WO2020214990A1

WO2020214990A1 - Abca4 cell line and methods of use

Info

Publication number: WO2020214990A1
Application number: PCT/US2020/028823
Authority: WO
Inventors: Philip R. Johnson; Laura Barrio REAL; Jasmine SHONG; Rebekka KRUMBACH; Scott J. DOOLEY; Bruce C. SCHNEPP
Original assignee: Ascidian Therapeutics, Inc.
Priority date: 2019-04-17
Filing date: 2020-04-17
Publication date: 2020-10-22

Abstract

Provided herein are cell lines having a modification allowing for expression of ABCA4 from a native locus. In some embodiments, the cell line contains a further modification that disrupts ABCA4 expression to allow screening of candidate agents for their ability to restore functional ABCA4 expression in the cell line. Such cell lines and screening methods can be used to characterize the efficacy of candidate agents on expression of ABCA4 in conditions mimicking disorders associated with mutations in ABCA4, such as Stargardt Disease.

Description

ABCA4 CELL LINE AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No. 62/835,446, filed April 17,

2019, the contents of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on April 16,

2020, is named 51502-014W02_Sequence_Listing_04.16.20_ST25 and is 340,766 bytes in size.

BACKGROUND

ABCA4 is a member of the ATP-binding cassette transporter gene sub-family A (ABC1). The gene was first cloned and characterized in 1997 as a gene that causes Stargardt disease, an autosomal recessive disease that causes macular degeneration. The ABCA4 gene transcribes a large retina- specific protein with two transmembrane domains (TMD), two glycosylated extracellular domains (ECD), and two nucleotide-binding domains (NBD). The ABCA4 protein is almost exclusively expressed in retina, localizing in outer segment disk edges of rod photoreceptors. Mutations in ABCA4 gene are known to cause the autosomal-recessive disease Stargardt macular dystrophy (STGD), which is a hereditary juvenile macular degeneration disease causing progressive loss of photoreceptor cells.

Additional diseases that can be attributed to mutations in ABCA4 include fundus flavimaculatus, cone-rod dystrophy, retinitis pigmentosa, and age related macular degeneration.

Currently, suitable in-vitro methods for pre-clinical testing of therapies for treating ABCA4- associated disorders are lacking. Thus, there is a need in the field for a recombinant cell modified to express ABCA4 from its native locus to facilitate such testing.

SUMMARY OF THE INVENTION

The present invention provides a cell line having a modification allowing it to express ABCA4 from its native locus. In some embodiments, the cell line contains a further modification that disrupts ABCA4 expression to allow screening of candidate agents for their ability to restore functional ABCA4 expression in the cell line. Such cell lines and screening methods can be used to characterize the efficacy of candidate agents on expression of ABCA4 in conditions mimicking disorders associated with mutations in ABCA4, such as Stargardt Disease.

In one aspect, the invention provides a cell (e.g., an isolated and/or recombinant cell) modified to express ABCA4. The cell includes an ABCA4 expression modification that increases expression of ABCA4 from a native ABCA4 locus. In some embodiments, the cell comprises a detectable level of pre- mRNA including one or more ABCA4 introns.

In some embodiments, the ABCA4 expression modification is a Transcription activator-like effector nuclease (TALEN). In some embodiments, the TALEN is fused to a viral trans-activator (e.g., VP16 or VP64). In some embodiments, the TALEN is expressed upstream of the ABCA4 transcriptional start site. In some embodiments, the TALEN includes a DNA binding domain that specifically binds to a target sequence in a 5’ UTR of the ABCA4 locus. In some embodiments, the target sequence is located within SEQ ID NO: 56 (e.g., any one of SEQ ID NOs: 57-59 or a portion therewithin). In some embodiments, the native ABCA4 locus further includes an endogenous promoter (e.g., wherein the chimeric protein drives expression of ABCA4 from the endogenous promoter at its native genomic locus).

In some embodiments, the expression modification is an exogenous eukaryotic promoter (e.g., a CAG promoter). In some embodiments, the CAG promoter includes (a) a cytomegalovirus (CMV) early enhancer element; (b) a promoter, first exon, and first intron of chicken beta-actin gene; and (c) a splice acceptor of a rabbit beta-globin gene. In some embodiments, the exogenous eukaryotic promoter (e.g., the CAG promoter) is located upstream of the ABCA4 gene. In some embodiments, the exogenous eukaryotic promoter (e.g., the CAG promoter) drives expression of the ABCA4 gene. In some embodiments, the cell further includes a mouse PGK promoter. In some embodiments, the mouse PGK promoter is located upstream of the CAG promoter.

