variant” as used herein, means a protein that differs in primary amino acid sequence from a reference (e.g., wild type) protein by one or more amino acids, wherein the one or more amino acid differences (e.g., one or more amino acid substitutions) impart an improved characteristic on the engineered protein, compared to the reference protein. The improved characteristic in some embodiments, is improved stability, production yield, improved tissue tropism, or improved immunogenicity profile (i.e., a deimmunized variant).

[0150] The AAV capsid proteins can be from the same or different AAV serotypes and can be wild-type or engineered. Vector genomes described herein can be replicated, and packaged into AAV capsids when introduced into a host cell also comprising one or more plasmids encodingthe respective rep and cap gene products. In one embodiment, a helper plasmid is also transfected into the host cell to aid in AAV particle production by the host cell. In one embodiment, the AAV capsid is an AAV8 capsid.

[0151] The terms “empty capsid,” “empty vial particle,” and “empty AAV” refer to an AAV capsid shell lacking a vector genome packaged within.AAV Particle Production

[0152] AAV particles can be produced by any standard method (see, for example, WO 2001 / 083692; Masic et al. 2014. Molecular Therapy, 22(11): 1900-1909; Carter, 1992, Current Opinions in Biotechnology, 1533-539; Muzyczka, 1992, Curr. Topics in Microbial, and Immunol., 158:97-129); Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81 :6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al, J. Virol, 62: 1963 (1988); and Lebkowski et al, Mol. Cell. Biol, 7:349 (1988). Samulski et al , J. Virol., 63:3822-3828 (1989); U.S. Patent No. 5,173,414; WO 95 / 13365; U.S. Patent No. 5,658.776; WO95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US 96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US 96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. Vaccine 13: 1244-1250 (1995); Paul et al. Human Gene Therapy 4:609-615 (1993); Clark et al. Gene Therapy 3: 1124- 1132 (1996); U.S. Patent. No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent. No. 6,258,595, herein incorporated by reference in their entireties).

[0153] In some embodiments, DNA plasmids comprising the transgenes described herein can be transformed into Escherichia coli to scale-up DNA production, purified using any standard method (for example, a Maxi-Prep K, Thermo Scientific), and verified by restriction digest or sequencing. Purified transgene plasmids can then be transfected using a standard method (e.g., calcium phosphate transfection, polyethyleneimine, electroporation, and the like) into an appropriate packaging cell line (e.g., HEK293, HeLa, or PerC.6, MRC-5, WI-38, Vera, and FRhL-2 cells) in combination with a plasmid comprising AAV rep and AAV cap genes, and an AAV helper plasmid.

[0154] As such, in one aspect of the disclosure, a packaging cell is provided comprising a DNA plasmid comprising one of the transgenes provided herein. In a further embodiment, the packaging cell comprises AAV replication (rep) and capsid (cap) genes, and a nucleic acid encoding helper virus protein sequences. The packaging cell, in a further embodiment, is a HEK293 packaging cell.

[0155] The AAV rep and cap genes may be from any AAV serotype and may be the same or different from that of the recombinant AAV vector ITRs including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.8, AAVrh.10, AAVrh.39, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13.

[0156] AAV particles can be produced, in one embodiment, by one of the methods described in U.S. Patent Application Publication No. 2021 / 0317474, the contents of which are incorporated by reference in their entirety. In various methods of recombinant AAV particles disclosed herein, AAV particles may be produced in packaging cell lines used to produce viral vectors, such as, immortalized human embryonic kidney 293 (HEK293) cell line and Sf9 insect cell lines.

[0157] In one embodiment, the present disclosure provides a method for manufacturing an AAV particle described herein, comprising, (i) culturing adherent cells, (ii) transfecting the adherent cells with plasmids for less than 60 minutes to enable production of the AAV particle, and optionally applying further processing, e.g., purification, formulation and filling steps to produce a finished product.

[0158] Production of AAV particles typically requires the presence of three elements in the packaging cell: (1) a vector genome comprising 5’ and 3’ AAV inverted terminal repeat (ITR) sequences, (2) AAV replication (rep) and capsid (cap) genes, and (3) nucleic acid encoding helper virus protein sequences. In one embodiment, a host cell, e.g., a HEK293 cell, is transfected with three plasmids encoding the aforementioned three components to produce one of the AAV particles described herein.

[0159] As such, in one aspect of the disclosure, a packaging cell is provided comprising a plasmid comprising one of the vector genomes described herein. The packaging cell, in one embodiment, comprises a vector genome comprising a nucleic acid sequence in Table 1 or Table 2

[0160] In one embodiment, the packaging cell comprises a plasmid comprising AAV replication (rep) and capsid (cap) gene sequences. In even a further embodiment, the packaging cell comprises a plasmid encoding helper AAV protein sequences. The packaging cell in one embodiment, is a HEK293 cell.

[0161] The plasmid comprising the vector genome is a plasmid that encodes the information for a recombinant AAV DNA vector genome that transcribe a fragment of the Abca4 coding sequence under the control of the chosen promoter and in some embodiments, an enhancerelement. The plasmid contains the vector genome components described herein. In embodiments described herein, the 5’ ITR is modified to preferentially package self- complementary AAV genomes into AAV capsids. Together, the regions between and including the ITRs are packaged into recombinant AAV capsids during the manufacture of the AAV particle. Plasmid components that are not intended for packaging into encapsidated vector genomes, in one embodiment, include an open reading frame encoding resistance to kanamycin (KanR) and an origin of replication (ori).

[0162] The second plasmid includes the rep and cap open reading frames. Rep and cap encode viral replication and capsid proteins, respectively. In the production of recombinant adeno- associated viral particles; the viral ITRs are the only elements used in cis while the viral rep and cap open reading frames are supplied in trans. Using the transfection of packaging cells to make AAV particles addresses the cis / trans roles for the different genetic elements by dividing them to separate plasmids.

[0163] The helper plasmid (also referred to as “pHELP,” component (3), above) contains the Trans acting adenoviral components necessary for recombinant adeno-associated virus production. The pHELP plasmid contains the regions of the adenovirus genome that provide factors that are important for AAV replication, namely E2A, E4, and VA RNA. The adenovirus El functions involved in AVV replication are provided by the transfection host cells. The pHELP plasmid does not, however, contain other adenovirus replication or structural genes. The adenovirus sequences do not contain the cis elements critical for replication, such as the inverted terminal repeats. Therefore, no infectious adenovirus is expected to be generated from such a production system.

[0164] In some embodiments, the step of producing the AAV particle further comprises a step of collecting the AAV particle from the cell. The AAV particle can be collected from the medium and / or by lysing the cells. The vector genome can be provided to the cell using any method known in the art, for example, a non-viral (e.g., plasmid) or viral vector. In some embodiments, the vector genome is supplied by a herpesvirus, an adenovirus vector, a baculovirus vector, or an Epstein-Barr virus (EBV)-derived vector.

[0165] The AAV rep and cap genes, and / or the helper virus protein sequences are provided in trans (e.g., from a vector, such as a plasmid, or by stably integrating the sequences into a packaging cell). For example, the AAV rep and cap genes and / or the helper virus protein sequences may be provided by a hybrid adenovirus, a hybrid herpesvirus vector (e.g., hybridherpes simplex virus type I (HSV-1) vector), an Epstein-Barr virus (EBV)-derived vector, a baculovirus vector, or a non-infectious adenovirus mini-plasmid. In some embodiments, helper virus functions may be provided by the packaging cell, which has helper sequences embedded in the chromosome, or maintained as a stable extrachromosomal element.

[0166] The term “transfection” is used to refer to the uptake of foreign DNA by a cell, and a cell has been “transfected” when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52: 456, Sambrook et al. (1989). Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986). Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981). Gene 13: 197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

[0167] Transfection can be performed using techniques known in the art, including but not limited to electroporation, lipofection, e.g., with lipofectamine, cationic polymers and cationic lipids. Any suitable transfection media may be used. In one embodiment, adherent human embryonic kidney (HEK293) cells are transfected with a triple DNA plasmid polyethylenimine (PEI) co-precipitation. In one embodiment, an AAV particle described herein is produced using triple DNA plasmid transfection into adherent cells using a PEI coprecipitation. In one embodiment, the DMEM growth medium used for cell expansion is replaced with a modified DMEM transfection media. This media is formulated without calcium and L-glutamine. In one embodiment, the transfection media is DMEM with no FBS, no calcium, no L-glutamine and 4.5 g / L glucose. In some embodiments, transfection media without serum (e.g., without FBS) improves transfection efficacy. In an embodiment, the transfection media is OptiMEM (Invitrogen / Thermo Fisher). In one embodiment, the three plasmids described herein are mixed together with PEI in transfection media and allowed to react. In some embodiments, the three plasmids are mixed together in about 1 : 1 : 1 molar ratio. In one embodiment, the plasmids and PEI are mixed in a ratio of 1 : 1 by weight of DNA:PEI. In one embodiment, the plasmids and PEI are mixed in a ratio of less than 1 : 1 by weight of DNA:PEI.

[0168] In some embodiments, AAV particles described herein can be harvested from packaging cells and purified by methods standard in the art (e.g., Clark et al, Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002); U.S. Patent No. 6,566,118 and WO 98 / 09657, incorporated herein in their entirety by reference) such as by cesium chloride ultracentrifugation gradient or column chromatography. In one embodiment, a harvesting or purification step provided in U.S. Patent Application PublicationNo. 2021 / 0317474 is used to harvest or purify one of the AAV particles described herein. The contents of U.S. Patent Application Publication No. 2021 / 0317474 are incorporated herein in their entirety.Pharmaceutical Compositions

[0169] One aspect of the disclosure is directed to a pharmaceutical composition comprising the dual AAV particles according to the disclosure, and a pharmaceutically acceptable carrier, excipient, diluent or buffer. By “pharmaceutically acceptable” it is meant a material that is not toxic or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects.

[0170] In one embodiment, the pharmaceutical composition further comprises an adjuvant or stabilizing agent.

[0171] Suitable administration forms of a pharmaceutical composition containing AAV vectors include, but are not limited to, injectable solutions or suspensions, eye lotions and ophthalmic ointment.

[0172] In one embodiment, the pharmaceutical composition is formulated for intraocular administration, including, e.g., subretinal, subfoveal, suprachoroidal and / or intravitreal administration. In a preferred embodiment, the pharmaceutical composition is formulated for subretinal injection. In another preferred embodiment, the pharmaceutical composition is formulated for subretinal injection with foveal placement.

[0173] In some embodiments, a pharmaceutical composition of the disclosure comprises dual AAV particles at a ratio of first AAV particle to second AAV particle. The ratio “about first value to about second value”, as used herein, can also be expressed as “about first value:about second value” — the first value and the second value can independently be a whole number or a number comprising a decimal component. The ratio of first AAV particle to second AAV particle, in embodiments described herein, is (i) a ratio of a number of first encapsidated transgenes to a number of second encapsidated transgenes, where the first encapsidated transgene is an encapsidated N-terminal transgene and the second encapsidated transgene is an encapsidated C-terminal transgene, or the first encapsidated transgene is an encapsidated C- terminal transgene and the second encapsidated transgene is an encapsidated N-terminal transgene, and / or (ii) a number of first AAV particles to a number of second AAV particle, where the first AAV particle comprises an N-terminal transgene and the second AAV particlecomprises a C-terminal transgene, or the first AAV particle comprises an C-terminal transgene and the second AAV particle comprises a N-terminal transgene.

[0174] In some embodiments, the ratio of first AAV particle to second AAV particle is about 1 to about 1, about 1 to about 1.5, about 1 to about 2, about 1 to about 2.5, about 1 to about 3, about 1 to about 3.5, about 1 to about 4, about 1 to about 4.5, about 1 to about 5, about 5 to about 1, about 4.5 to about 1, about 4 to about 1, about 3.5 to about 1, about 3 to about 1, about2.5 to about 1, 2 to about 1, or about 1.5 to about 1. In some embodiments, the ratio of first AAV particle: second AAV particle is from about 1 to about 5 (first AAV particles): about 1 (second AAV particle). In some embodiments, the ratio of first AAV particle: second AAV particle is about 1 (first AAV particle):from about 1 to about 5 (second AAV particles).

[0175] In some embodiments, the ratio of the number of first encapsidated transgenes to the number of second encapsidated transgenes is about 1 to about 1, about 1 to about 1.5, about 1 to about 2, about 1 to about 2.5, about 1 to about 3, about 1 to about 3.5, about 1 to about 4, about 1 to about 4.5, about 1 to about 5, about 5 to about 1, about 4.5 to about 1, about 4 to about 1, about 3.5 to about 1, about 3 to about 1, about 2.5 to about 1, 2 to about 1, or about1.5 to about 1. In some embodiments, the ratio of the number of encapsidated N-terminal transgenes to the number of encapsidated C-terminal transgenes is about 1 to about 1, about 1 to about 1.5, about 1 to about 2, about 1 to about 2.5, about 1 to about 3, about 1 to about 3.5, about 1 to about 4, about 1 to about 4.5, about 1 to about 5, about 5 to about 1, about 4.5 to about 1, about 4 to about 1, about 3.5 to about 1, about 3 to about 1, about 2.5 to about 1, 2 to about 1, or about 1.5 to about 1. In some embodiments, the ratio of the number of first encapsidated transgene:the number of second encapsidated transgenes is from about 1 to about 5 (first encapsidated transgene): about 1 (second encapsidated transgene). In some embodiments, the ratio of the number of first encapsidated transgene:the number of second encapsidated transgene is about 1 (first encapsidated transgene) :from about 1 to about 5 (second encapsidated transgene). In some embodiments, the ratio of the number of encapsidated N-terminal transgenes :the number of encapsidated C-terminal transgenes is from about 1 to about 5 (encapsidated N-terminal transgenes): about 1 (encapsidated C-terminal transgene). In some embodiments, the ratio of the number of encapsidated N-terminal transgenes :the number of encapsidated C-terminal transgenes is about 1 (encapsidated N- terminal transgene): from about 1 to about 5 (encapsidated C-terminal transgenes).

[0176] In a preferred embodiment, the pharmaceutical composition comprises dual AAV particles at a ratio of N-terminal AAV particle to C-terminal AAV particle at about 1 to about1. In some embodiments, the about 1 to about 1 ratio is a ratio of the number of encapsidated N-terminal transgenes to the number of encapsidated C-terminal transgenes. In yet another embodiment, the ratio of about 1 to about 1 is a ratio of the number of first AAV particles (comprising N-terminal transgenes) to the number of second AAV particles (comprising C- terminal transgenes). In some embodiments, the about 1 to about 1 ratio is a ratio of the number of first encapsidated transgenes (N-terminal transgenes) to the number of second encapsidated transgenes (C-terminal transgenes). As used and described herein, the term “encapsidated transgene” can be used interchangeably with the term “vector genome”.

[0177] In some embodiments, the dual AAV vector composition is administered by intravitreal administration. In a preferred embodiment, the dual AAV vector composition is administered by subretinal administration. In some embodiments, the subretinal administration includes subfoveal administration. In another embodiment, the subretinal administration includes subretinal administration with foveal placement.

[0178] In one embodiment, the pharmaceutical composition comprises dual AAV particles suspended in a buffer comprising 1 x phosphate buffered saline (PBS). In one embodiment, the pharmaceutical composition comprises dual AAV particles suspended in a buffer comprising poloxamer 188. In a further embodiment, the poloxamer 188 is present at a weight per volume (w / v) of from about 0.001% to about 0.009%. In a further embodiment, the poloxamer 188 is present at a weight per volume (w / v) of about 0.005%.

[0179] In one embodiment, the pharmaceutical composition comprises dual AAV particles suspended in a buffer comprising Tris (pH 8.0), magnesium chloride (MgCL), sodium chloride (NaCl), and poloxamer 188. In a further embodiment, the pharmaceutical composition comprises an effective amount of one of the AAV particles suspended in a buffer comprising 20 mM Tris (pH 8.0), 1 mM magnesium chloride (MgCh), 200 mM sodium chloride (NaCl), 0.005% poloxamer 188 weight per volume (w / v).

[0180] In one embodiment, the pharmaceutical composition comprises a total vg titer of about | / | 0l ()total vg / mL to about H IO14total vg / mL. In one embodiment, the pharmaceutical composition comprises a total vg titer of about 2 1013total vg / mL (i.e., about 1.0 1013vg / mL of a first particle and about l.Ox lO13vg / mL of a second particle). In another embodiment, the total vg titer of the dual AAV particles is about H IO10total vg / mL, about 1.25 1010total vg / mL, about 2x lO10total vg / mL, about 5x l010total vg / mL, about I / I O" total vg / mL, about 1.25X 1011total vg / mL, about 2 1011total vg / mL, about 5 1011total vg / mL, about H IO12totalvg / mL, about I .25 / I 012total vg / mL, about 2 / I 012total vg / mL, about 5 / I 012total vg / mL, about I x lO13total vg / mL, about 1.25>< 1013total vg / mL, about 2x l013total vg / mL, about 5x !013total vg / mL, about 1 x 1014total vg / mL, or a total vg titer in a range defined by any two of these total vg titers. In some embodiments, the total vg titer of the dual AAV particles is diluted (vg titerdiluent) at a 1 :2, 1:3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 1 :20, 1 :30, 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, 1 : 100, 1 :125, 1 : 150, 1 : 175, 1 :200 dilution, or at a dilution within a range defined by any two of these dilutions. In some embodiments, the vg titer of 2x l013total vg / mL (e.g., I x lO13vg / mL INS1203-RD and I x lO13vg / mL INS1203-RA) is diluted (total vg titer: diluent) at 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1:7, 1 :8, 1 :9, 1 : 10, 1:20, 1 :30, 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, 1 : 100, 1 : 125, 1 : 150, 1 : 175, 1 :200 dilution, or at a dilution within a range defined by any two of these dilutions. In some embodiments, the diluent comprises 0.001% w / v poloxamer 188. In some embodiments, an injection volume of the total vg titer is from about 0.05 mL to about 0.8 mL per eye treated. In some embodiments, an injection volume of the total vg titer is about 0.05 mL, about 0.06 mL, about 0.07 mL, about 0.08 mL, about 0.09 mL, about 0.1 mL, about 0.15 mL, about 0.2 mL, about 0.25 mL, about 0.3 mL, about 0.35 mL, about 0.4 mL, about 0.45 mL, about 0.5 mL, about 0.55 mL, about 0.6 mL, about 0.65 mL, about 0.7 mL, about 0.75 mL, about 0.8 mL per eye treated, or a volume in a range between any two volumes thereof.

[0181] In some embodiments, pharmaceutical compositions provided herein comprise sterile aqueous and non-aqueous injection solutions, which are optionally isotonic with the blood of the subject to whom the pharmaceutical composition is to be delivered. Pharmaceutical compositions can contain antioxidants, buffers, bacteriostats and solutes, which render the composition isotonic with the blood of the intended subject to be administered. Aqueous and non-aqueous sterile suspensions, solutions and emulsions can include suspending agents and thickening agents. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. In some embodiments pharmaceutical compositions comprise pharmaceutically acceptable vehicles and can include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0182] In some embodiments, pharmaceutical compositions can be presented in unit / dose or multi-dose containers, for example, in sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-inj ection immediately prior to use.RE J Efficiency

[0183] The disclosed compositions / systems / methods allows for the efficient RNA recombination between individual fragments. In some embodiments, reconstitution (i.e., splicing or recombination) efficiency achieved using the compositions, systems or methods of the disclosure is determined using any suitable method known to one of skill in the art. In some embodiments, reconstitution efficiency is represented by a measure of correctly joined RNA relative to a control RNA, or a measure of full-length protein or protein activity relative to that of a control protein. In some embodiments, the control RNA is the unjoined RNA, wherein reconstitution efficiency is represented by a measure of joined RNA relative to unjoined RNA. This measurement can be made by detecting and comparing junction RNA and the unjoined C- terminal RNA species (e.g., junction RNA : unjoined C-terminal RNA). In some embodiments, reconstitution efficiency is represented by a measure of full-length or active protein relative to a protein fragment or inactive protein.

[0184] In some embodiments, the reconstitution, recombination or splicing efficiency (a measure of the correct joining of the two different coding sequences present on different vector genome relative to a control RNA, and / or the production of the desired full-length protein relative to a control protein) is about 10% to about 200%. In some embodiments, the reconstitution efficiency is about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 100%, about 10% to about 110%, about 10% to about 120%, about 10% to about 130%, about 10% to about 140%, about 10% to about 150%, about 10% to about 160%, about 10% to about 170%, about 10% to about 180%, about 10% to about 190%, about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 60%, about 15% to about 70%, about 15% to about 80%, about 15% to about 90%, about 15% to about 100%, about 15% to about 110%, about 15% to about 120%, about 15% to about 130%, about 15% to about 140%, about 15% to about 150%, about 15% to about 160%, about 15% to about 170%, about 15% to about 180%, about 15% to about 190%, about 15% to about 200%, about 20% to about 25%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20%to about 100%, about 20% to about 110%, about 20% to about 120%, about 20% to about 130%, about 20% to about 140%, about 20% to about 150%, about 20% to about 160%, about 20% to about 170%, about 20% to about 180%, about 20% to about 190%, about 20% to about 200%, about 25% to about 30%, about 25% to about 40%, about 25% to about 50%, about 25% to about 60%, about 25% to about 70%, about 25% to about 80%, about 25% to about 90%, about 25% to about 100%, about 25% to about 110%, about 25% to about 120%, about 25% to about 130%, about 25% to about 140%, about 25% to about 150%, about 25% to about 160%, about 25% to about 170%, about 25% to about 180%, about 25% to about 190%, about 25% to about 200%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 100%, about 30% to about 110%, about 30% to about 120%, about 30% to about 130%, about 30% to about 140%, about 30% to about 150%, about 30% to about 160%, about 30% to about 170%, about 30% to about 180%, about 30% to about 190%, about 30% to about 200%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 100%, about 40% to about 110%, about 40% to about 120%, about 40% to about 130%, about 40% to about 140%, about 40% to about 150%, about 40% to about 160%, about 40% to about 170%, about 40% to about 180%, about 40% to about 190%, about 40% to about 200%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 100%, about 50% to about 110%, about 50% to about 120%, about 50% to about 130%, about 50% to about 140%, about 50% to about 150%, about 50% to about 160%, about 50% to about 170%, about 50% to about 180%, about 50% to about 190%, about 50% to about 200%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 100%, about 60% to about 110%, about 60% to about 120%, about 60% to about 130%, about 60% to about 140%, about 60% to about 150%, about 60% to about 160%, about 60% to about 170%, about 60% to about 180%, about 60% to about 190%, about 60% to about 200%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 70% to about 110%, about 70% to about 120%, about 70% to about 130%, about 70% to about 140%, about 70% to about 150%, about 70% to about 160%, about 70% to about 170%, about 70% to about 180%, about 70% to about 190%, about 70% to about 200%, about 80% to about 90%, about 80% to about 100%, about 80% to about 110%, about 80% to about 120%, about 80% to about 130%, about 80% to about 140%, about 80% to about 150%, about 80% to about 160%, about 80% to about 170%, about 80% to about 180%, about 80% to about 190%, about 80% to about 200%, about 90% to about 100%, about 90% to about 110%, about 90% to about 120%, about90% to about 130%, about 90% to about 140%, about 90% to about 150%, about 90% to about 160%, about 90% to about 170%, about 90% to about 180%, about 90% to about 190%, about 90% to about 200%, about 100% to about 110%, about 100% to about 120%, about 100% to about 130%, about 100% to about 140%, about 100% to about 150%, about 100% to about 160%, about 100% to about 170%, about 100% to about 180%, about 100% to about 190%, about 100% to about 200%, about 110% to about 120%, about 110% to about 130%, about 110% to about 140%, about 110% to about 150%, about 110% to about 160%, about 110% to about 170%, about 110% to about 180%, about 110% to about 190%, about 110% to about 200%, about 120% to about 130%, about 120% to about 140%, about 120% to about 150%, about 120% to about 160%, about 120% to about 170%, about 120% to about 180%, about 120% to about 190%, about 120% to about 200%, about 130% to about 140%, about 130% to about 150%, about 130% to about 160%, about 130% to about 170%, about 130% to about 180%, about 130% to about 190%, about 130% to about 200%, about 140% to about 150%, about 140% to about 160%, about 140% to about 170%, about 140% to about 180%, about 140% to about 190%, about 140% to about 200%, about 150% to about 160%, about 150% to about 170%, about 150% to about 180%, about 150% to about 190%, about 150% to about 200%, about 160% to about 170%, about 160% to about 180%, about 160% to about 190%, about 160% to about 200%, about 170% to about 180%, about 170% to about 190%, about 170% to about 200%, about 180% to about 190%, about 180% to about 200%, or about 190% to about 200%. In some embodiments, the reconstitution efficiency is about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%. In some embodiments, the reconstitution efficiency is at least about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%. In some embodiments, the reconstitution efficiency is at most about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%.

[0185] In some embodiments, the compositions, systems or methods of the disclosure are evaluated by determining an RNA production level using any suitable method known to one of skill in the art. In some embodiments, the RNA production level is represented by a measureof correctly joined RNA relative to a control RNA. In some embodiments, the control RNA is a corresponding mutant RNA or an endogenous RNA. For example, the ratio of the amount of joined RNA to the amount of mutant or endogenous RNA produced in the transfected cell is compared with same ratio in non-transfected cells, to determine the production level of the correctly joined RNA. In some embodiments, the control RNA is the corresponding endogenous RNA produced in a non-transfected cell from a normal subject that expresses the corresponding full-length protein, and the RNA production level is determined by measuring the amount of the joined RNA in the transfected cell and comparing it to that of the control RNA in a non-transfected cell from a normal subject.

[0186] In some embodiments, the RNA production level (i.e., a measure of the amount of correctly joined RNA relative to that of a control RNA) achieved is about 5% to about 200%. In some embodiments, the RNA production level achieved is about 5% to about 10%, about 5% to about 20%, about 5% to about 25%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 5% to about 70%, about 5% to about 80%, about 5% to about 90%, about 5% to about 100%, about 10% to about 110%, about 10% to about 120%, about 10% to about 130%, about 10% to about 140%, about 10% to about 150%, about 10% to about 160%, about 10% to about 170%, about 10% to about 180%, about 10% to about 190%, about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 60%, about 15% to about 70%, about 15% to about 80%, about 15% to about 90%, about 15% to about 100%, about 15% to about 110%, about 15% to about 120%, about 15% to about 130%, about 15% to about 140%, about 15% to about 150%, about 15% to about 160%, about 15% to about 170%, about 15% to about 180%, about 15% to about 190%, about 15% to about 200%, about 20% to about 25%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 100%, about 20% to about 110%, about 20% to about 120%, about 20% to about 130%, about 20% to about 140%, about 20% to about 150%, about 20% to about 160%, about 20% to about 170%, about 20% to about 180%, about 20% to about 190%, about 20% to about 200%, about 25% to about 30%, about 25% to about 40%, about 25% to about 50%, about 25% to about 60%, about 25% to about 70%, about 25% to about 80%, about 25% to about 90%, about 25% to about 100%, about 25% to about 110%, about 25% to about 120%, about 25% to about 130%, about 25% to about 140%, about 25% to about 150%, about 25% to about 160%, about 25% to about 170%, about 25% to about 180%, about25% to about 190%, about 25% to about 200%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 100%, about 30% to about 110%, about 30% to about 120%, about 30% to about 130%, about 30% to about 140%, about 30% to about 150%, about 30% to about 160%, about 30% to about 170%, about 30% to about 180%, about 30% to about 190%, about 30% to about 200%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 100%, about 40% to about 110%, about 40% to about 120%, about 40% to about 130%, about 40% to about 140%, about 40% to about 150%, about 40% to about 160%, about 40% to about 170%, about 40% to about 180%, about 40% to about 190%, about 40% to about 200%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 100%, about 50% to about 110%, about 50% to about 120%, about 50% to about 130%, about 50% to about 140%, about 50% to about 150%, about 50% to about 160%, about 50% to about 170%, about 50% to about 180%, about 50% to about 190%, about 50% to about 200%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 100%, about 60% to about 110%, about 60% to about 120%, about 60% to about 130%, about 60% to about 140%, about 60% to about 150%, about 60% to about 160%, about 60% to about 170%, about 60% to about 180%, about 60% to about 190%, about 60% to about 200%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 70% to about 110%, about 70% to about 120%, about 70% to about 130%, about 70% to about 140%, about 70% to about 150%, about 70% to about 160%, about 70% to about 170%, about 70% to about 180%, about 70% to about 190%, about 70% to about 200%, about 80% to about 90%, about 80% to about 100%, about 80% to about 110%, about 80% to about 120%, about 80% to about 130%, about 80% to about 140%, about 80% to about 150%, about 80% to about 160%, about 80% to about 170%, about 80% to about 180%, about 80% to about 190%, about 80% to about 200%, about 90% to about 100%, about 90% to about 110%, about 90% to about 120%, about 90% to about 130%, about 90% to about 140%, about 90% to about 150%, about 90% to about 160%, about 90% to about 170%, about 90% to about 180%, about 90% to about 190%, about 90% to about 200%, about 100% to about 110%, about 100% to about 120%, about 100% to about 130%, about 100% to about 140%, about 100% to about 150%, about 100% to about 160%, about 100% to about 170%, about 100% to about 180%, about 100% to about 190%, about 100% to about 200%, about 110% to about 120%, about 110% to about 130%, about 110% to about 140%, about 110% to about 150%, about 110% to about 160%, about 110% to about 170%, about 110% to about 180%, about 110% to about190%, about 110% to about 200%, about 120% to about 130%, about 120% to about 140%, about 120% to about 150%, about 120% to about 160%, about 120% to about 170%, about 120% to about 180%, about 120% to about 190%, about 120% to about 200%, about 130% to about 140%, about 130% to about 150%, about 130% to about 160%, about 130% to about 170%, about 130% to about 180%, about 130% to about 190%, about 130% to about 200%, about 140% to about 150%, about 140% to about 160%, about 140% to about 170%, about 140% to about 180%, about 140% to about 190%, about 140% to about 200%, about 150% to about 160%, about 150% to about 170%, about 150% to about 180%, about 150% to about 190%, about 150% to about 200%, about 160% to about 170%, about 160% to about 180%, about 160% to about 190%, about 160% to about 200%, about 170% to about 180%, about 170% to about 190%, about 170% to about 200%, about 180% to about 190%, about 180% to about 200%, or about 190% to about 200%. In some embodiments, the RNA production level achieved is about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%. In some embodiments, the RNA production level achieved is at least about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%. In some embodiments, the RNA production level achieved is at most about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%.

[0187] In some embodiments, the protein expression level is represented by a measure of the amount of full-length protein or protein activity relative to that of a control protein. In some embodiments, the control protein is a corresponding mutant protein or an endogenous protein. For example, the ratio of the amount of full-length protein or protein activity to the amount of mutant or endogenous protein produced in the transfected cell is compared with same ratio in non-transfected cells. In some embodiments, the control protein is the full-length protein produced in, e.g., a cell that is engineered to express a control full-length protein (wherein the cell is not transfected with the inventive constructs) or a non-transfected cell from a normal subject that expresses a control full-length protein, and the protein expression level is determined by measuring the amount or activity of the protein in the transfected cell andcomparing it to that of the control protein. In some embodiments, the control protein is a mutant form of the protein, produced in a cell that is transfected or non-transfected with the construct, and the amount of full-length protein or protein activity is compared with that of the control protein to determine the protein expression level. In some embodiments, the amount of full- length protein or protein activity is compared with that of an endogenous, or housekeeping, protein to determine the protein production level.