In some embodiments, the cell is a eukaryotic cell, e.g., a mammalian cell (e.g., a HEK293T cell or an ARPE-19 cell) in some embodiments, the cell is a retinal-derived cell (e.g., a cell derived from the retinal pigment epithelium, e.g., an ARPE-19 celi).

in some embodiments, the cell further includes an ABCA4 disruption modification that decreases expression of functional ABCA4 from a native ABCA4 locus in some embodiments, the ABCA4 disruption modification causes a disruption in one or more ABCA4 exons (e.g., two or more ABCA4 exons, e.g., two and oniy two ABCA4 exons). In some embodiments, the ABCA4 disruption modification causes a disruption in ABCA4 exons 3 and 4. in some embodiments, the ABCA4 disruption modification is a CRISPR/Cas9-mediated ablation.

in another aspect, the invention provides a method of generating the cell of any of the preceding embodiments. For example, in some embodiments, the method includes inserting an ABCA4 expression modification in the native genome of a host cell, wherein the ABCA4 expression modification increases expression of ABCA4 from a native ABCA4 locus.

In another aspect, provided herein are methods of testing a candidate agent for treatment of an ABCA4-associated disorder. For example, in some embodiments, such a method includes: (a) providing a candidate agent for testing; (b) contacting the candidate agent with the cell of any one of any of the preceding embodiments (e.g., a cell having an ABCA4 expression modification and an ABCA4 disruption modification) for a time and condition suitable for change in genetic and/or protein expression in the cell; and (c) measuring expression of ABCA4 expressed by the cell after step (b). Based on the

aforementioned measurement, the method can further include determining whether the candidate agent restores ABCA4 expression (fully or partially). In some embodiments, the candidate agent is an ABCA4- restoring agent, such as an ABCA4 trans-splicing molecule. In some embodiments, the candidate agent is for treatment of an ABCA-associated disorder. In some embodiments, the ABCA4-associated disorder is selected from the group consisting of Stargardt macular dystrophy (STGD), fundus flavimaculatus, cone-rod dystrophy, retinitis pigmentosa, and age-related macular degeneration.

In some embodiments, the method further includes comparing the ABCA4 expression by the cell after contact with the agent with ABCA4 expression by the cell before contact with the agent (e.g., determining that the candidate agent restores ABCA4 expression based on at least a one and one-half fold or at least two-fold (e.g., at least three-fold, at least four-fold, at least five-fold, at least ten-fold, or greater) restoration of ABCA4 expression by the cell after contact compared to ABCA4 expression by the cell before contact). In some embodiments, the method further includes comparing the ABCA4 expression by the cell after contact with the agent with ABCA4 expression by a control cell contacted with a reference agent (e.g., determining that the candidate agent restores ABCA4 expression based on at least a two-fold (e.g., at least three-fold, at least four-fold, at least five-fold, at least ten-fold, or greater)_restoration of ABCA4 expression by the cell after contact compared to ABCA4 expression by the control cell). In some embodiments, the candidate agent is a candidate ABCA4 trans-splicing molecule and the reference agent is a reference ABCA4 trans-splicing molecule.

In another embodiment, the method further includes administering the candidate agent to a subject. In some embodiments, the subject has an ABCA4-assoicated disorder. In some embodiments, the ABCA4-associated disorder is selected from the group consisting of Stargardt macular dystrophy (STGD), fundus flavimaculatus, cone-rod dystrophy, retinitis pigmentosa, and age-related macular degeneration. In some embodiments, the subject is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing showing a TALEN protein consisting of a DNA binding domain linked to a transcription activation domain. A VP64 transcription activation domain is shown. The right panel shows a portion of the 5’ untranslated region (5’-UTR) of ABCA4. The TATA box and the putative transcription start site are shown. The sequences targeted the by the three different DNA binding domains of TALENs are also shown. TALEN 1 binds to the first underlined sequence, TALEN 2 binds to the second underlined sequence, and TALEN 3 binds to the third underlined sequence, as indicated.

FIG. 2 is a gel showing HEK293T cells were transfected with TALEN constructs designed to induce endogenous ABCA4 expression. All three TALENS from FIG. 1 were stably introduced into HEK293T cells and single cell clones were picked and analyzed by western blot. The positive control (+) indicates cells transfected with a plasmid expressing an ABCA4 cDNA. Cell lysates were made 48 hours after transfection and the membrane fractions were examined for ABCA4 expression using antibody ab72955 (Abeam). Clones ZT-22 and ZT-48 showed ABCA4 protein expression.