[0188] In some embodiments, the protein expression level (i.e., a measure of the amount of full-length protein or protein activity relative to that of a control protein) achieved is about 1% to about 200%. In some embodiments, the protein expression level achieved is about 10% to about 200%. In some embodiments, the protein expression level achieved is about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 75%, about 10% to about 80%, about 10% to about 85%, about 10% to about 90%, about 10% to about 100%, about 10% to about 110%, about 10% to about 120%, about 10% to about 130%, about 10% to about 140%, about 10% to about 150%, about 10% to about 160%, about 10% to about 170%, about 10% to about 180%, about 10% to about 190%, about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 60%, about 15% to about 70%, about 15% to about 80%, about 15% to about 90%, about 15% to about 100%, about 15% to about 110%, about 15% to about 120%, about 15% to about 130%, about 15% to about 140%, about 15% to about 150%, about 15% to about 160%, about 15% to about 170%, about 15% to about 180%, about 15% to about 190%, about 15% to about 200%, about 20% to about 25%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 100%, about 20% to about 110%, about 20% to about 120%, about 20% to about 130%, about 20% to about 140%, about 20% to about 150%, about 20% to about 160%, about 20% to about 170%, about 20% to about 180%, about 20% to about 190%, about 20% to about 200%, about 25% to about 30%, about 25% to about 40%, about 25% to about 50%, about 25% to about 60%, about 25% to about 70%, about 25% to about 80%, about 25% to about 90%, about 25% to about 100%, about 25% to about 110%, about 25% to about 120%, about 25% to about 130%, about 25% to about 140%, about 25% to about 150%, about 25% to about 160%, about 25% to about 170%, about 25% to about 180%, about 25% to about 190%, about 25% to about 200%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about80%, about 30% to about 90%, about 30% to about 100%, about 30% to about 110%, about 30% to about 120%, about 30% to about 130%, about 30% to about 140%, about 30% to about 150%, about 30% to about 160%, about 30% to about 170%, about 30% to about 180%, about 30% to about 190%, about 30% to about 200%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 100%, about 40% to about 110%, about 40% to about 120%, about 40% to about 130%, about 40% to about 140%, about 40% to about 150%, about 40% to about 160%, about 40% to about 170%, about 40% to about 180%, about 40% to about 190%, about 40% to about 200%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 100%, about 50% to about 110%, about 50% to about 120%, about 50% to about 130%, about 50% to about 140%, about 50% to about 150%, about 50% to about 160%, about 50% to about 170%, about 50% to about 180%, about 50% to about 190%, about 50% to about 200%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 100%, about 60% to about 110%, about 60% to about 120%, about 60% to about 130%, about 60% to about 140%, about 60% to about 150%, about 60% to about 160%, about 60% to about 170%, about 60% to about 180%, about 60% to about 190%, about 60% to about 200%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 70% to about 110%, about 70% to about 120%, about 70% to about 130%, about 70% to about 140%, about 70% to about 150%, about 70% to about 160%, about 70% to about 170%, about 70% to about 180%, about 70% to about 190%, about 70% to about 200%, about 80% to about 90%, about 80% to about 100%, about 80% to about 110%, about 80% to about 120%, about 80% to about 130%, about 80% to about 140%, about 80% to about 150%, about 80% to about 160%, about 80% to about 170%, about 80% to about 180%, about 80% to about 190%, about 80% to about 200%, about 90% to about 100%, about 90% to about 110%, about 90% to about 120%, about 90% to about 130%, about 90% to about 140%, about 90% to about 150%, about 90% to about 160%, about 90% to about 170%, about 90% to about 180%, about 90% to about 190%, about 90% to about 200%, about 100% to about 110%, about 100% to about 120%, about 100% to about 130%, about 100% to about 140%, about 100% to about 150%, about 100% to about 160%, about 100% to about 170%, about 100% to about 180%, about 100% to about 190%, about 100% to about 200%, about 110% to about 120%, about 110% to about 130%, about 110% to about 140%, about 110% to about 150%, about 110% to about 160%, about 110% to about 170%, about 110% to about 180%, about 110% to about 190%, about 110% to about 200%, about 120% to about 130%, about 120% to about 140%, about 120% to about 150%, about 120% to about 160%, about 120% to about 170%,about 120% to about 180%, about 120% to about 190%, about 120% to about 200%, about 130% to about 140%, about 130% to about 150%, about 130% to about 160%, about 130% to about 170%, about 130% to about 180%, about 130% to about 190%, about 130% to about 200%, about 140% to about 150%, about 140% to about 160%, about 140% to about 170%, about 140% to about 180%, about 140% to about 190%, about 140% to about 200%, about 150% to about 160%, about 150% to about 170%, about 150% to about 180%, about 150% to about 190%, about 150% to about 200%, about 160% to about 170%, about 160% to about 180%, about 160% to about 190%, about 160% to about 200%, about 170% to about 180%, about 170% to about 190%, about 170% to about 200%, about 180% to about 190%, about 180% to about 200%, or about 190% to about 200%. In some embodiments, the protein expression level achieved is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%. In some embodiments, the protein expression level achieved is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%. In some embodiments, the protein expression level achieved is at most about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%.

[0189] In some embodiments, the protein activity level (i.e. a measure of protein activity of full-length protein relative to that of a control protein) achieved is about 50% to about 200%. In some embodiments, the protein activity level achieved is about 50% to about 200%. In some embodiments, the protein activity level achieved is about 50% to about 55%, about 50% to about 60%, about 50% to about 65%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 50% to about 110%, about 50% to about 120%, about 50% to about 130%, about 50% to about 140%, about 50% to about 150%, about 50% to about 160%, about 50% to about 170%, about 50% to about 180%, about 50% to about 190%, about 50% to about 200%, about 55% to about 60%, about 55% to about 65%, about 55% to about 70%, about 55% to about 75%, about 55% to about 80%, about 55% to about 85%, about 55% to about 90%, about 55% to about 95%, about 55% to about 100%, about 55% to about 110%,about 55% to about 120%, about 55% to about 130%, about 55% to about 140%, about 55% to about 150%, about 55% to about 160%, about 55% to about 170%, about 55% to about 180%, about 55% to about 190%, about 55% to about 200%, about 60% to about 65%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 100%, about 60% to about 110%, about 60% to about 120%, about 60% to about 130%, about 60% to about 140%, about 60% to about 150%, about 60% to about 160%, about 60% to about 170%, about 60% to about 180%, about 60% to about 190%, about 60% to about 200%, about 65% to about 70%, about 65% to about 75%, about 65% to about 80%, about 65% to about 85%, about 65% to about 90%, about 65% to about 95%, about 65% to about 100%, about 65% to about 110%, about 65% to about 120%, about 65% to about 130%, about 65% to about 140%, about 65% to about 150%, about 65% to about 160%, about 65% to about 170%, about 65% to about 180%, about 65% to about 190%, about 65% to about 200%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 70% to about 110%, about 70% to about 120%, about 70% to about 130%, about 70% to about 140%, about 70% to about 150%, about 70% to about 160%, about 70% to about 170%, about 70% to about 180%, about 70% to about 190%, about 70% to about 200%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 75% to about 100%, about 75% to about 110%, about 75% to about 120%, about 75% to about 130%, about 75% to about 140%, about 75% to about 150%, about 75% to about 160%, about 75% to about 170%, about 75% to about 180%, about 75% to about 190%, about 75% to about 200%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 80% to about 110%, about 80% to about 120%, about 80% to about 130%, about 80% to about 140%, about 80% to about 150%, about 80% to about 160%, about 80% to about 170%, about 80% to about 180%, about 80% to about 190%, about 80% to about 200%, about 85% to about 90%, about 85% to about 95%, about 85% to about 100%, about 85% to about 110%, about 85% to about 120%, about 85% to about 130%, about 85% to about 140%, about 85% to about 150%, about 85% to about 160%, about 85% to about 170%, about 85% to about 180%, about 85% to about 190%, about 85% to about 200%, about 90% to about 95%, about 90% to about 100%, about 90% to about 110%, about 90% to about 120%, about 90% to about 130%, about 90% to about 140%, about 90% to about 150%, about 90% to about 160%, about 90% to about 170%, about 90% to about 180%, about 90% to about 190%, about 90% to about 200%, about 95% to about 100%, about 95% to about 110%, about 95% to about 120%, about 95% to about 130%, about 95% to about140%, about 95% to about 150%, about 95% to about 160%, about 95% to about 170%, about 95% to about 180%, about 95% to about 190%, about 95% to about 200%, about 100% to about 110%, about 100% to about 120%, about 100% to about 130%, about 100% to about 140%, about 100% to about 150%, about 100% to about 160%, about 100% to about 170%, about 100% to about 180%, about 100% to about 190%, about 100% to about 200%, about 110% to about 120%, about 110% to about 130%, about 110% to about 140%, about 110% to about 150%, about 110% to about 160%, about 110% to about 170%, about 110% to about 180%, about 110% to about 190%, about 110% to about 200%, about 120% to about 130%, about 120% to about 140%, about 120% to about 150%, about 120% to about 160%, about 120% to about 170%, about 120% to about 180%, about 120% to about 190%, about 120% to about 200%, about 130% to about 140%, about 130% to about 150%, about 130% to about 160%, about 130% to about 170%, about 130% to about 180%, about 130% to about 190%, about 130% to about 200%, about 140% to about 150%, about 140% to about 160%, about 140% to about 170%, about 140% to about 180%, about 140% to about 190%, about 140% to about 200%, about 150% to about 160%, about 150% to about 170%, about 150% to about 180%, about 150% to about 190%, about 150% to about 200%, about 160% to about 170%, about 160% to about 180%, about 160% to about 190%, about 160% to about 200%, about 170% to about 180%, about 170% to about 190%, about 170% to about 200%, about 180% to about 190%, about 180% to about 200%, or about 190% to about 200%. In some embodiments, the protein activity level achieved is about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, or about 200%. In some embodiments, the protein activity level achieved is at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the protein activity level achieved is at most about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, or about 200%.

[0190] In some embodiments, the amount of correctly joined RNA or full-length protein produced in a cell is sufficient to ameliorate or cure a condition or disease in a subject, asunderstood by one of skill in the art for the particular condition or disease. In some embodiments, the amount of correctly joined RNA or full-length protein produced in a cell is an effective amount. In some embodiments, this amount is equivalent to about 50% to 200% the amount of the RNA or protein produced in a normal cell. In some embodiments, this amount is equivalent to about 40% to about 200% the amount of the RNA or protein produced in a normal cell. In some embodiments, this amount is equivalent to about 40% to about 45%, about 40% to about 50%, about 40% to about 55%, about 40% to about 60%, about 40% to about 65%, about 40% to about 70%, about 40% to about 75%, about 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 100%, about 40% to about 110%, about 40% to about 120%, about 40% to about 130%, about 40% to about 140%, about 40% to about 150%, about 40% to about 160%, about 40% to about 170%, about 40% to about 180%, about 40% to about 190%, about 40% to about 200%, about 45% to about 50%, about 45% to about 55%, about 45% to about 60%, about 45% to about 65%, about 45% to about 70%, about 45% to about 75%, about 45% to about 80%, about 45% to about 85%, about 45% to about 90%, about 45% to about 100%, about 45% to about 110%, about 45% to about 120%, about 45% to about 130%, about 45% to about 140%, about 45% to about 150%, about 45% to about 160%, about 45% to about 170%, about 45% to about 180%, about 45% to about 190%, about 45% to about 200%, about 50% to about 55%, about 50% to about 60%, about 50% to about 65%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 100%, about 50% to about 110%, about 50% to about 120%, about 50% to about 130%, about 50% to about 140%, about 50% to about 150%, about 50% to about 160%, about 50% to about 170%, about 50% to about 180%, about 50% to about 190%, about 50% to about 200%, about 55% to about 60%, about 55% to about 65%, about 55% to about 70%, about 55% to about 75%, about 55% to about 80%, about 55% to about 85%, about 55% to about 90%, about 55% to about 100%, about 55% to about 110%, about 55% to about 120%, about 55% to about 130%, about 55% to about 140%, about 55% to about 150%, about 55% to about 160%, about 55% to about 170%, about 55% to about 180%, about 55% to about 190%, about 55% to about 200%, about 60% to about 65%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 100%, about 60% to about 110%, about 60% to about 120%, about 60% to about 130%, about 60% to about 140%, about 60% to about 150%, about 60% to about 160%, about 60% to about 170%, about 60% to about 180%, about 60% to about 190%, about 60% to about 200%, about 65% to about 70%, about 65% to about 75%, about 65% to about 80%, about 65% to about 85%, about 65%to about 90%, about 65% to about 100%, about 65% to about 110%, about 65% to about 120%, about 65% to about 130%, about 65% to about 140%, about 65% to about 150%, about 65% to about 160%, about 65% to about 170%, about 65% to about 180%, about 65% to about 190%, about 65% to about 200%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 100%, about 70% to about 110%, about 70% to about 120%, about 70% to about 130%, about 70% to about 140%, about 70% to about 150%, about 70% to about 160%, about 70% to about 170%, about 70% to about 180%, about 70% to about 190%, about 70% to about 200%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 100%, about 75% to about 110%, about 75% to about 120%, about 75% to about 130%, about 75% to about 140%, about 75% to about 150%, about 75% to about 160%, about 75% to about 170%, about 75% to about 180%, about 75% to about 190%, about 75% to about 200%, about 80% to about 85%, about 80% to about 90%, about 80% to about 100%, about 80% to about 110%, about 80% to about 120%, about 80% to about 130%, about 80% to about 140%, about 80% to about 150%, about 80% to about 160%, about 80% to about 170%, about 80% to about 180%, about 80% to about 190%, about 80% to about 200%, about 85% to about 90%, about 85% to about 100%, about 85% to about 110%, about 85% to about 120%, about 85% to about 130%, about 85% to about 140%, about 85% to about 150%, about 85% to about 160%, about 85% to about 170%, about 85% to about 180%, about 85% to about 190%, about 85% to about 200%, or about 90% to about 100%, about 90% to about 110%, about 90% to about 120%, about 90% to about 130%, about 90% to about 140%, about 90% to about 150%, about 90% to about 160%, about 90% to about 170%, about 90% to about 180%, about 90% to about 190%, about 90% to about 200%, about 95% to about 100%, about 95% to about 110%, about 95% to about 120%, about 95% to about 130%, about 95% to about 140%, about 95% to about 150%, about 95% to about 160%, about 95% to about 170%, about 95% to about 180%, about 95% to about 190%, about 95% to about 200%, about 100% to about 110%, about 100% to about 120%, about 100% to about 130%, about 100% to about 140%, about 100% to about 150%, about 100% to about 160%, about 100% to about 170%, about 100% to about 180%, about 100% to about 190%, about 100% to about 200%, about 110% to about 120%, about 110% to about 130%, about 110% to about 140%, about 110% to about 150%, about 110% to about 160%, about 110% to about 170%, about 110% to about 180%, about 110% to about 190%, about 110% to about 200%, about 120% to about 130%, about 120% to about 140%, about 120% to about 150%, about 120% to about 160%, about 120% to about 170%, about 120% to about 180%, about 120% to about 190%, about 120% to about 200%, about 130% to about 140%, about 130% toabout 150%, about 130% to about 160%, about 130% to about 170%, about 130% to about 180%, about 130% to about 190%, about 130% to about 200%, about 140% to about 150%, about 140% to about 160%, about 140% to about 170%, about 140% to about 180%, about 140% to about 190%, about 140% to about 200%, about 150% to about 160%, about 150% to about 170%, about 150% to about 180%, about 150% to about 190%, about 150% to about 200%, about 160% to about 170%, about 160% to about 180%, about 160% to about 190%, about 160% to about 200%, about 170% to about 180%, about 170% to about 190%, about 170% to about 200%, about 180% to about 190%, about 180% to about 200%, or about 190% to about 200% the amount of the RNA or protein produced in a normal cell. In some embodiments, this amount is equivalent to about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 100%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, or about 200% the amount of the RNA or protein produced in a normal cell. In some embodiments, this amount is equivalent to about at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% the amount of the RNA or protein produced in a normal cell. In some embodiments, this amount is equivalent to about at most about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 100%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, or about 200% the amount of the RNA or protein produced in a normal cell.

[0191] The measurements of RNA or protein used to determine recombination efficiency or production level can be made by any suitable method known to those of skill in the art. In some embodiments, recombination efficiency or production level is determined by measuring an amount of functional protein expressed, for example by Western blotting. In some embodiments, recombination efficiency or production level is determined by measuring the RNA transcript, for example using two probe based quantitative real-time PCR. For example, the first assay spans a sequence fully contained in the 3’ exonic coding sequence (labelled 3’ probe). The second assay spans the junction between the 5’ and the 3’ exonic coding sequence (labelled junction probe). Reconstitution efficiency can be calculated as the ratio of (junctionprobe count) / (3’ probe count). “Reconstitution efficiency,” “recombination efficiency,” and “splicing efficiency” are used interchangeably herein.

[0192] Achieving efficient recombination between multiple RNA molecules allows for packaging and delivery of transgenes into AAVs, which exceed the packaging limit of a single AAV. AAV packaging limits represent a major hurdle for gene therapy approaches for diseases caused by the absence / defect of large genes. One application of this composition / system is expression of large disease-causing genes using viral vectors with restricted packaging capacity.Methods of Treatment

[0193] One aspect of the present disclosure relates to a method of treating Stargardt Disease (STGD) in a subject in need thereof, comprising administering to the subject, a composition comprising an effective amount of a dual AAV particle composition described herein.

[0194] STGD is characterized by reduction of central vision and visual acuity starting, in some cases, as early as the first decade of life and in others in early, mid-, or late adulthood. The majority of individuals with STGD are diagnosed with type 1 STGD (STGD1), which is caused by mutations in the ABCA4 gene (6.8 kb) encoding a 210-kDa ATP-dependent flippase importer. Mutated ABCA4 loses its normal function of transporting toxic all-trans-retinal conjugates across disc membranes, resulting in accumulation of toxic bis-retinoids, including N-retinylidene-N-retinylethanolamine (A2E), and deposition of lipofuscin in the retinal pigment epithelium (RPE), which eventually causes retinal degeneration and vision loss.

[0195] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms include, but are not limited to, mammals such as humans, simians, murines, equines, bovines, porcines, canines, felines, and the like. In some embodiments, the subject or patient is human.

[0196] In embodiments where the subject is a human, the subject is a newborn, from about 1 month to about 5 years old, from about 1 month to about 1 year old, from about 1 month to about 3 years old, or from about 1 month to about 10 years old. In one embodiment, the subject is from about 10 years old to about 80 years old, e.g., from about 10 years old to about 60 years old, from about 10 years old to about 50 years old or from about 10 years old to about 40 years old. In one embodiment, the subject is from about 1 month old to about 10 years old, e.g., from about 1 month old to about 9 years old, 1 month old to about 8 years old, 1 month old to about 7 years old, 1 month old to about 6 years old, 1 month old to about 5 years old, 1 month old toabout 4 years old, 1 month old to about 3 years old, 1 month old to about 2 years old, or 1 month old to about 1 years old. In yet even another embodiment, the subject is a human from about 2 years old to about 12 years old, 3 years old to about 12 years old, 4 years old to about 12 years old or about 5 years old to about 12 years old.

[0197] In one embodiment of a method of treating STGD, the subject is a human. In a further embodiment, the subject is a neonate. In another embodiment, the subject is a human and the subject is an infant. In even another embodiment, the subject is a human and is an adolescent. In yet even another embodiment, the subject is a human and an adult.

[0198] As used herein, the term “effective amount” or “effective dose”, refers to the amount of a substance (e.g., an AAV particle of the present disclosure) sufficient to effect beneficial or desired results (e.g., expression of a protein, or a desired prophylactic or therapeutic effect). An effective amount can be administered in one or more administration(s), application(s) or dosage(s) and is not intended to be limited to a particular formulation or administration route. Where a dose is provided in “vector genomes”, an “effective dose” may be referred to herein as an “effective vector genome dose”.

[0199] In some embodiments, a dose is provided as vg per AAV vector. A “total dose,” as used herein, refers to the total number of vg administered to a subject. The total number of vgs administered to a subject in embodiments described herein, comprises the sum of (i) the dose of a first AAV particle (a first AAV vector) and a (ii) the dose of a second AAV particle (a second AAV vector). In preferred embodiments of the disclosure, the first AAV particle and the second AAV particle are administered at about equal vg doses per eye treated. In a preferred embodiment, treatment comprises administering a composition of the disclosure to both eyes of a subject (i.e., bilaterial treatment). In another embodiment, the treatment comprises administering a composition of the disclosure to one eye of the subject (i.e., unilateral treatment).

[0200] As used herein, the term “treating” includes any effect, e.g., preventing, lessening, reducing, modulating, ameliorating or eliminating, that results in the improvement of the condition, disease, disorder, improving the clinical phenotype of the condition, and the like, or ameliorating a symptom of the condition. “Treating” can thus include one or more of reducing onset of A STGD, reducing the progression of STGD, preventing STGD, reducing the severity of the STGD symptoms, retarding STGD symptom progression, delaying progression of STGD.

[0201] In one embodiment, treating comprises restoring the Abca4 protein level in a patient to the normal range or to a non-STGD level. In another embodiment, treating comprises increasing the Abca4 protein level in a patient to at least about 200%, about 195%, about 190%, about 185%, about 180%, about 175%, about 170%, about 165%, about 160%, about 155%, about 150%, about 145%, about 140%, about 135%, about 130% about 125%, about 120%, about 115%, about 110%, about 105%, about 100%, about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30% about 25%, about 20%, about 15%, about 10%, about 5%, about 2%, or about 1% of the normal or the pre-treatment level.

[0202] Administration routes include intraocular delivery, including, e.g., subretinal, subfoveal, and / or intravitreal administration, to allow for direct delivery to photoreceptors and / or retinal pigment epithelium (RPE). In another embodiment, administration is via parenteral administration. In a further embodiment, the parenteral administration is intravenous administration. Alternatively, other routes of administration may be selected (e.g., intranasal, intratracheal, intraarterial, intramuscular). The AAV particles described herein may be delivered in a single composition or multiple compositions.

[0203] In a preferred embodiment, the pharmaceutical composition is administered via subretinal injection with foveal placement.

[0204] In one embodiment of a STGD treatment method provided herein, the AAV capsid is an AAV8 capsid. In one embodiment, the AAV capsid is an engineered variant of an AAV8 capsid.

[0205] In another embodiment of the treatment methods provided herein, treating comprises decreasing the number of symptoms, or a reducing the severity of one or more symptoms in the subject being treated, compared to the symptoms exhibited prior to treatment. In one embodiment, treating comprises reducing the severity of one or more STGD symptoms in the patient. Such symptoms may include, but are not limited to: gray, black or hazy spots in the center of the vision, sensitivity to light, needing more time for eyes to adjust between light and dark places, color blindness, loss of visual acuity, central loss of vision, peripheral loss of vision, a visual acuity of 20 / 200 or less, a visual field of 20 degrees or less, or any combination thereof. In many jurisdictions, legal blindness is defined as having either a visual acuity of 20 / 200 or less in the better eye or a visual field of 20 degrees or less in the better eye.

[0206] According to the embodiments described herein, coding sequence expression may refer to gene expression (i.e., by measuring mRNA levels) or expression of the corresponding protein. It will be understood by those of ordinary skill in the art that in order to determine levels of coding sequence expression in different tissue types, substantially the same amount of tissue, or substantially the same number of cells should be compared for gene expression levels.

[0207] In some embodiments, the effective amount of the AAV particles in the composition is from about 1.0* 109vg to about l><1014vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is from about l.OxlO9vg to about IxlO13vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is from about l.OxlO9vg to about IxlO12vg of each the first AAV vector and the second AAV vector, per eye.

[0208] In some embodiments, the dose per eye of the first and second particle of a dual AAV particle composition, per particle, is about IxlO9to about IxlO14vector genomes (vg), about lxiolovg to about lxl014vg, about IxlO11vg to about lxl014vg, about lxl012vg to about lxl014vg, about lxl013vgto about lxl014vg, about lxl09vgto about lxl013vg, about IxlO9vg to about IxlO12vg, about 1 x 109vg to about 1 x 1011vg, about 1 x 109vg to about IxlO10vg, about lxl010vg to about lxl013vg, about 2.5 xl010vg to about lxl013vg, about 3.0xl010vg to about IxlO13vg, about 5xl010vg to about lxl013vg, about 7.5xlO10vg to about IxlO13vg, about IxlO11vg to about lxl013vg, about 2.5xlOnto about lxl013vg, about 5xlOnto about IxlO13vg, about 7.5xlOnvg to about IxlO13vg, about lxl010vg to about 7.5xl012vg, about 2.5xlO10vg to about 7.5xl012vg, about 3.0xl010vg to about 7.5xl012vg, about 5xl010vg to about 7.5 xl012vg, about 7.5 xlO10vg toabout7.5xl012vg, about lxl010vg to about lxl012vg, about 2.5xl010vg to about lxl012vg, about 3.0xl010vg to about lxl012vg, about 5xl010vg to about IxlO12vg, about 7.5xlO10vg to about lxl012vg. In yet another embodiment, the dose per eye of a first or second particle of a dual AAV particle composition, per particle, is about 5xl09vg to about 2.5xl012vg, about IxlO10vg to about 5xl012vg, about 2.5xlOlovg to about 5xl012vg, about 3.0xl010vg to about 5xl012vg, about 5xl010vg to about 5xl012vg, about 7.5xlO10vg to about 5xl012vg.

[0209] In some embodiments, the effective amount of the AAV particles in the composition is from about l.OxlO9vg to about 5xl012vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in thecomposition is from about l.Ox lO9vg to about l >< 1012vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is from about l.Ox lO9vg to about I x lO11vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is from about 1.Ox 109vg to about 1 x IO10vg of each the first AAV vector and the second AAV vector, per eye. In a preferred embodiment, the effective amount of the AAV particles in the composition is from about 5x l09vg to about 2.5 x lO12vg of each the first AAV vector and the second AAV vector, per eye.

[0210] The dual particle composition can be delivered as either a single or dual administrations, e.g., as a single injection or multiple injections. In some embodiments, the effective amount of the AAV particles in the composition is about I x lO9vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about 5x l09vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about I x lO10vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about 1 ,5x IO10vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about 5x lO10vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about I x lO11vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about 1.5xl0nvg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about 5x l0nvg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about I x lO12vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about 1 ,5x 1012vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about 2x l012vg of each the first AAV vector and the second AAV vector, per eye. In some embodiments, the effective amount of the AAV particles in the composition is about 2.5 x lO12vg of each the first AAV vector and the second AAV vector, per eye. In even anotherembodiment, the effective amount of the AAV particles in the composition is about 5* 1012vg of each the first AAV vector and the second AAV vector, per eye.

[0211] In some embodiments, the total vg delivered per eye by subretinal injection is from about I M O10total vg to about 5>< 1012total vg of the first and second AAV particles combined. The about 1 x 1010total vg comprises about 5 * 109vg of the first AAV particle and about 5 * 109vg of the second AAV particle. The about 5* 1012total vg comprises about 2.5* 1012vg of the first AAV particle and about 2.5* 1012vg of the second AAV particle.SEQUENCE TABLESEXAMPLES

[0212] The disclosure now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the disclosure.Example 1 - Production of full length ABCA4 protein in vivo

[0213] The dual AAV particle composition were tested in vivo and the reconstitution of full- length Abca4 protein in the retina was evaluated. The dual AAV8-REJ-Abca4 vectors tested comprises an N-terminal transgene as provided in Table 1 (INS1203-RD) and a C-terminal transgene as provided in Table 2 (INS1203-RA), respectively.

[0214] INS1203-RD and INS1203-RA were produced by triple transfection of HEK293 cells and subsequently purified by CsCl gradient ultracentrifugation. A combined titer of 2.42E+13 total vg / mL (1.24E+13 vg / mL INS1203-RD; 1.18E+13 vg / mL INS1203-RA) was determined by droplet digital PCR (ddPCR).

[0215] INS1203-RD and INS1203-RA were administered to Abca4 knockout mice (Abca^' , a Stargardt Disease mouse model) bilaterally via subretinal injection at a 1 : 1 ratio at a dose of 2E+10 vg total dose per eye (e.g., about 1E+10 vg of INS1203-RD and about 1E+10 vg of INS1203-RA). Two weeks post-injection, eyecups were harvested, and retinal sections of treated eyes were prepared for immunostaining.

[0216] Eye tissues were fixed and embedded in paraffin. Immunohistochemistry was performed to visualized Abca4 protein. All sections were counterstained with DAPI. Histology experiments were conducted according to established RNAScope and BaseScope protocols provided by Advanced Cell Diagnostics. Abca4 protein was expressed at high levels in the knockout mouse retina, confirming the efficiency of the REJ-approach (FIG. 4A). High magnification imaging of the outer nuclear layer (ONL) revealed that Abca4 protein expressionwas localized in the outer segments of photoreceptors, which is consistent with where Abca4 protein is found for WT animals and the intended biodistribution (FIG. 4A and 4B). These data demonstrate that INS 1203 is effective in restoring ABCA4 protein expression specifically to photoreceptors, and that ABCA4 protein generated by INS 1203 localizes to the subcellular compartment as found for WT animals.

[0217] In situ hybridization probes were designed to target either the N-terminal fragment (FIG. 5A), C-terminal fragment (FIG. 5B), or the junction of the two fragments (full-length mRNA, FIG. 5C). Individual fragment RNA as well as the joined junction between fragments can be specifically visualized with RNA in situ hybridization (RNA- / Base-scope). Localization of the in situ hybridization signal was specific to nuclei in the outer nuclear layer (where photoreceptor cell bodies are located). See FIGs. 5A to 5C. At the margins of the subretinal bleb, where coverage tapers off, cells positive for both N- and C-terminal RNA were typically also positive for the Abca4 protein, suggesting that Abca4 expression can be readily observed in cells that are double transduced. See FIGs. 5D to 5F. The dual AAV particle composition formulated as 1 : 1 mixture of the two individual vectors resulted in equivalent transduction of retinal photoreceptors with N- and C-terminal fragments respectively.

[0218] Whole eye sections were collected from slides directly adjacent to those used for histology. Tissue was lysed and DNA was extracted. Droplet Digital PCR (ddPCR) was performed to quantify the number of N-terminal fragment DNA (8002) or C-terminal fragment DNA (8003). Data was normalized to a reference gene and expressed as the number of vector genomes per diploid genome. Table 3 provides the ddPCR results.

[0219] Specific ddPCR to each construct showed a 1 : 1 ratio of genomic transduction in the photoreceptor cells and RT-ddPCR confirmed high expression and RNA-joining. REJ-junction specific in situ hybridization demonstrates widespread distribution within the target cells, underlining the efficiency of this approach.

[0220] A second cohort of Abca-T mice (p56, n=4) was dosed bilaterally with 1 pL of INS1203 (2.0E+10 vg total dose per eye) and scAAV9-CBA-GFP (2.5E+09 vg / eye) and analyzed 2 weeks post-inj ection. The goal of this study cohort was to demonstrate that INS 1203enabled retinal expression of full-length ABCA4 protein using a capillary Western immunoassay.

[0221] Two weeks post-injection, retinal lysates were prepared from transduced eyes and ABCA4 protein was analyzed and quantified relative to a wild-type reference material, diluted to 0.25X, 0.5X, or 1.0X concentration relative to lysate volume. Using this reference material, physiological levels of ABCA4 protein were approximated at the 1.0X dilution. Both N- terminal and C-terminal immunoassays were used to detect the presence of full-length ABCA4 protein as well as INS 1203 -RD and INS1203-RA protein fragments by using an N-terminal specific antibody and C-termina specific antibody. Full-length ABCA4 protein was observed at physiological or supraphy si ologi cal levels (relative to wild-type retinal lysates), with undetectable levels of either INS1203-RD or INS1203-RA protein fragment (FIGs. 6A-6F).

[0222] Abca4 is expected at ~230kDa whereas N- or C-terminal (un-joined fragments) are predicted in the ~140kDa range for N-, and ~100kDa range for C-terminal fragments. Abca4 protein expression was restored to physiological or even supraphy si ologi cal levels. See FIGS. 6A-6B and FIGS. 6D-6E. The full-length Abca4 protein (~230kDa) was specifically produced. See FIG. 6C and FIG. 6F. Un-joined RNA derived protein fragments (at ~140kDa for N-, and ~100kDa for C-terminus) were not observed (arrows in FIG. 6C and FIG. 6F).

[0223] INS 1203 achieved Abca4 expression at ~ 120-300% of the wild type level (FIG. 6B and FIG. 6E). In contrast, intein dual AAV approach achieved only -10-15% of the wild type level of Abca4 expression at the comparable dose. See Supplement Fig. S20 of Tomabene (Tornabene et al. (2019). Sci Transl Med. 11(492): doi: 10.1126 / scietranslmed.aav4523, “Tomabene”). INS 1203 -mediated Abca4 levels are more than lOx above the threshold for efficacy observed in Tomabene. REI’s higher efficiency allows for reduction in dose which improves the safety profile of a potential Stargardt therapeutic candidate.

[0224] Taken together, these results indicate that INS 1203 may successfully and efficiently restore Abca4 expression in a subject having Stargardt Disease, which may enable a curative gene replacement therapy to prevent or halt Stargardt Disease progression.Example 2 - Histology and replacement of physiological levels of Abca4.

[0225] Ten (10) Stargardt Disease mice (Abca4- / -) received subretinal injection of the two different AAV8-REI-Abca4 vectors as used in Example 1 at a 1 : 1 ratio (e.g., -1E+10 vg per vector). Table 4 provides the estimated targeting of each mouse.

[0226] Sagittal sections (whole eye) were dissected. 5 gm paraffin sections were collected in pairs. The sections were stained for Abca4 protein expression. FIG. 7A shows robust Abca4 protein expression in the eye of the animal VTU1437 with -90% targeting. FIG. 7B shows minimal Abca4 protein expression in the eye of the animal VTU1441 with -5% targeting. FIG. 7C shows moderate Abca4 protein expression in the eye of the animal VTU1443 with -50% targeting.

[0227] FIGs. 8A-8D show significant Abca4 protein expression in the eye of the animal VTU1433 with 90% targeting. FIG. 8B is a higher power image of FIG. 8A; and FIG. 8D is a higher power image of FIG. 8C.