FIG. 3 is a schematic drawing showing the native ABCA4 transcription start site and flanking 1 kb homology arms.

FIGS. 4A and 4B show the CRISPR guide RNA sequences (FIG. 4A) that were designed to insert the CAG promoter and a puromycin selectable marker using homology arms (FIG. 4B). FIG. 4C shows the primer sequences used to validate CAG insertion.

FIG. 5 is a schematic drawing showing a CAG promoter construct.

FIGS. 6A and 6B are a graph and a gel, respectively, showing expression results from several clonal lines that were selected for further analyses. FIG. 6A shows RNA expression and FIG. 6B shows protein expression of the cell lines. Membrane preparations of the indicated cells lines were probed for ABCA4 protein using a rabbit polyclonal antibody to ABCA4 (Abeam, ab72955). Exposure time is 23 seconds. HEK293T cells are the parental cell that does not express ABCA4. The top band is nonspecific background present in all cells.

FIG. 7 is a schematic drawing showing CRISPR guide RNA for targeting exons 3 and 4.

FIG. 8 is a graph showing RNA expression and a gel showing protein profiles of single cell clones derived after treatment with CRISPR/Cas9, as depicted in FIG. 7. FIGS. 9A and 9B are schematic drawings showing PCR for mutation analyses on gDNA (FIG. 9A) and PCR for genotyping on gDNA (FIG. 9B), confirming that exons 3 and 4 were targeted and interrupted.

FIG. 10 is a set of tables showing that the mutation analyses from FIGS. 9A and 9B confirmed that exons 3 and 4 were targeted and interrupted in alleles in the 17+06 and 17+21 cell lines.

FIG. 11 is a gel showing ABCA4 expression by ARPE-19 cells with and without genetic modifications.

FIG. 12 is a gel showing trans-splicing molecule-mediated rescue of ABCA4 expression in HEK293T cells and ARPE-19 cells. NBD refers to control trans-splicing molecules that lack a binding domain. RTM refers to trans-splicing molecules that include a binding domain.

DETAILED DESCRIPTION

Pre-clinical testing of certain gene-based therapies, such as trans-splicing molecules (e.g., those described in WO 2017/087900 and WO 2019/204514, each of which is incorporated herein by reference in its entirety), requires an in vitro system to evaluate the efficiency of editing the native ABCA4 pre- mRNA. Thus, a cell line expressing ABCA4 from its endogenous locus is necessary. The full-length ABCA4 pre-mRNA (containing all exons/introns) is desirable in order to evaluate trans-spicing candidates that span the entire transcript. A cell line that simply expresses an ABCA4 cDNA is inadequate for such purposes, because it contains no introns to target. Currently, there are no cell lines to the Inventors’ knowledge that can reproducibly express levels of ABCA4 protein detectable by western blot. The present invention provides a cell line that expresses native ABCA4 protein at levels sufficient for use as a preclinical in vitro system for screening therapeutic candidates, such as ABCA4 trans-splicing molecules.

Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application. In the event of any conflicting definitions between those set forth herein and those of a referenced publication, the definition provided herein shall control.

As used herein, the term“ABCA4” refers to a polynucleotide (e.g., RNA (e.g., pre-mRNA or mRNA) or DNA) that encodes retinal-specific ATP-binding cassette transporter. An exemplary genomic DNA sequence of a functional (wildtype) human ABCA4 gene is given by NCBI Reference Sequence: NG_009073. The amino acid sequence of an exemplary ABCA4 protein is given by Protein Accession No. P78363. ABCA4 refers to ABCA4 from any organism having a functional ABCA4 gene, such as a mammal, e.g., a human.

As used herein, an“ABCA4 expression modification” refers to a non-native element (an element that is either never expressed in the host cell or is normally expressed in a different manner, e.g., at a different genetic location) in the genome of the host cell that increases expression of functional ABCA4 from a native ABCA4 locus when present in the host cell. Increased expression can be measured at the RNA level, at the protein level, or both, according to any of the RNA or protein detection methods known in the art or described herein.

As used herein, an“ABCA4 disruption modification” refers to a non-native element (an element that is either never expressed in the host cell or is normally expressed in a different manner, e.g., at a different genetic location) in the genome of the host cell that decreases expression of (e.g., ablates) functional ABCA4 from a native ABCA4 locus when present in the host cell. Decreased expression can be measured at the RNA level, at the protein level, or both, according to any of the RNA or protein detection methods known in the art or described herein. Decreased expression includes, for example, expression of a non-functional ABCA4 protein.