[0228] FIG. 9A shows strong Abca4 protein expression in the eye of the Abca4- / - animal treated with a high dose of dual AAV8-REJ-Abca4 particles. FIG. 9B is a higher power image of FIG. 9A. FIG. 9C shows the staining of Abca4 expression in a wildtype mouse.Example 3 - INS1203 Functional Efficacy by A2E Reduction in the Abca4- / ~ Mouse

[0229] ABCA4 protein expressed in retinal photoreceptors functions to transport retinoids such as N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine from the luminal to the cytoplasmic side of photoreceptor disc membranes (Plotter E, McClements ME, MacLaren RE. Therapy Approaches for Stargardt Disease. Biomolecules. 2021 Aug 9; 11(8): 1179). When this process is disrupted in Stargardt disease due to an absence of ABCA4, a build-up of retinoids forms in the disc membrane, leading to the formation of bisretinoid fusion products, such as A2E. At a biochemical level, ABCA4 transports lipid bonded all-trans retinal (the light sensing pigment in the retina) from the photoreceptor disc lumen to the cytosolic side. Once in the cytosol, all-trans retinal is released and enters a recycling pathway (Molday et al. Biochim Biophys Acta. 2009 Jul;1791(7):573-83). In theabsence of ABCA4, this initial transport into the recycling cycle is defective. As a consequence, all-trans retinal accumulates in the disc lumen, which forms a reaction vessel where a lipid bonded all-trans retinal can react with an additional all-trans retinal to form A2E, which is essentially an all-trans retinal dimer (Liu et al. J Biol Chem. 2000 Sep 22;275(38):29354-60). A2E accumulates in the photoreceptor disks and is ultimately deposited in the RPE, accumulating as insoluble lipofuscin depositions (Plotter et al. Biomolecules. 2021 Aug 9; 11(8): 1179). Lipofuscin depositions in the RPE are composed of various chemical constituents that have been characterized chromatographically and structurally (Wu et al. J Biol Chem. 2009 Jul 24;284(30):20155-66).

[0230] A2E was the first characterized lipofuscin constituent of these components (Wu et al. J Biol Chem. 2009 Jul 24;284(30):20155-66). In support of the validity of A2E as a biochemical marker of disease progression, it was found that, as in the Abca4~'~ mouse retina, A2E levels are reported to be 6- to 12-fold higher in the retinae of Stargardt patients relative to that in undiseased control retinae (Mata et al. Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7154-9).

[0231] As reported in literature, exposure of cultured human RPE cells to physiological levels of A2E produced cellular disease phenotypes and disrupted cell morphology (Sparrow et al. Invest Ophthalmol Vis Sci. 1999 Nov;40(12):2988-95). Likewise, induced pluripotent stem cell (iPSC)-derived RPE cells exposed to A2E also demonstrated disease characteristics including cell death, demonstrating that A2E plays an important role in the pathogenesis of retinal degenerative diseases in humans (Parmar et al. Stem Cell Res. 2018 Mar; 27:95-104). Besides RPE and photoreceptor degeneration, another hallmark of Stargardt disease is RPE autofluorescence that accompanies lipofuscin accumulation.

[0232] A2E is the major orange-emitting fluorophore of RPE lipofuscin (Kennedy et al. Eye (Lond). 1995;9 ( Pt 6):763-71), which is associated with a strong emission peak at 570 nm when excited with 488 nm light (Sparrow et al. Invest Ophthalmol Vis Sci. 1999 Nov;40(12):2988-95). Taken together, a body of literature demonstrates that the loss-of- function of ABCA4 results in defective clearance of retinoids from the photoreceptor disc lumen, leading to accumulation of the bisretinoid A2E as lipofuscin depositions in the RPE, resulting in increased autofluorescence, RPE, and photoreceptor damage (Sparrow et al. Invest Ophthalmol Vis Sci. 1999 Nov;40(12):2988-95; Parmar et al. Stem Cell Res. 2018 Mar;27:95- 104; Plotter et al. Biomolecules. 2021 Aug 9; 11(8): 1179).

[0233] Quantification of A2E in the eyecup has been widely used as a biochemical marker of disease progression in the Abca4~'~ mouse model of Stargardt disease (Plotter et al. Biomolecules. 2021 Aug 9; 11(8): 1179). It is hypothesized that an intervention that shows efficacy for halting or preventing the accumulation of A2E is likely to result in the therapeutic effect of halting or preventing disease progression in the human disease context. Therefore, the prevention of A2E accumulation is widely accepted and used as the nonclinical efficacy endpoint in the Abca4~'~ mouse model and is particularly relevant for an ABCA4 gene replacement therapy, such as INS 1203.

[0234] This study was designed to test the hypothesis that subretinal delivery of INS 1203 resulting in successful DNA transduction in the retina (as measured by ddPCR), and leading to functional ABCA4 protein expression in photoreceptors (as measured by capillary Western immunoassay), can prevent A2E accumulation in treated eyecups (as measured by mass spectrometry), thus effectively correcting a key driver for disease phenotypes in the Abca4~ / ~ mouse.

[0235] Housing Requirements

[0236] All animals received an ear tag with a 5-digit ID number for tracking and all animal information will be stored in a local MS Access database. All animals were housed in groups of 3-5 in large cages kept in ventilated shelves under standard animal care conditions. A refuge, gnawing material, and nesting material was present in every cage. Newborn pups were housed with their mothers until P21.

[0237] Mice had access to irradiated food (Picolab rodent diet 20) and reverse osmosis (RO) water, ad libitum. The room was kept on a 12-hour light / 12-hour dark cycle. All animal room conditions were set according to the Guide for the Care and Use of Laboratory Animals (8th edition).

[0238] Animals were monitored daily for gross behavioral changes as a humane endpoint, including lack of activity, lack of grooming, and intolerability. Any animals demonstrating severe changes to due intolerability were referred for veterinary consultation and may be unenrolled from the study based upon recommendation by the attending veterinarian.

[0239] EXPERIMENTAL DESIGN

[0240] Abca4~'~ mice (p28) were treated with a single dose of either 2.0E+10 vg INS1203 (n=13) or vehicle control (0.001% w / v poloxamer 188 in IX PBS; n=12) via unilateral subretinal injection at 1 pL / eye. INS1203 comprised a 1 : 1 ratio of INS1203-RD and INS1203-RA vectors. Naive, untreated wild-type control animals (n=10) were also included in the study. Table 5. Approximately 10 weeks post-injection, eyecups were harvested, and retinal lysates were prepared for genomic DNA (gDNA) quantification by ddPCR, ABCA4 protein detection by capillary Western immunoassay, and A2E reduction by mass spectrometry.

[0241] For all INS 1203 -treated eyecups, INS1203-RD and INS1203-RA vector genome (vg) quantities were determined by ddPCR and normalized to the count of total diploid genomes (dg) in the sample. Two ABCA4 probe sets were designed to be specific to the codon-optimized human ABCA4 gene coding sequence for INS1203-RD and INS1203-RA, respectively.

[0242] Table 5 shows the study arms.

[0243] Test articles (i.e., vehicle or INS1203) were stored in 4°C upon receipt until the day of experiment. On the day of injection, test article was kept on ice for the duration of the procedure. Vehicle was 0.001% w / v poloxamer 188 in IX PBS.

[0244] Anesthesia

[0245] Ketamine and Xylazine are administered via intraperitoneal injection at 85 mg / kg and 14 mg / kg, respectively.

[0246] Subretinal administration

[0247] Mouse eyes were dilated, and the animals were anesthetized according to standard operating procedures. The mice were placed on a regulated heating pad, and the posterior pole was visualized under magnification. A 12.7mm 30-gauge insulin syringe was used to puncture the cornea just above the corneal limbus, avoiding any contact with the sclera and lens. The transvitreal subretinal injections were performed using a 10 pL Hamilton syringe with a 33- gauge blunt needle inserted through the corneal puncture across the vitreous, with the shaft aimed at the back of the eyecup, avoiding any trauma to the lens or iris. A total volume of 1 pLwas delivered. Following injection, a small amount of Neomycin, polymyxin B, and dexa. 3.5g (Bausch and Lomb) was applied.

[0248] A range of vg / dg was observed across treated eyecups (Table 6). These data support the notion that the technical variability in the accuracy of subretinal delivery across individual injected eyes would result in variability of vector genomes quantified across eyecups. This presents the opportunity to use INS 1203 gDNA quantitation to evaluate the efficiency of delivery for each individual eyecup.

[0249] The ratio of vector genome quantities of the INS 1203 -RD and INS1203-RA vectors within individual eyecups were compared. Within each individual eyecup, the two individual vectors were consistently present at a near equal ratio (Table 6: 1 : 1.138 ± 0.06046 ratio, Figure 10, R2=0.9989). These data establish a strong correlation between transduction of the REI vectors that comprise INS 1203, supporting that the 1 : 1 formulation of INS 1203 results in reasonably equivalent transduction of both viruses.

[0250] INS 1203 -treated eyecups were processed for ABCA4 protein quantification by less capillary Western immunoassay. ABCA4 protein expression was normalized to a referencemurine wild-type lysate (FIG. 11). Three samples had low or near undetectable levels of ABCA4 protein (< 10% of wild-type reference levels of ABCA4 protein), consistent with the expected surgical success rate for subretinal injections (Qi et al. PLoS One. 2015 Aug 28; 10(8):e0136523). All remaining samples had between 15.0% and 51.3% of wild-type levels of ABCA4 protein expression. Of note, the samples that had undetectable or near undetectable quantification of INS1203 vector genomes (Sample # 9: 0.57 and 0.56 for INS1203-RD and INS1203-RA, respectively; Sample # 10: 0.00 and 0.00 for INS1203-RD and INS1203-RA, respectively) also had near undetectable levels of ABCA4 protein (FIG. 11).

[0251] Retinal lysates were then analyzed for A2E quantification by mass spectrometry. The eyecups of vehicle-treated Abca4~'~ animals showed significant accumulation of A2E at 14 weeks of age when compared to age-matched WT controls, demonstrating the expected disease progression (vehicle-treated Abca4~'~ A2E: 868.2 ± 34.64 ng / mL; wild-type A2E: 60.2 ± 5.598 ng / mL, p < 0.0001). INS 1203 -treated Abca4~'~ eyecups showed a significant (p < 0.001) 30.7% reduction of A2E levels compared with vehicle-treated controls (INS1203, 2.0E+10 vg dose: 601.6 ± 67.7 ng / mL) (FIG. 12). These data demonstrate that INS1203 effectively reduces A2E accumulation in the Abca4~'~ mouse model.

[0252] An analysis comparing INS1203-RD and INS1203-RA gDNA levels with A2E concentration was conducted to understand the relationship between dual vector transduction efficiency and efficacy. The relationship between gDNA quantity and A2E concentration was assessed across all INS 1203 -treated eyecups. A negative association was observed between INS1203-RD gDNA and A2E concentration, (R2= 0.854), and between INS1203-RA gDNA and A2E concentration (R2= 0.854). An increase in viral transduction of the retina is therefore associated with increased efficacy as measured by A2E reduction.

[0253] Results from this study concluded that subretinal delivery of INS 1203 in Abca4~ / ~ mice resulted in a significant reduction of the Stargardt disease biochemical marker A2E, and that INS 1203 vector genome concentration levels appear to coincide with a reduction in A2E levels.Example 4 - Dose Ranging Study for Demonstration of Efficacy of INS1203 by A2E Reduction

[0254] A study was conducted in p28 Abca4~'~ mice using INS1203-RA and INS1203-RD research grade vectors. This study aimed to test the efficacy of four dose levels of INS 1203 in correcting the disease phenotype of the Abca4~ / ~ mouse model for Stargardt disease.

[0255] Abca4 mice (p28) were administered INS1203 or vehicle control (0.001% poloxamer 188 in IX PBS) with a single subretinal injection at a total dose of 2.0E+10 vg, 6.0E+09 vg, 2.0E+09 vg, or 6.0E+08 vg (total dose includes an equal vg contribution of INS1203-RD and INS1203-RA diluted in 0.001% poloxamer 188 in IX PBS). Approximately 10 weeks postinjection, eyecups were harvested, and prepared for gDNA quantification by droplet digital PCR (ddPCR), ABCA4 protein detection by capillary Western immunoassay, and A2E reduction by mass spectrometry.

[0256] To quantify INS1203 gDNA for vector biodistribution, the number of viral vector genomes (vg) for both INS 1203 -RD and INS1203-RA was determined by ddPCR for all INS1203 injected eyecups. These vector genome counts are expressed normalized relative to the count of total diploid genomes (dg) in the same sample. A dose-dependent increase in vector genomes was observed for both INS1203-RD (FIG. 13A) and INS1203-RA (FIG. 13B). Abca4~ / _mice injected with INS1203 at a dose of 2.0E+10 vg had 40.55 vg / dg and 33.65 vg / dg for INS1203-RD and INS1203-RA, respectively. At a dose of 6.0E+09 vg, the tissue contained 16.11 vg / dg and 13.56 vg / dg; at a dose of 2.0E+09 vg, the tissue contained 8.98 vg / dg and 7.49 vg / dg, and at a dose of 6.0E+08 vg, the tissue contained 2.73 vg / dg and 2.30 vg / dg, respectively. Data are summarized in Table 7.

[0257] Results demonstrated that the INS 1203 -RD and INS1203-RA were seen at approximately equal levels within the tissue (Table 7, FIG. 14) which is in line with expectations based on the 1 :1 mixture composition of the INS 1203 vector used for dosing.

[0258] The eyecups of wild-type, vehicle-treated Abca-l-and INS 1203 -treated Abca4- / ~ animals were processed for A2E quantification by mass spectrometry (FIG. 15). Data is presented as nanogram (ng) of A2E per mL extract, an arbitrary unit that is expressed in relation to a standard curve prepared in a wild type eyecup reference matrix. The eyecups of vehicle- treated Abca4- / - animals show a significant accumulation of A2E at 14 weeks of age when compared to age-matched WT controls, demonstrating the expected disease progression. WT mice, which have a functional Abca4 gene, exhibited substantially lower A2E levels compared to Abca4~ / ~ mice. WT samples resulted in 315.6 ± 40.77 ng / mL A2E. In contrast, more than fivefold higher A2E levels were observed in the vehicle treated Abca4~ / ~ mice (1,656.0 ± 143.0 ng / mL) (FIG. 15).

[0259] Results showed that a subretinal delivery of INS 1203 results in a dose-dependent reduction of total A2E in the eyecup, with 1,538.0 ± 209.1 ng / mL A2E in the lowest dose (6.0E+08 vg / eye), 1,513.0 ± 175.6 ng / mL A2E in the next higher dose (2.0E+09 vg / eye), 1,334.0 ± 227.9 ng / mL A2E in the second to highest (6.0E+09 vg / eye), and 1,099.0 ± 187.7 ng / mL A2E in the highest dose tested (2.0E+10 vg / eye).

[0260] The lower doses (6.0E+08 vg / eye; 8.8% reduction compared to vehicle) and 2.0E+09 vg / eye; 10.7% reduction) showed a modest decrease in A2E that did not reach statisticalsignificance, while higher doses (6.0E+09 vg / eye: 24.0% reduction, p<0.01; and 2.0E+10 vg / eye: 41.6% reduction, p<0.0001) led to more pronounced reductions that were statistically significant. The most significant decrease in A2E was observed with the highest dose, which reduced A2E levels of 41.6% compared to untreated Abca-T mice. Results are also summarized in Table 8.

[0261] Collectively, these data demonstrate a dose-dependent increase in viral transduction of INS1203-RA and INS 1203 -RD, and a dose-dependent decrease in A2E, a marker for Stargardt Disease progression. Efficacy of INS 1203 was also demonstrated at multiple dose levels (2.0E+10 vg and 6.0E+09 vg), and a sub-efficacious dose was identified (2.0E+09 vg).

[0262] To contextualize the treatment effect size, it is important to note that the overall reduction in A2E levels measured in the entire eyecup sample does not fully reflect the local treatment efficacy within the retinal areas that were successfully treated with INS 1203. Subretinal injections typically result in a partial coverage of the retinal surface. Therefore, the observed reduction in A2E levels in the INS 1203 -treated retina is likely underestimated due to the dilution of the treatment effect across the entire eyecup (i.e., mixing of treated and untreatedareas of the retina). With this limitation, the data suggest that INS 1203 has the potential to effectively and substantially reduce A2E levels in all transduced cells of the retina.Example 5 - INS1203 ABCA4 mRNA Localization and Expression in the Non-Human Primate

[0263] The subretinal route of administration in the non-human primate (NHP) (both to the subfoveal retina as well as the extra-foveal retina) was chosen to align with the intended clinical route of administration. A cone photoreceptor-dense structure in the center of the macula, called the fovea, is a human- and NHP-specific structure responsible for heightened visual acuity (Bringmann et al., Prog Retin Eye Res. 2018 Sep;66:49-84; Zhang et al. Am J Ophthalmol. 2015 Aug;160(2):290-300.el). Retinal development in the NHP is complete by 6 months of age and human retinal development is complete by 1 year of age (Hendrickson. Eye (Lond). 1992;6 ( Pt 2): 136-44). Therefore, NHPs selected for INS1203 nonclinical studies are 6 months of age or older to represent a fully developed human eye.

[0264] Target tissue transduction of INS 1203 and Abca4 transgene expression in an anatomically representative primate model was assessed. A female non-human primate (NHP) (African green monkey; Chlorocebus sabaeus, 4 years old) was administered a single subretinal injection of INS1203. Eight weeks post-injection, ocular and non-ocular tissues were collected and evaluated for histology, immunohistochemistry, vector genome biodistribution, and RNA expression.

[0265] INS 1203 test article was prepared by triple transfection of HEK293 cells and subsequently purified by CsCl gradient ultracentrifugation. A combined titer of 2.42E+13 total vg / mL (1.24E+13 vg / mL INS1203-RD; 1.18E+13 vg / mL INS1203-RA) was determined by droplet digital PCR (ddPCR).

[0266] The study animal received a single subretinal injection into both eyes at the time of surgery. A dose of either 1.0E+12 vg / eye (high dose, left eye) or 3.0E+11 vg / eye (low dose, right eye) was delivered by subretinal injection with foveal placement. Vitrectomy ports were placed at the level of the ora serrata prior to the subretinal injection. The first was placed in the superotemporal quadrant and the second in the inferotemporal quadrant. Intraocular illumination and placement of a lens on the cornea were used to visually guide a cannula through the second vitrectomy port. 100 pL of test article was administered subretinally with foveal placement. Injection volume was controlled with a microfluid injector. Bleb formation was visually confirmed at the time of the procedure. FIGS. 16A and 16B. Systemic steroidtreatment (methylprednisolone, 8 mg / kg) was administered beginning the day prior to test article administration, and then weekly thereafter for 6 weeks. Ocular exams were performed weekly, and ophthalmic imaging (optical coherence tomography [OCT] and confocal scanning laser ophthalmoscope [cSLO]) was performed at baseline as well as monthly post-injection. Necropsy and tissue collection were performed 8 weeks (day 56 ± 1) post-injection. Table 9 provides the study schedule. Preliminary tolerability of the test article was demonstrated with OCT imaging of the treated NHP at baseline (FIG. 17 A) and 4-weeks post-injection (FIG. 17B), which showed no notable observations other than those expected with the subretinal injection procedure.

[0267] Slit Lamp Exam: At designated time points (Table 9) both eyes, oculus uterque (OU), will be examined by slit lamp biomicroscopy and retinoscopy using a 90-diopter lens. Scoring will be applied to qualitative clinical ophthalmic findings using a nonhuman primate ophthalmic scoring system and summary score derived from exam components.

[0268] Tonometry: At designated time points (Table 9) intraocular pressure (IOP) will be measured OU using a TonoVet Plus tonometer set to the dog (d) calibration setting. Three measures will be taken from each eye and the mean IOP calculated.

[0269] Fundus Photography: At designated time points (Table 9) color anterior segment and fundus photography will be performed OU with a 50° field of view centered on the macula using a Topcon TRC-50EX retinal camera with Canon 6D digital imaging hardware and New Vision Fundus Image Analysis System software.

[0270] Confocal Scanning Laser Ophthalmoscopy (cSLO): At designated time points (Table 9), infrared (IR) and autofluorescence (AF) cSLO images will be captured OU using a Heidelberg Spectralis OCT HRA (or OCT Plus) with a 55° lens, employing the Heyex TruTrack and AutoRescan follow-up imaging function referenced to the baseline images.

[0271] Optical Coherence Tomography (OCT): An OCT volume scan of the entire macula will be obtained at a dense scan interval. Image maps will be qualitatively evaluated, and inner retinal thickness will be quantified, if deemed necessary following qualitative evaluation.

[0272] Physical Exam and Physiology: At designated time points (Table 9) the head, torso, limbs, and integument of each monkey will be evaluated, auscultation performed, and vitals assessed, including heart and respiratory rate measured manually over a 15 second interval. Body temperature will be determined using a digital rectal thermometer.

[0273] Clinical Observations: General wellbeing will be assessed twice daily by cage side observation.

[0274] Body Weights: Body weights will be collected at ophthalmic exam intervals (Table 9).

[0275] Clinical Pathology: At designated time points (Table 9) 7 mL blood will be collected for clinical pathology:

[0276] Complete blood count: 1 mL blood will be transferred to K2EDTA lavender top vacutainers, gently inverted several times and retained on ice until CBC with differential analysis on an Abaxis VetScan HM5 hematology system. Clinical chemistry: 3 mL blood will be collected and transferred to green top vacutainers (containing lithium heparin) inverted 3-5times and maintained refrigerated until clinical chemistry analysis on an Abaxis VetScan VS2 clinical chemistry system.

[0277] Neutralizing Antibodies (NAbs) Serum: Whole blood (3 mL) will be collected via the femoral or saphenous vein at designated time points (Table 9). Blood will be transferred to vacutainer tubes (in the absence of anticoagulant) and incubated at room temperature for approximately 1 hour before centrifugation 4000 rpm for 10 min at 4 °C and isolation of serum aliquots (~0.5 mL x 2 aliquots per time point). Individual aliquots will be stored separately and shipped below -70 °C to a Sponsor designated laboratory for NAb analysis.

[0278] Aqueous and Vitreous Humor Collection: At designated time points (Table 9) topical proparacaine 0.5% will be administered, allowing at least 30 seconds to take effect, an eye speculum placed, then the ocular surface rinsed with 5% Betadine solution followed by a sterile 0.9% saline rinse. Aqueous humor (50 pL) will be sampled OU with a 0.3 mL insulin syringe with a 31 -gauge needle advance into the anterior chamber -2 mm anterior to the temporal limbus. Vitreous humor (100 pL) will be sampled with a 0.5 mL insulin syringe with a 28- gauge needle advance into the vitreous chamber -2 mm posterior to the temporal limbus at the level of the ora serrata. Aqueous and vitreous aliquots will be transferred to pre-labeled cryotubes and stored and shipped below -70 °C to the Sponsor, or a Sponsor designated laboratory’ for analysis.

[0279] Necropsy: At study terminus (Table 9) monkeys will be sedated with ketamine (8 mg / kg) and xylazine (1.6 mg / kg) IM and euthanized with sodium pentobarbital (100 mg / kg IV) to effect and exsanguinated by severing the femoral vessels. A necropsy will be performed and gross lesions and indicated tissues (Table 9) collected, trimmed and flash frozen for vector genome (qPCR) or transgene expression (RT-PCR), or fixed in 10% neutral buffered formalin (NBF), for hematoxylin and eosin (H&E) and immunohistochemistry (IHC) analysis, guided by other findings.

[0280] Eye Collection: Aqueous humor (-100 pL) will be collected OU with a 0.3 mL insulin syringe with a 31-gauge needle, and vitreous (-100 mL) will be collected OU with a 28-gauge needle, and samples flash frozen and stored below -70 °C. Eyes will be enucleated with connected optic nerve. Excess orbital tissue will be trimmed, extraocular muscle collected (Table 9), and optic nerve collected, leaving portion attached to globe, and the vitreous chamber injected with 200 mL 4% paraformaldehyde and the globe transferred to 4% paraformaldehyde for 18-24 hours and then stored and shipped refrigerated in phosphate buffered saline (PBS)with 0.05% sodium azide for sectioning and H&E staining. Stepped sections, including fovea for central region, will be generated for H&E, and adjacent duplicate sections generated for IHC for shipment to the sponsor.

[0281] Eight (8) weeks post-injection, the following tissues were processed for analysis: eyes, optic nerves, extraocular muscles, heart, liver, brain (frontal cortex, lateral geniculate nucleus, and occipital cortex), ovaries, spleen, mandibular lymph nodes, cervical spinal cord, cervical dorsal root ganglion, and gross lesions.

[0282] Sections for each tissue level within the eye (optic disk, nasal macula, fovea, and temporal macula) were prepared. H&E-stained slides of the macula were evaluated by an independent histopathologist. No overt changes to the retinal layers were observed, and appearance of the retina was consistent with subretinal injection procedure, indicating that INS 1203 to the macula was generally well-tolerated. An RNA probe spanning the junction of the donor and acceptor REJ domains of the human INS 1203 ABCA4 gene was designed for species-specific detection of INS 1203 mRNA by fluorescent in situ hybridization (FISH). Positive and negative control slides were included to confirm probe specificity. For histological comparison, retinal sections from a naive African green monkey were obtained from the testing laboratory to serve as the control.

[0283] At 8 weeks post treatment, human ABCA4 mRNA expression was observed in the photoreceptors in the outer nuclear layer (ONL) of both INS 1203 -treated eyes: the high dose of INS1203 in the left eye (OS) shown in FIG. 18A; the low dose of INS1203 in the right eye (OD) shown in FIG. 18B. No expression was observed in the photoreceptors of the naive untreated NHP retina (FIG. 18C). These data also demonstrate that vector manufactured using the REJ technology effectively delivers full-length ABCA4 mRNA to the primate retina, and that the hGRKl promoter confers cell type specificity for ABCA4 expression in photoreceptors.

[0284] To evaluate the extent of coverage within the NHP retina, a set of sections was selected to represent each tissue level (optic disk, nasal macula, fovea, and temporal macula) and processed for FISH as described above for human ABCA4 mRNA expression. FIG. 19A (high dose) and FIG. 19B (low dose) show sections representing the fovea. Retinal coverage was quantified by measuring the total retinal distance (in mm) as well as the retinal distance with positive ABCA4 expression. These values are represented as a percent (ABCA4+ retinal distance / total retinal distance).

[0285] Positive expression was observed across all sections included in the analysis. In the high dose (1.0E+12 vg INS 1203: 5E+11 vg INS 1203 -RD and 5E+11 vg INS 1203 -RA) treated eye, a range of retinal coverage between 36% and 45% was observed. In the low dose (3.0E+11 vg INS 1203: 1.5E+11 vg INS 1203 -RD and 1.5E+11 vg INS1203-RA) treated eye, a range of retinal coverage between 15% and 42% was observed.

[0286] Quantification of gDNA was conducted by ddPCR with two ABCA4 probe sets designed to be specific to the codon-optimized gene coding sequences of each of the INS 1203 complement vectors. INS 1203 gDNA was detected in a dose-dependent manner in the retina for both the high (43.06 and 39.28 vg / dg for INS1203-RD and INS1203-RA, respectively) and low dose treated eyes (24.25 and 22.30 vg / dg for INS1203-RD and INS1203-RA, respectively). Importantly, this demonstrates that the 1 : 1 vector genome titer ratio of INS1203 vectors results in equivalent levels of transduction in the treated tissue. INS 1203 was also detected at low levels (0.01 to 1.61 vg / dg) in the optic nerves and the extraocular muscles of both eyes. Given the ocular immune privilege afforded by the blood-ocular barrier, the proportional vector genome counts in the retina and extraocular tissues were consistent with the expected biodistribution, as well as previous reports in which subretinal injection of AAV8 was used at similar dose levels (Ferla et al. Mol Ther Methods Clin Dev. 2023 Feb 11 ;28:396-411). INS 1203 biodistribution in systemic tissues was minimal; average INS 1203 gDNA quantities were greatest in the spleen (0.08 vg / dg for INS1203-RA and INS1203-RD). All other systemic tissues were below this level and in most instances were undetected (Table 10).

[0287] Vector biodistribution of aqueous and vitreous humor was also evaluated by ddPCR both at baseline and following necropsy (Table 11). No gDNA was detected at baseline across the four samples tested. At the time of necropsy, both INS 1203 -RD and INS1203-RA were detected in aqueous and vitreous humor.

[0288] Tissues that were positive for vector genomes were then quantified for INS 1203 mRNA by reverse transcription (RT) ddPCR with two ABCA4 RNA probe sets designed to target the 5’ coding sequence of INS1203-RD and the 3’ coding sequence of INS1203-RA. For RNA extracted from tissue, RNA ddPCR is expressed as copies / pg RNA. For RNA extracted from sections, RNA ddPCR is expressed as copies / pL of lysate. Full-length (i.e., joined) INS1203 mRNA was detected in the retina for both the high and low dose treated eyes (1.6E+03 and 1.3E+03 copies / pL lysate, respectively). INS1203-RD mRNA was detected in the retina at 1.3E+04 and 1.1E+04 copies / pL lysate in the high and low doses, respectively. INS1203-RA mRNA was detected in the retina at 7.8E+03 and 8.1E+03 copies / pL lysate in the high and low doses, respectively.

[0289] Individual vector mRNA was detected at low levels, and full-length INS1203 mRNA was undetected in the analyzed systemic tissues. Individual vector mRNA as well as full-length (i.e., joined) INS1203 mRNA were detected in the extraocular tissues of both high and low dose treated eyes (Table 12). INS 1203 mRNA was undetectable in the aqueous and vitreous humor, both at baseline and at the time of necropsy.

[0290] Results from this study conclude that a single subretinal delivery of INS 1203 at a dose of 1.0E+12 vg and 3.0E+11 vg was generally well -tolerated, with observed findings consistent of those expected following a subretinal injection and no overt changes to the retinal structure (FIG. 13, FIG. 14). INS 1203 administration resulted in ABCA4 mRNA expression specifically in photoreceptor cells, with extensive coverage of the desired treatment area within the fovea. Vector biodistribution and subsequent ABCA4 mRNA expression were observed in ocular tissues, with low mRNA expression in extraocular tissues. No mRNA detection was seen in the analyzed systemic tissues (e.g., spleen, liver). These data also demonstrate that INS 1203 effectively delivers full-length ABCA4 mRNA in a cell type-specific manner to photoreceptors of the NHP.INCORPORATION BY REFERENCE

[0291] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.EQUIVALENTS

[0292] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the disclosure described herein. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMSWhat is claimed is:

1. A dual adeno-associated virus (AAV) particle composition comprising:(a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises from 5’ to 3’ :(i) a first 5 ’-inverted terminal repeat (5’-ITR) sequence;(ii) a first promoter sequence;(iii) a Kozak sequence;(iv) an N-terminal coding sequence encoding an N-terminal portion of a target protein, said N-terminal coding sequence being operably linked to and under control of said first promoter;(v) a nucleic acid sequence of a splicing donor;(vi) a nucleic acid sequence of a first dimerization domain;(vii) a first poly(A) signal sequence; and(viii) a first 3’-ITR sequence; and(b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’ :(i) a second 5’ -ITR sequence;(ii) a second promoter sequence;(iii) a nucleic acid sequence of a second dimerization domain, said second dimerization domain being operably linked to and under control of said second promoter;(iv) a nucleic acid sequence of a splicing acceptor;(v) a C-terminal coding sequence encoding a C-terminal portion of the target protein;(vi) a second poly(A) signal sequence; and(vii) a second 3 ’-ITR sequence; wherein the first dimerization domain comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 9; and wherein the second dimerization domain comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:

162. A dual adeno-associated virus (AAV) particle composition comprising:(a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises from 5’ to 3’ :(i) a first 5 ’-inverted terminal repeat (5’-ITR) sequence;(ii) a first promoter sequence;(iii) a Kozak sequence;(iv) an N-terminal coding sequence encoding an N-terminal portion of a target protein, said N-terminal coding sequence being operably linked to and under control of said first promoter;(v) a nucleic acid sequence of a splicing donor;(vi) a nucleic acid sequence of a first dimerization domain;(vii) a first poly(A) signal sequence; and(viii) a first 3 ’-inverted terminal repeat (3’-ITR) sequence; and(b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’:(i) a second 5’ -ITR sequence;(ii) a second promoter sequence;(iii) a nucleic acid sequence of a second dimerization domain, said second dimerization domain being operably linked to and under control of said second promoter;(iv) a nucleic acid sequence of a splicing acceptor;(v) a C-terminal coding sequence encoding a C-terminal portion of the target protein;(vi) a second poly(A) signal sequence; and(vii) a second 3 ’-ITR sequence; wherein the N-terminal coding sequence comprises a first intervening intron sequence within the N-terminal coding sequence; and wherein the C-terminal coding sequence comprises a second intervening intron sequence within the C-terminal coding sequence.