As used herein, a“mutation” refers to any aberrant nucleic acid sequence that causes a defective (e.g., non-functional, reduced function, aberrant function, less than normal amounts produced) protein product. Mutations include base pair mutations (e.g., single nucleotide polymorphisms), missense mutations, frameshift mutations, deletions, insertions, and splice mutations. In some embodiments, a mutation refers to a nucleic acid sequence that is different in one or more portions of its sequence than a corresponding wildtype nucleic acid sequence or functional variant thereof. In some embodiments, a mutation refers to a nucleic acid sequence that encodes a protein having an amino acid sequence that is different than a corresponding wildtype protein or functional variant thereof. A“mutated exon” (e.g., a mutated ABCA4 exon) refers to an exon containing a mutation or an exon sequence that reflects a mutation in a different region, such as a cryptic exon resulting from a mutation in an intron.

A“recombinant cell,”“recombinant cell line,”“isolated recombinant cell line,” or“host cell” refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells.

Host cells include“transformants” and“transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

As used herein, the term“treatment,” or a grammatical derivation thereof, is defined as reducing the progression of a disease, reducing the severity of a disease symptom, retarding progression of a disease symptom, removing a disease symptom, or delaying onset of a disease.

A“nucleic acid trans-splicing molecule” or“trans-splicing molecule” has three main elements: (a) a binding domain that confers specificity by tethering the trans-splicing molecule to its target gene (e.g., pre-mRNA); (b) a splicing domain (e.g., a splicing domain having a 3’ or 5’ splice site); and (c) a coding sequence configured to be trans-spliced onto the target gene, which can replace one or more exons in the target gene (e.g., one or more mutated exons). A“pre-mRNA trans-splicing molecule” or“RTM” refers to a nucleic acid trans-splicing molecule that targets pre-mRNA. In some embodiments, a transsplicing molecule, such as an RTM, can include cDNA, e.g., as part of a functional exon (e.g., a functional ABCA4 exon, e.g., a codon-optimized exon) for replacement or correction of a mutated ABCA4 exon).

As used herein, an“ABCA4 trans-splicing molecule” is a trans-splicing molecule that has a binding domain that confers specificity to an ABCA4 target gene (e.g., ABCA4 pre-mRNA) and a coding sequence configured to be trans-spliced onto the ABCA4 target gene, e.g., the coding sequence includes one or more ABCA4 exons.

As used herein, a“candidate ABCA4 trans-splicing molecule” is an ABCA4 trans-splicing molecule being tested for trans-splicing efficacy as measured by binding function (e.g., association constant or binding affinity) and/or trans-splicing efficacy (e.g., trans-splicing efficiency, functional mRNA production, or functional protein expression), or any downstream effect thereof (e.g., restoration of biological function resulting from increase in trans-splicing function). In some instances, the candidate ABCA4 trans-splicing molecule has one or more structural changes relative to a reference ABCA4 transsplicing molecule, and the effect of the structural change(s) is being tested. In some embodiments, the binding domains of the candidate ABCA4 trans-splicing molecule and the reference ABCA4 trans-splicing molecule are the same. In other embodiments, the binding domains of the candidate ABCA4 transsplicing molecule and the reference ABCA4 trans-splicing molecule are different.

As used herein, a“reference ABCA4 trans-splicing molecule” is an ABCA4 trans-splicing molecule used as a control in a test for trans-splicing efficiency of a candidate ABCA4 trans-splicing molecule. In some instances, the reference ABCA4 trans-splicing molecule has a known level of transsplicing efficacy to which the trans-splicing efficacy of the candidate ABCA4 trans-splicing is compared.

By“trans-splicing” is meant joining of a nucleic acid molecule containing one or more exons (e.g., exogenous exons, e.g., exons that are part of a coding domain of a trans-splicing molecule) to a first portion of a separate RNA molecule (e.g., a pre-mRNA molecule, e.g., an endogenous pre-mRNA molecule) by replacing a second portion of the RNA molecule through a spliceosome-mediated mechanism.

“Binding” between a binding domain and a target intron, as used herein, refers to hydrogen bonding between the binding domain and the target intron in a degree sufficient to mediate trans-splicing by bringing the trans-splicing molecule into association with the target gene (e.g., pre-mRNA). In some embodiments, the hydrogen bonds between the binding domain and the target intron are between nucleotide bases that are complementary to and in antisense orientation from one another (e.g., hybridized to one another).

As used herein, an“artificial intron” refers to a nucleic acid sequence that links (directly or indirectly) a binding domain to a coding domain. An artificial intron includes a splicing domain and may further include one or more spacer sequences and/or other regulatory elements.

A“splicing domain,” as used herein, refers to a nucleic acid sequence having motifs that are recognized by the spliceosome and mediate trans-splicing. A splicing domain includes a splice site (e.g., a single splice site, i.e. , one and only one splice site), which can be a 3’ splice site or a 5’ splice site. A splicing domain may include other regulatory elements. For example, in some embodiments, a splicing domain includes splicing enhancers (e.g., exonic splicing enhancers (ESE) or intronic splicing enhancers (ISE)). In some embodiments, a splicing domain includes a branch point (e.g., a strong conserved branch point) or branch site sequence and/or a polypyrimidine tract (PPT). In some embodiments, a splicing domain of a 5’ trans-splicing molecule does not contain the branch point or PPT, but comprises a 5’ splice acceptor or a 3’ splice donor.

Exons and introns of ABCA4 are identified herein as set forth in Table 1 , below, which can be mapped onto the ABCA4 pre-mRNA molecule of SEQ ID NO: 6. Each exon and intron of ABCA4 are identified herein according to the reference number in the first (left-hand) column. The size of each exon and intron (base pairs; bp) are indicated in the second and third columns. The fourth column indicates the length of a cDNA molecule corresponding to exons 5’ to the corresponding intron number. The fifth column indicates the length of a cDNA molecule corresponding to mRNA 3’ to the corresponding intron number. Table 1. ABCA4 exon and intron summary

As used herein, a“target ABCA4 intron” refers to one of the 49 ABCA4 introns identified in Table 1 , above. Nucleic acid sequence identifiers for each ABCA4 intron sequence are provided in Table 2, below. It will be understood that the scope of the term“target ABCA4 intron” encompasses variants of

ABCA4 introns provided herein, such as intron sequences having 90-100% homology with the sequences provided herein (e.g., 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology with the sequences provided herein), where the location of the variant intron on the ABCA4 gene corresponds with that provided herein (e.g., in relation to its adjacent exons as set forth in Table 1). Table 2. ABCA4 intron sequences

As used herein, the term“subject” includes any mammal in need of these methods of treatment or prophylaxis, including humans. Other mammals in need of such treatment or prophylaxis include dogs, cats, or other domesticated animals, horses, livestock, laboratory animals, including non-human primates, etc. The subject may be male or female. In one embodiment, the subject has a disease or disorder caused by a mutation in the ABCA4 gene (e.g., Stargardt Disease, e.g., Stargardt Disease 1) or the CEP290 gene (e.g., an autosomal recessive disorder, such as LCA 10). In another embodiment, the subject is at risk of developing a disease or disorder caused by a mutation in the ABCA4 gene or the CEP290 gene. In another embodiment, the subject has shown clinical signs of a disease or disorder caused by a mutation in the ABCA4 gene (such as Stargardt Disease) or the CEP290 gene (such as LCA 10). The subject may be any age during which treatment or prophylactic therapy may be beneficial. For example, in some embodiments, the subject is 0-5 years of age, 5-10 years of age, 10-20 years of age, 20-30 years of age, 30-50 years of age, 50-70 years of age, or more than 70 years of age. In another embodiment, the subject is 12 months of age or older, 18 months of age or older, 2 years of age or older, 3 years of age or older, 4 years of age or older, 5 years of age or older, 6 years of age or older, 7 years of age or older, 8 years of age or older, 9 years of age or older, or 10 years of age or older. In another embodiment, the subject has viable retinal cells.

As used herein, the terms“disorder associated with a mutation” or“mutation associated with a disorder” refer to a correlation between a disorder and a mutation. In some embodiments, a disorder associated with a mutation is known or suspected to be wholly or partially, or directly or indirectly, caused by the mutation. For example, a subject having the mutation may be at risk of developing the disorder, and the risk may additionally depend on other factors, such as other (e.g., independent) mutations (e.g., in the same or a different gene), or environmental factors.

As used herein, the term“prevention” of a disorder, or a grammatical derivation thereof, is defined as reducing the risk of onset of a disease, e.g., as a prophylactic therapy for a subject who is at risk of developing a disorder associated with a mutation. A subject can be characterized as“at risk” of developing a disorder by identifying a mutation associated with the disorder, according to any suitable method known in the art or described herein. In some embodiments, a subject who is at risk of developing a disorder has one or more ABCA4 or CEP290 mutations associated with the disorder.

Additionally or alternatively, a subject can be characterized as“at risk” of developing a disorder if the subject has a family history of the disorder.