3. A dual adeno-associated virus (AAV) particle composition comprising:(a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises from 5’ to 3’ :(i) a first 5’ -ITR sequence;(ii) a first promoter sequence;(iii) a Kozak sequence;(iv) an N-terminal coding sequence encoding an N-terminal portion of an Abca4 protein, said N-terminal coding sequence being operably linked to and under control of said first promoter;(v) a nucleic acid sequence of a splicing donor;(vi) a nucleic acid sequence of a first dimerization domain;(vii) a first poly(A) signal sequence; and(viii) a first 3 ’-ITR sequence; and(b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’ :(i) a second 5 ’-ITR sequence;(ii) a second promoter sequence;(iii) a nucleic acid sequence of a second dimerization domain, said second dimerization domain being operably linked to and under control of said second promoter;(iv) a nucleic acid sequence of a splicing acceptor;(v) a C-terminal coding sequence encoding a C-terminal portion of the Abca4 protein;(vi) a second poly(A) signal sequence; and(vii) a second 3 ’-ITR sequence; wherein the N-terminal coding sequence comprises a nucleic acid sequence that is at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 7; and wherein the C-terminal coding sequence comprises a nucleic acid sequence that is at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 20.

4. A dual adeno-associated virus (AAV) particle composition comprising:(a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises from 5’ to 3’ :(i) a first 5’-ITR sequence;(ii) a first promoter sequence;(iii) a Kozak sequence;(iv) an N-terminal coding sequence encoding an N-terminal portion of an Abca4 protein, said N-terminal coding sequence being operably linked to and under control of said first promoter;(v) a nucleic acid sequence of a splicing donor;(vi) a nucleic acid sequence of a first dimerization domain;(vii) a first poly(A) signal sequence; and(viii) a first 3’-ITR sequence; and(b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’ :(i) a second 5’ -ITR sequence;(ii) a second promoter sequence;(iii) a nucleic acid sequence of a second dimerization domain, said second dimerization domain being operably linked to and under control of said second promoter;(iv) a nucleic acid sequence of a splicing acceptor;(v) a C-terminal coding sequence encoding a C-terminal portion of the Abca4 protein;(vi) a second poly(A) signal sequence; and(vii) a second 3 ’-ITR sequence; wherein a first transcript of the first transgene and a second transcript of the second transgene can be spliced and joined to form a full-length coding sequence that encodes the Abca4 protein; and wherein the full-length Abca4 coding sequence comprises a nucleic acid sequence that is at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 22.

5. The composition of claim 1 or 2, wherein the target protein is an Abca4 protein.

6. The composition of claim 1 or 2, wherein the N-terminal coding sequence is codon optimized.

7. The composition of any one of claims 1, 2, 5, and 6, wherein the N-terminal coding sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 7.

8. The composition of any one of claims 1, 2, and 5-7, wherein the C-terminal coding sequence is codon optimized.

9. The composition of any one of claims 1, 2, and 5-8, wherein the C-terminal coding sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 20.

10. The composition of any one of claims 1-3 and 5-9, wherein a first transcript of the first transgene and a second transcript of the second transgene can be spliced and joined to form a full-length coding sequence that encodes an Abca4 protein.

11. The composition of claim 10, wherein the full-length Abca4 coding sequence is codon optimized.

12. The composition of claim 10 or 11, wherein the full-length Abca4 coding sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 22.

13. The composition of any one of claims 1 and 5-12, wherein the N-terminal coding sequence comprises a first intervening intron sequence within the N-terminal coding sequence.

14. The composition of claim 2 or 13, wherein the first intervening intron sequence is a modified mouse beta-actin (Actb) intron 2 sequence.

15. The composition of claim 14, wherein the first intervening intron sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 5.

16. The composition of any one of claims 1 and 5-15, wherein the C-terminal coding sequence comprises a second intervening intron sequence within the C-terminal coding sequence.

17. The composition of claim 2 or 16, wherein the second intervening intron sequence is a modified mouse beta-actin (Actb) intron 2 sequence.

18. The composition of claim 17, wherein the second intervening intron sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 5.

19. The composition of any one of the preceding claims, wherein the splicing donor comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 8.

20. The composition of any one of the preceding claims, wherein the splicing acceptor comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 17.

21. The composition of any one of the preceding claims, wherein the first dimerization domain and the second dimerization domain comprise sequences that are complementary to each other.

22. The composition of claim 21, wherein the first dimerization domain and the second dimerization domain contain complementary RNA stem loops.

23. The composition of claim 22, wherein the complementary RNA stem loops form a central kissing loop interaction with four or more loop base pairs for intermolecular pairing.

24. The composition of claim 23, wherein the positioning of the complementary RNA stem loops is offset by at least 1 nt, at least 2nt, at least 3 nt, at least 4nt, at least 5nt, at least 6 nt, at least 7nt, at least 8 nt, at least 9nt, or at least lOnt so that the respective stem regions of the complementary RNA stem loops base pair in trans through strand invasion.

25. The composition of claim 24, wherein the stem regions of the complementary RNA stem loops contain about 1% to about 30% mismatches, but match to the other dimerization domain.

26. The composition of claim 24 or 25, wherein the first dimerization domain and the second dimerization domain form an extended duplex through strand invasion.I l l27. The composition of any one of claims 2-26, wherein the first dimerization domain comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 9.

28. The composition of any one of claims 2-27, wherein the second dimerization domain comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 16.

29. The composition of any one of the preceding claims, wherein the first promoter is an ocular tissue-specific promoter.

30. The composition of any one of the preceding claims, wherein the first promoter is a human rhodopsin kinase (hGRKl) promoter.

31. The composition of claim 30, wherein the first promoter comprises the nucleic acid sequence of SEQ ID NO: 2.

32. The composition of any one of the preceding claims, wherein the second promoter is an ocular tissue-specific promoter.

33. The composition of any one of the preceding claims, wherein the second promoter is a human rhodopsin kinase (hGRKl) promoter.

34. The composition of claim 33, wherein the second promoter comprises the nucleic acid sequence of SEQ ID NO: 2.

35. The composition of any one of the preceding claims, wherein the first 5’-ITR is a 5’ AAV2 ITR.

36. The composition of claim 35, wherein the first 5’-ITR comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 1.

37. The composition of any one of the preceding claims, wherein the first 3’-ITR is a 3’ AAV2 ITR.

38. The composition of claim 37, wherein the first 3’-ITR comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 1139. The composition of any one of the preceding claims, wherein the second 5’-ITR is a 5’ AAV2 ITR.

40. The composition of any one of claim 39, wherein the second 5’-ITR comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 1.

41. The composition of any one of the preceding claims, wherein the second 3 ’-ITR is a 3’ AAV2 ITR.

42. The composition of claim 41, wherein the second 3 ’-ITR comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 1143. The composition of any one of the preceding claims, wherein the first poly(A) signal sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 10.

44. The composition of any one of the preceding claims, wherein the second poly(A) signal sequence comprises a late SV40 poly(A) signal sequence.

45. The composition of claim 44, wherein the second poly(A) signal sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO:21.

46. The composition of any one of the preceding claims, wherein the Kozak sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to a sequence of SEQ ID NO:3, 23, 24, or 25.

47. A dual adeno-associated virus (AAV) particle composition comprising:(a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises from 5’ to 3’:(i) a 5 ’-inverted terminal repeat (5’-ITR) sequence of SEQ ID NO: 1;(ii) a promoter sequence of SEQ ID NO: 2;(iii) a Kozak sequence of SEQ ID NO: 3;(iv) an N-terminal coding sequence of SEQ ID NO: 7 encoding an N- terminal portion of an Abca4 protein, said N-terminal coding sequence being operably linked to and under control of said promoter;(v) a nucleic acid sequence of a splicing donor comprising the sequence of SEQ ID NO: 8;(vi) a nucleic acid sequence of a dimerization domain comprising the sequence of SEQ ID NO: 9;(vii) a poly(A) signal sequence of SEQ ID NO: 10; and (viii) a 3’-ITR sequence of SEQ ID NO: 11; and(b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’:(i) a 5’-ITR sequence of SEQ ID NO: 1;(ii) a promoter sequence of SEQ ID NO: 2;(iii) a nucleic acid sequence of a dimerization domain comprising a sequence of SEQ ID NO: 16, said dimerization domain being operably linked to and under control of said promoter;(iv) a nucleic acid sequence of a splicing acceptor comprising a sequence of SEQ ID NO: 17;(v) a C-terminal coding sequence of SEQ ID NO: 20 encoding a C- terminal portion of an Abca4 protein;(vi) a poly(A) signal sequence of SEQ ID NO: 21; and(vii) a 3’-ITR sequence of SEQ ID NO: 11.

48. The composition of any one of the preceding claims, wherein the AAV capsid of the first AAV particle comprises one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.8, AAVrh.10, AAVrh.39, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 capsid proteins.

49. The composition of claim 48, wherein the AAV particle is an AAV8 particle and the AAV capsid comprises the one or more AAV8 capsid proteins.

50. The composition of claim 49, wherein the one or more AAV8 capsid proteins comprise AAV8 capsid protein VP1.

51. The composition of claim 49 or 50, wherein the one or more AAV8 capsid proteins comprise AAV8 capsid protein VP2.

52. The composition of any one of claims 49-51, wherein the one or more AAV8 capsid proteins comprise AAV8 capsid protein VP3.

53. The composition of claim 48, wherein the AAV capsid of the first AAV particle comprises one or more engineered variants of the AAV8 capsid protein.

54. The composition of claim 53, wherein the one or more engineered variants of the AAV8 capsid protein comprises an engineered variant of AAV8 capsid protein VP1.

55. The composition of claim 53 or 54, wherein the one or more engineered variants of the AAV8 capsid protein comprises an engineered variant of AAV8 capsid protein VP2.

56. The composition of any one of claims 53-55, wherein the one or more engineered variants of the AAV8 capsid protein comprises an engineered variant of AAV8 capsid protein VP3.

57. The composition of any one of claims 53-56, wherein the one or more engineered variants of an AAV8 capsid protein are one or more deimmunized variants of an AAV8 capsid protein.

58. The composition of any one of the preceding claims, wherein the AAV capsid of the second AAV particle comprises one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.8, AAVrh.10, AAVrh.39, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 capsid proteins.

59. The composition of claim 58, wherein the second AAV particle is an AAV8 particle and the AAV capsid of the second AAV particle comprises the one or more AAV8 capsid proteins.

60. The composition of claim 59, wherein the one or more AAV8 capsid proteins comprises AAV8 capsid protein VP1.

61. The composition of claim 59 or 60, wherein the one or more AAV8 capsid proteins comprises AAV8 capsid protein VP2.

62. The composition of any one of claims 59-61, wherein the one or more AAV8 capsid proteins comprise AAV8 capsid protein VP3.

63. The composition of claim 58, wherein the AAV capsid of the second AAV particle comprises one or more engineered variants of an AAV8 capsid protein.

64. The composition of claim 63, wherein the one or more engineered variants of the AAV8 capsid protein comprises an engineered variant of AAV8 capsid protein VP1.

65. The composition of claim 63 or 64, wherein the one or more engineered variants of the AAV8 capsid protein comprises an engineered variant of AAV8 capsid protein VP2.

66. The composition of any one of claims 63-65, wherein the one or more engineered variants of the AAV8 capsid protein comprises an engineered variant of AAV8 capsid protein VP3.

67. The composition of any one of claims 63-66, wherein the one or more engineered variants of the AAV8 capsid protein are one or more deimmunized variants of an AAV8 capsid protein.

68. The composition of any one of claims 1-48, wherein the first AAV particle is a first AAV8 particle and the second AAV particle is a second AAV8 particle.

69. A dual adeno-associated virus (AAV) particle composition comprising:(a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises from 5’ to 3’ :(i) a 5 ’-inverted terminal repeat (5’-ITR) sequence of SEQ ID NO: 1;(ii) a promoter sequence of SEQ ID NO: 2;(iii) a Kozak sequence of SEQ ID NO: 3;(iv) an N-terminal coding sequence of SEQ ID NO: 7 encoding an N- terminal portion of an Abca4 protein, said N-terminal coding sequence being operably linked to and under control of said promoter;(v) a nucleic acid sequence of a splicing donor comprising the sequence of SEQ ID NO: 8;(vi) a nucleic acid sequence of a dimerization domain comprising the sequence of SEQ ID NO: 9;(vii) a poly(A) signal sequence of SEQ ID NO: 10; and(viii) a 3’-ITR sequence of SEQ ID NO: 11; and(b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’:(i) a 5’-ITR sequence of SEQ ID NO: 1;(ii) a promoter sequence of SEQ ID NO: 2;(iii) a nucleic acid sequence of a dimerization domain comprising a sequence of SEQ ID NO: 16, said dimerization domain being operably linked to and under control of said promoter;(iv) a nucleic acid sequence of a splicing acceptor comprising a sequence of SEQ ID NO: 17;(v) a C-terminal coding sequence of SEQ ID NO: 20 encoding a C- terminal portion of the Abca4 protein;(vi) a poly(A) signal sequence of SEQ ID NO: 21; and(vii) a 3’-ITR sequence of SEQ ID NO: 11; wherein the first AAV particle comprises AAV8 capsid proteins; and wherein the second AAV particle comprises AAV8 capsid proteins.

70. A pharmaceutical composition comprising an effective amount of the composition of any one of the preceding claims and a pharmaceutically acceptable carrier, excipient, diluent, or buffer.

71. The pharmaceutical composition of claim 70 comprising an effective amount of the first AAV particle and the second AAV particle at a ratio of about 1 to about 1.

72. The pharmaceutical composition of claim 71, wherein the ratio of about 1 to about 1 is a ratio of the number of first encapsidated transgenes to the number of second encapsidated transgenes.

73. The pharmaceutical composition of claim 72, wherein the ratio of about 1 to about 1 is a ratio of the number of first AAV particles to the number of second AAV particles.

74. The pharmaceutical composition of any one of claims 70-73, wherein the pharmaceutical composition is suspended in a buffer at a concentration of from about 1 >< 1O10total vg / mL to about 1 x 1014total vg / mL.

75. The pharmaceutical composition of any one of claims 70-74, wherein the pharmaceutical composition is suspended in a buffer at a concentration of from about 1 x lO10total vg / mL to about 2x 1013total vg / mL.

76. The pharmaceutical composition of any one of the claims 70-74, wherein the pharmaceutical composition is suspended in a buffer at a concentration of about I x lO10total vg / mL, about 1.25x lO10total vg / mL, about 2x lO10total vg / mL, about 5x l010total vg / mL, about I x lO11total vg / mL, about 1.25xlOntotal vg / mL, about 2x lOntotal vg / mL, about 5x l0ntotal vg / mL, about I x lO12total vg / mL, about 1 ,25x 1012total vg / mL, about 2x 1012total vg / mL, about 5 x lO12total vg / mL, about I x lO13total vg / mL, about 1.25x l013total vg / mL, about 2x l013total vg / mL, about 5x 1013total vg / mL, or about I x lO14total vg / mL.

77. A method for treating Stargardt Disease (STGD) in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of any one of claims 70-76.

78. The method of claim 77, wherein the administering comprises subretinal administration.

79. The method of claim 77, wherein the administering comprises subfoveal administration.

80. The method of claim 77, wherein the administering comprises intravitreal administration.

81. The method of claim 78, wherein the subretinal administration comprises subretinal administration with foveal placement.

82. A method for treating STGD in a subject in need thereof, comprising: administering to the subject, by subretinal injection, an effective amount of a pharmaceutical composition comprising first and second AAV particles, wherein the first AAV particle comprises an AAV capsid encapsidating a first transgene, the first transgene comprising from 5’ to 3’: a 5’-ITR sequence of SEQ ID NO: 1; a first promoter sequence comprising an ocular tissue-specific promoter of SEQ ID NO: 2; a Kozak sequence of SEQ ID NO: 3; an N-terminal coding sequence of SEQ ID NO: 7 encoding an N-terminal portion of an Abca4 protein, the N-terminal coding sequence being operably linked to and under control of the first promoter; a nucleic acid sequence of a splicing donor of SEQ ID NO: 8; a nucleic acid sequence of a first dimerization domain of SEQ ID NO: 9; a poly(A) signal sequence of SEQ ID NO: 10; and a 3’-ITR sequence of SEQ ID NO: 11, wherein the second AAV particle comprises an AAV capsid encapsidating a second transgene, the second transgene comprising from 5’ to 3’ : the 5’-ITR sequence of SEQ ID NO: 1;a second promoter sequence comprising the ocular tissue-specific promoter of SEQ ID NO: 2; a nucleic acid sequence of a second dimerization domain comprising a sequence of SEQ ID NO: 16, said second dimerization domain being operably linked to and under control of said second promoter; a nucleic acid sequence of a splicing acceptor comprising a sequence of SEQ ID NO: 17; a C-terminal coding sequence of SEQ ID NO: 20 encoding a C-terminal portion of the Abca4 protein; a poly(A) signal sequence of SEQ ID NO: 21; and the 3’-ITR sequence of SEQ ID NO: 11, and wherein a first transcript of the first transgene and a second transcript of the second transgene is spliced and joined to form a full-length coding sequence of SEQ ID NO: 22 that encodes the Abca4 protein.

83. The method of any one of claims of 77-82, wherein the method comprises administering the pharmaceutical composition to one eye of the subject.

84. The method of any one of claims of 77-82, wherein the method comprises administering the pharmaceutical composition to both eyes of the subject.

85. The method of any one of claims 77-84, wherein the subject is a neonatal human subject.

86. The method of any one of claims 77-84, wherein the subject is an adolescent human subject.

87. The method of any one of claims 77-84, wherein the subject is an adult human subject.

88. The method of any one of claims 77-87, wherein the effective amount of the AAV particle in the pharmaceutical composition is from about I x lO9vg to about l >< 1014vg of the first AAV particle and about 1 x 109vg to about 1 x 1014vg of the second AAV particle per eye treated.

89. The method of any one of claims 77-87, wherein the effective amount of the AAV particle in the pharmaceutical composition is from about I x lO9vg to about I x lO13vg of thefirst AAV particle about l >< 109vg to about l >< 1013vg of the second AAV particle per eye treated.

90. The method of any one of claims 77-87, wherein the effective amount of the AAV particle in the pharmaceutical composition is from about I x lO9vg to about I x lO12vg of the first AAV particle and about 1 x 109vg to about I x lO12vg of the second AAV particle per eye treated.

91. The method of any one of claims 77-87, wherein the effective amount of the AAV particle in the pharmaceutical composition is from about I x lO9vg to about I x lO11vg of the first AAV particle and about 1 x 109vg to about 1 x 1011vg of the second AAV particle per eye treated.

92. The method of any one of claims 77-87, wherein the effective amount of the AAV particle in the pharmaceutical composition is from about I x lO9vg to about I x lO10vg of the first AAV particle and about 1 x 109vg to about I x lO10vg of the second AAV particle per eye treated.

93. The method of any one of claims 77-87, wherein the effective amount of the AAV particles in the pharmaceutical composition is about 5x l09vg of the first AAV particle and about 5x 109vg of the second AAV particle per eye treated.

94. The method of any one of claims 77-87, wherein the effective amount of the AAV particles in the pharmaceutical composition is about I x lO10vg of the first AAV particle and about I x lO10vg of the second AAV particle per eye treated.

95. The method of any one of claims 77-87, wherein the effective amount of the AAV particles in the pharmaceutical composition is about 1.5 x lO10vg of the first AAV particle and about 1.5 x lO10vg of the second AAV particle per eye treated.

96. The method of any one of claims 77-87, wherein the effective amount of the AAV particles in the pharmaceutical composition is about 5x lO10vg of the first AAV particle and about 5 x IO10vg of the second AAV particle per eye treated.

97. The method of any one of claims 77-87, wherein the effective amount of the AAV particles in the pharmaceutical composition is about I x lO11vg of the first AAV particle and about 1 x 1011vg of the second AAV particle per eye treated.

98. The method of any one of claims 77-87, wherein the effective amount of the AAV particles in the pharmaceutical composition is about 1.5xlOnvg of the first AAV particle and about 1.5* 1011vg of the second AAV particle per eye treated.

99. The method of any one of claims 77-87, wherein the effective amount of the AAV particles in the pharmaceutical composition is about 5x lOnvg of the first AAV particle and about 5* 1011vg of the second AAV particle per eye treated.

100. The method of any one of claims 77-87, wherein the effective amount of the AAV particles in the pharmaceutical composition is about I x lO12vg of the first AAV particle and about 1 * 1012vg of the second AAV particle per eye treated.

101. The method of any one of claims 77-87, wherein the effective amount of the AAV particles in the pharmaceutical composition is about 1.5x l012vg of the first AAV particle and about 1.5 x lO12vg of the second AAV particle per eye treated.

102. The method of any one of claims 77-87, wherein the effective amount of the AAV particles in the pharmaceutical composition is about 2x l012vg of the first AAV particle and about 2x 1012vg of the second AAV particle per eye treated.

103. The method of any one of claims 77-87, wherein the effective amount of the AAV particles in the pharmaceutical composition is about 2.5 x 1012vg of the first AAV particle and about 2.5 x 1012vg of the second AAV particle per eye treated.

104. The method of any one of claims 77-87, wherein the effective amount of the AAV particles in the pharmaceutical composition is from about I x lO10total vg to about 5x l012total vg of the first and second AAV particles combined, per eye treated.

105. The method of claim 104, wherein the about I x lO10total vg of the AAV particles comprises about 5x l09vg of the first AAV particle and about 5x l09vg of the second AAV particle.

106. The method of claim 104, wherein the 5x 1012total vg of the AAV particles comprises about 2.5x l012vg of the first AAV particle and about 2.5x l012vg of the second AAV particle.

107. The method of any one of claims 77-87, wherein the pharmaceutical composition is administered in a volume of about 0.05 mL, about 0.06 mL, about 0.07 mL, about 0.08 mL,about 0.09 mL, about 0.1 mL, about 0.15 mL, about 0.2 mL, about 0.25 mL, about 0.3 mL, about 0.35 mL, about 0.4 mL, about 0.45 mL, about 0.5 mL, about 0.55 mL, about 0.6 mL, about 0.65 mL, about 0.7 mL, about 0.75 mL, or about 0.8 mL per eye treated.

108. The method of any one of claims 77-87, wherein the pharmaceutical composition is administered to one or more eyes of the subject in a volume of from about 0.05 mL to about 0.8 mL per eye treated.

109. The method of any one of claims 77-87, wherein the pharmaceutical composition is administered to one or more eyes of the subject in a volume of from about 0.3 mL to about 0.8 mL per eye treated.

110. The method of any one of claims 77-109, wherein the effective amount of the AAV particle is administered as a single dose per eye.

111. The method of any one of claims 77-110, wherein the treating comprises restoring the Abca4 protein level in the subject to a normal range or to a non-STGD level.

112. The method of any one of claims 77-111, wherein the treating comprises increasing the Abca4 protein level in the subject to at least about 200%, about 195%, about 190%, about 185%, about 180%, about 175%, about 170%, about 165%, about 160%, about 155%, about 150%, about 145%, about 140%, about 135%, about 130% about 125%, about 120%, about 115%, about 110%, about 105%, about 100%, about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30% about 25%, about 20%, about 15%, about 10%, about 5%, about 2%, or about 1% of the normal or the pre-treatment level.

113. The method of any one of claims 77-112, wherein the treating comprises reducing the severity of one or more STGD symptoms in the subject.

114. The method of claim 113, wherein one or more STGD symptoms comprise gray, black, or hazy spots in the center of the vision, sensitivity to light, needing more time for eyes to adjust between light and dark places, color blindness, loss of visual acuity, central loss of vision, peripheral loss of vision, visual acuity of about 20 / 200 or less in a better eye, a visual field of about 20 degrees or less in the better eye, or any combination thereof.

115. A DNA plasmid comprising an N-terminal transgene construct, wherein the transgene construct comprises from 5’ to 3’ :(i) a 5 ’-ITR sequence;(ii) a promoter sequence;(iii) a Kozak sequence;(iv) an N-terminal coding sequence encoding an N-terminal portion of a target protein, said N-terminal coding sequence being operably linked to and under control of said promoter;(v) a nucleic acid sequence of a splicing donor;(vi) a nucleic acid sequence of a dimerization domain;(vii) a poly(A) signal sequence; and(viii) a 3 ’-ITR sequence; wherein the dimerization domain comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 9.

116. A DNA plasmid comprising an N-terminal transgene construct, wherein the transgene construct comprises from 5’ to 3’ :(i) a 5 ’-ITR sequence;(ii) a promoter sequence;(iii) a Kozak sequence;(iv) an N-terminal coding sequence encoding an N-terminal portion of a target protein, said N-terminal coding sequence being operably linked to and under control of said promoter;(v) a nucleic acid sequence of a splicing donor;(vi) a nucleic acid sequence of a dimerization domain;(vii) a poly(A) signal sequence; and(viii) a 3 ’-ITR sequence; wherein the N-terminal coding sequence comprises an intervening intron sequence within the N-terminal coding sequence.

117. A DNA plasmid comprising an N-terminal transgene construct, wherein the transgene construct comprises from 5’ to 3’ :(i) a 5 ’-ITR sequence;(ii) a promoter sequence;(iii) a Kozak sequence;(iv) an N-terminal coding sequence encoding an N-terminal portion of an Abca4 protein, said N-terminal coding sequence being operably linked to and under control of said promoter;(v) a nucleic acid sequence of a splicing donor;(vi) a nucleic acid sequence of a dimerization domain;(vii) a poly(A) signal sequence; and(viii) a 3 ’-ITR sequence; wherein the N-terminal coding sequence comprises a nucleic acid sequence that is at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 7.

118. The DNA plasmid of claim 115 or 116, wherein the target protein is an Abca4 protein.

119. The DNA plasmid of any one of claims 115-118, wherein the N-terminal coding sequence is codon optimized.

120. The DNA plasmid of claim 115 or 116, wherein the N-terminal coding sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 7.

121. The DNA plasmid of claim 115, wherein the N-terminal coding sequence comprises an intervening intron sequence within the N-terminal coding sequence.

122. The DNA plasmid of claim 116 or 121, wherein the intervening intron sequence is a modified mouse beta-actin (Actb) intron 2 sequence.

123. The DNA plasmid of claim 122, wherein the intervening intron sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 5.

124. The DNA plasmid of any one of claims 115-123, wherein the splicing donor comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 8.

125. The DNA plasmid of any one of claims 116-124, wherein the dimerization domain comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 9.

126. The DNA plasmid of any one of claims 115-125, wherein the promoter is an ocular tissue-specific promoter.

127. The DNA plasmid of any one of claims 115-126, wherein the promoter is a human rhodopsin kinase (hGRKl) promoter.

128. The DNA plasmid of claim 127, wherein the promoter comprises the nucleic acid sequence of SEQ ID NO: 2.

129. The DNA plasmid of any one of claims 115-128, wherein the 5’-ITR is a 5’ AAV2 ITR.

130. The DNA plasmid of any one of claims 129, wherein the 5’-ITR comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 1.

131. The DNA plasmid of any one of claims 115-130, wherein the 3 ’-ITR is a 3’ AAV2 ITR.

132. The DNA plasmid of claim 131, wherein the 3 ’-ITR comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 11133. The DNA plasmid of any one of claims 115-132, wherein the poly(A) signal sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 10.

134. The DNA plasmid of any one of claims 115-133, wherein the Kozak sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a sequence of SEQ ID NO:3, 23, 24, or 25.

135. A DNA plasmid comprising an N-terminal transgene construct, wherein the transgene construct comprises from 5’ to 3’ :(i) a 5’-ITR sequence of SEQ ID NO:1;(ii) a promoter sequence of SEQ ID NO:2;(iii) a Kozak sequence of SEQ ID NO:3;(iv) an N-terminal coding sequence of SEQ ID NO:7 encoding an N-terminal portion of an Abca4 protein, said N-terminal coding sequence being operably linked to and under control of said promoter;(v) a nucleic acid sequence of a splicing donor comprising the sequence of SEQ ID NO:8;(vi) a nucleic acid sequence of a dimerization domain comprising the sequence of SEQ ID NO:9;(vii) a poly(A) signal sequence of SEQ ID NO: 10; and(viii) a 3’-ITR sequence of SEQ ID NO:11.

136. A DNA plasmid comprising a C-terminal transgene construct, wherein the transgene construct comprises from 5’ to 3’ :(i) a 5 ’-ITR sequence;(ii) a promoter sequence;(iii) a nucleic acid sequence of a dimerization domain, said dimerization domain being operably linked to and under control of said promoter;(iv) a nucleic acid sequence of a splicing acceptor;(v) a C-terminal coding sequence encoding a C-terminal portion of a target protein;(vi) a poly(A) signal sequence; and(vii) a 3 ’-ITR sequence; wherein the dimerization domain comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 16.

137. A DNA plasmid comprising a C-terminal transgene construct, wherein the transgene construct comprises from 5’ to 3’ :(i) a 5 ’-ITR sequence;(ii) a promoter sequence;(iii) a nucleic acid sequence of a dimerization domain, said dimerization domain being operably linked to and under control of said promoter;(iv) a nucleic acid sequence of a splicing acceptor;(v) a C-terminal coding sequence encoding a C-terminal portion of a target protein;(vi) a poly(A) signal sequence; and(vii) a 3 ’-ITR sequence; wherein the C-terminal coding sequence comprises an intervening intron sequence within the C-terminal coding sequence.

138. A DNA plasmid comprising a C-terminal transgene construct, wherein the transgene construct comprises from 5’ to 3’ :(i) a 5 ’-ITR sequence;(ii) a promoter sequence;(iii) a nucleic acid sequence of a dimerization domain, said dimerization domain being operably linked to and under control of said promoter;(iv) nucleic acid sequence of a splicing acceptor;(v) a C-terminal coding sequence encoding a C-terminal portion of a target protein;(vi) a poly(A) signal sequence; and(vii) a 3 ’-ITR sequence; wherein the C-terminal coding sequence comprises a nucleic acid sequence that is at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 20.

139. The DNA plasmid of claim 136 or 137, wherein the target protein is an Abca4 protein.

140. The DNA plasmid of claim 136 or 137, wherein the C-terminal coding sequence is codon optimized.

141. The DNA plasmid of claim 136 or 137, wherein the C-terminal coding sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 20.

142. The DNA plasmid of any one of claims 136 and 139-141, wherein the C-terminal coding sequence comprises an intervening intron sequence within the C-terminal coding sequence.

143. The DNA plasmid of claim 137 or 142, wherein the intervening intron sequence is a modified mouse beta-actin (Actb) intron 2 sequence.

144. The DNA plasmid of claim 143, wherein the intervening intron sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 5.

145. The DNA plasmid of any one of claims 136-144, wherein the splicing acceptor comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 17.

146. The DNA plasmid of any one of claims 136-145, wherein the dimerization domain comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 16.

147. The DNA plasmid of any one of claims 136-146, wherein the promoter is an ocular tissue-specific promoter.

148. The DNA plasmid of any one of claims 136-147, wherein the promoter is a human rhodopsin kinase (hGRKl) promoter.

149. The DNA plasmid of claim 148, wherein the promoter comprises the nucleic acid sequence of SEQ ID NO: 2.

150. The DNA plasmid of any one of claims 136-149, wherein the 5’ -ITR is a 5’ AAV2 ITR.

151. The DNA plasmid of any one of claims 150, wherein the 5 ’-ITR comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 1.

152. The DNA plasmid of any one of claims 136-151, wherein the 3 ’-ITR is a 3’ AAV2 ITR.

153. The DNA plasmid of claim 152, wherein the 3 ’-ITR comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical toSEQ ID NO: 11154. The DNA plasmid of any one of claims 136-153, wherein the poly(A) signal sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:21.

155. A DNA plasmid comprising a C-terminal transgene construct, wherein the transgene construct comprises from 5’ to 3’ :(i) a 5’-ITR sequence of SEQ ID NO:1;(ii) a promoter sequence of SEQ ID NO:2;(iii) a nucleic acid sequence of a dimerization domain comprising a sequence of SEQ ID NO: 16, said dimerization domain being operably linked to and under control of said promoter;(iv) a nucleic acid sequence of a splicing acceptor comprising a sequence of SEQ ID NO: 17;(v) a C-terminal coding sequence of SEQ ID NO:20 encoding a C-terminal portion of an Abca4 protein;(vi) a poly(A) signal sequence of SEQ ID NO:21; and(vii) a 3’-ITR sequence of SEQ ID NO:11.

156. A packaging cell comprising one or more DNA plasmids of any one of claims 115-155.

157. The packaging cell of claim 156, comprising the DNA plasmid comprising an N- terminal transgene construct and the DNA plasmid comprising a C-terminal transgene construct.

158. The packaging cell of claim 156 or 157, further comprising a helper plasmid containing adenoviral components necessary for recombinant adeno-associated virus production.

159. The packaging cell of any one of claims 156-158, further comprising a plasmid comprising AAV replication (rep) and capsid (cap) genes.

160. The packaging cell of any one of claims 156-159, wherein the packaging cell is a HEK293 cell.

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