Treating or preventing a disorder in a subject can be performed by directly administering the trans-splicing molecule (e.g., within an AAV vector or AAV particle) to the subject. Alternatively, host cells containing the trans-splicing molecule may be administered to the subject.

The term“administering,” or a grammatical derivation thereof, as used in the methods described herein, refers to delivering the composition, or an ex vivo-treated cell, to the subject in need thereof, e.g., having a mutation or defect in the targeted gene. For example, in one embodiment in which ocular cells are targeted, the method involves delivering the composition by subretinal injection to the photoreceptor cells or other ocular cells. In another embodiment, intravitreal injection to ocular cells or injection via the palpebral vein to ocular cells may be employed. In another embodiment, the composition is administered intravenously. Still other methods of administration may be selected by one of skill in the art, in view of this disclosure.

Codon optimization refers to modifying a nucleic acid sequence to change individual nucleic acids without any resulting change in the encoded amino acid. Sequences modified in this way are referred to herein as“codon-optimized.” This process may be performed on any of the sequences described in this specification to enhance expression or stability. Codon optimization may be performed in a manner such as that described in, e.g., U.S. Patent Nos. 7,561 ,972, 7,561 ,973, and 7,888,112, each of which is incorporated herein by reference in its entirety. The sequence surrounding the translational start site can be converted to a consensus Kozak sequence according to known methods. See, e.g., Kozak et al, Nucleic Acids Res. 15 (20): 8125-8148, incorporated herein by reference in its entirety.

The term“homologous” refers to the degree of identity between sequences of two nucleic acid sequences. The homology of homologous sequences is determined by comparing two sequences aligned under optimal conditions over the sequences to be compared. The sequences to be compared herein may have an addition or deletion (for example, gap and the like) in the optimum alignment of the two sequences. Such a sequence homology can be calculated by creating an alignment using, for example, the ClustalW algorithm (Nucleic Acid Res., 1994, 22(22): 4673 4680). Commonly available sequence analysis software, such as, Vector NTI, GENETYX, BLAST, or analysis tools provided by public databases may also be used.

The term“pharmaceutically acceptable” means safe for administration to a mammal, such as a human. In some embodiments, a pharmaceutically acceptable composition is approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The term“carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic molecule (e.g., a trans-splicing molecule or a trans-splicing molecule including a vector or cell of the present invention) is administered. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA., 2^nd edition, 2005.

The terms“a” and“an” mean“one or more of.” For example,“a modification” is understood to represent one or more such modifications. As such, the terms“a” and“an,”“one or more of a (or an),” and“at least one of a (or an)” are used interchangeably herein.

As used herein, the term“about” refers to a value within ± 10% variability from the reference value, unless otherwise specified.

For any conflict in definitions between various sources or references, the definition provided herein shall control.

Recombinant Cell Lines

The invention includes a cell (e.g., an isolated and/or recombinant cell, e.g., a mammalian cell) modified to express ABCA4. In some embodiments, the cell line contains a further modification that disrupts ABCA4 expression to allow screening of candidate agents for their ability to restore functional ABCA4 expression in the cell line. Such cell lines and screening methods can be used to characterize the efficacy of candidate agents on expression of ABCA4 in conditions mimicking disorders associated with mutations in ABCA4, such as Stargardt Disease. In some instances, the cell includes an expression modification that increases expression of ABCA4 from a native ABCA4 locus (e.g., chromosome 1 (1 p22.1 )) having a polynucleotide having an ABCA4 gene. In some instances, the ABCA4 expression modification is a Transcription activator-iike effector nuclease (TALEN). For example, a TALEN can be fused to a viral trans-activator (e.g., a VP16 or VP64). In some embodiments, the TALEN is expressed upstream of the ABCA4 transcriptional start site. The TALEN may include a DNA binding domain that specifically binds to (e.g., is complementary to e.g., hybridizes with) a target sequence in a 5’ UTR of the ABCA4 locus. For example, the target sequence in a 5’ UTR of the ABCA4 locus may be located within SEQ ID NO: 56, be!ow:

In particular instances, the target sequence is any of the underlined sequences of SEQ ID NO: 56, i.e., any one of SEQ ID NOs: 57-59, reproduced below:

Thus, in some instances, the DNA binding domain binds specifically to a target sequence having SEQ ID NO: 56. in some instances, the binding domain is 95% complementary to 20-30 consecutive nucleic acids within SEQ ID NO: 56 (e.g., 96% complementary to 20-30 consecutive nucleic acids within SEQ ID NO: 56, 97% complementary to 20-30 consecutive nucleic acids within SEQ ID NO: 56, 98% complementary to 20-30 consecutive nucleic acids within SEQ ID NO: 56, 99% complementary to 20-30 consecutive nucleic acids within SEQ ID NO: 56, or 100% complementary to 20-30 consecutive nucleic acids within SEQ ID NO: 56; e.g., to 21-29 consecutive nucleic acids within SEQ ID NO: 56, to 22-28 consecutive nucleic acids within SEQ ID NO: 56, to 23-27 consecutive nucleic acids within SEQ ID NO:

56, to 24-26 consecutive nucleic acids within SEQ ID NO: 56, or to 25 consecutive nucleic acids within SEQ ID NO: 56, e.g., to 20 consecutive nucleic acids within SEQ ID NO: 56, to 21 consecutive nucleic acids within SEQ ID NO: 56, to 22 consecutive nucleic acids within SEQ ID NO: 56, to 23 consecutive nucleic acids within SEQ ID NO: 56, to 24 consecutive nucleic acids within SEQ ID NO: 56, to 25 consecutive nucleic acids within SEQ ID NO: 56, to 26 consecutive nucleic acids within SEQ ID NO: 56, to 27 consecutive nucleic acids within SEQ ID NO: 56, to 28 consecutive nucleic acids within SEQ ID NO: 56, to 29 consecutive nucleic acids within SEQ ID NO: 56, or to 30 consecutive nucleic acids within SEQ ID NO: 56). In some instances, the DNA binding domain binds specifically to a target sequence having SEQ ID NO: 57. In some instances, the binding domain is 95% complementary to SEQ ID NO: 57 (e.g., 96% complementary to SEQ ID NO: 57, 97% complementary to SEQ ID NO: 57, 98% complementary to SEQ ID NO: 57, 99% 95% complementary to SEQ ID NO: 57, or 100% complementary to SEQ ID NO: 57). in some instances, the DNA binding domain binds specifically to a target sequence having SEQ ID NO: 58. In some instances, the binding domain is 95% complementary to SEQ ID NO: 58 (e.g., 96% complementary to SEQ ID NO: 58, 97% complementary to SEQ ID NO: 58, 98% complementary to SEQ ID NO: 58, 99% complementary to SEQ ID NO: 58, or 100% complementary to SEQ ID NO: 58).

in some instances, the DNA binding domain binds specifically to a target sequence having SEQ ID NO: 59. In some instances, the binding domain is 95% complementary to SEQ ID NO: 59 (e.g., 96% complementary to SEQ ID NO: 59, 97% complementary to SEQ ID NO: 59, 98% complementary to SEQ ID NO: 59, 99% complementary to SEQ ID NO: 59, or 100% complementary to SEQ ID NO: 59).

The native ABCA4 locus in any of the recombinant cells of the invention can further include an endogenous promoter. For example, the chimeric protein may drive expression of ABCA4 from the endogenous promoter at its native genomic locus.

In some instances, the expression modification is an exogenous eukaryotic promoter, such as a CAG promoter. In particular instances, for example, the CAG promoter can include (a) a cytomegalovirus (CMV) early enhancer element; (b) a promoter, first exon, and first intron of chicken beta-actin gene; and (c) a splice acceptor of a rabbit beta-globin gene. The exogenous eukaryotic promoter (e.g., the CAG promoter) can be located upstream of the ABCA4 gene and may drive expression of the ABCA4 gene. In some embodiments, the polynucleotide further comprises a mouse PGK promoter (e.g., a PGK promoter located upstream of the CAG promoter).

in any of the preceding embodiments of the recombinant ceil, the cell can further include an ABCA4 disruption modification that decreases expression of functional ABCA4 from a native ABCA4 locus. In some embodiments, the ABCA4 disruption modification causes a disruption in one or more ABCA4 exons (e.g., two or more ABCA4 exons, e.g., two and only two ABCA4 exons). For example, exemplary ceils of the invention have an ABCA4 disruption modification that causes a disruption in ABCA4 exons 3 and 4. In some embodiments, the ABCA4 disruption modification is a CRISPR/Cas9- mediated ablation, as describe in the example below.

Any suitable methods known in the art or described herein can be used for generating any of the ceils of the invention, e.g , as described above. For example, the methods of generating such cells include inserting an ABCA4 expression modification (e.g., a TALEN, such as those described herein, or an exogenous eukaryotic promoter (e.g., a CAG promoter)) in the native genome of a host cell (e.g., a mammalian host cell, e.g., a HEK293T cell), wherein the ABCA4 expression modification increases expression of ABCA4 from a native ABCA4 locus. In some embodiments, methods of producing any of the recombinant cells of the invention further include inserting an ABCA4 disruption modification that decreases expression of functional ABCA4 from a native ABCA4 locus, e.g., using CRISPR, e.g., CRISPR/Cas9.

Screening of ABCA4-Restoring Candidate Agents

The invention provides cell lines that can be used to screen or test agents that restore ABCA4 protein expression (e.g., for ABCA4-assoicated disorders, such as Stargardt macular dystrophy (STGD, or Stargardt Disease), fundus flavimaculatus, cone-rod dystrophy, retinitis pigmentosa, and age related macular degeneration). Such agents include therapies that rely on the detection of (e.g., by binding, or hybridizing to) an ABCA4 intron (e.g., intron 19, 22, 23, or 24). For example, an ABCA4-restoring agent that can be screened or tested is an ABCA4 trans-splicing molecule, which can replace a defective exon or a plurality of defective exons by binding to an intron. Such ABCA4 trans-splicing molecules are described, for example, in WO 2017/087900.

The presence and/or expression level/amount of ABCA4 in a cell or culture of cells of the invention can be analyzed by a number of methodologies, many of which are known in the art and understood by the skilled artisan, including, but not limited to, immunohistochemistry (“IHC”), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting (“FACS”), MassARRAY, proteomics, quantitative blood based assays (e.g., Serum ELISA), biochemical enzymatic activity assays, in situ hybridization, fluorescence in situ hybridization (FISH), Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction (PCR) including quantitative real time PCR (qRT-PCR) and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like, RNA-seq, microarray analysis, gene expression profiling, and/or serial analysis of gene expression (“SAGE”), as well as any one of the wide variety of assays that can be performed by protein, gene, and/or tissue array analysis. Typical protocols for evaluating the status of genes and gene products are found, for example in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15

(Immunoblotting) and 18 (PCR Analysis). Multiplexed immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery (“MSD”) may also be used.

Any suitable method for measuring ABCA4 protein expression can be used as part of the methods provided herein, or in the generation of a cell line disclosed herein. Such methods are known in the art or provided herein. For example, a protein expression level of ABCA4 can be determined using a method selected from the group consisting of flow cytometry (e.g., fluorescence-activated cell sorting (FACS)), Western blot, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation,

immunohistochemistry (IHC), immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, and HPLC.

Methods for the evaluation of mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled riboprobes specific for the one or more genes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for one or more of the genes, and other amplification type detection methods, such as, for example, branched DNA, SISBA,

TMA and the like). In addition, such methods can include one or more steps that allow one to determine the levels of mRNA in a culture (e.g., by simultaneously examining the levels a comparative control mRNA sequence of a“housekeeping” gene such as an actin family member).

In some instances, the expression level of ABCA4 in the cell or culture of cells that have been contacted with an ABCA4-restoring agent is an average expression (e.g., mean expression or median expression) of ABCA4, and the reference expression level of the ABCA4 is an average expression (e.g., mean expression or median expression) of ABCA4 by the cell or culture of the cells without treatment (e.g., of the same agent or of any ABCA4-restoring agent), or before treatment. In some embodiments of any of the preceding methods, elevated or increased expression refers to an overall increase of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%,

97%, 98%, 99% or greater, in the level of ABCA4 protein or nucleic acid (e.g., gene (DNA or mRNA))), detected by standard art-known methods such as those described herein, as compared to a reference (e.g., control, such as a housekeeping gene). In some embodiments, elevated expression refers to an overall increase of greater than about 1 .5 fold, about 1 .75 fold, about 2 fold, about 2.25 fold, about 2.5 fold, about 2.75 fold, about 3.0 fold, or about 3.25 fold as compared to a reference (e.g., control, such as a housekeeping gene).

ABCA4 Trans-splicing molecules

The cell lines of the present invention are useful for screening candidate agents for restoration of ABCA4 expression (e.g., in a subject having a disease associated with an ABCA4 defect), such as ABCA4 trans-splicing molecules. Such ABCA4 trans-splicing molecules are described in

WO 2017/087900 and WO 2019/204514, each of which is incorporated herein by reference in its entirety. Thus, in some instances, methods of the present invention involve using an ABCA4 cell line as described herein to test an ABCA4 trans-splicing molecules as described herein.

ABCA4 trans-splicing molecules are useful for treating diseases and disorders associated with a mutation in an ABCA4 gene by replacing one or more exons in the ABCA4 gene (e.g., an ABCA4 gene having a mutated ABCA4 exon). In some embodiments, the nucleic acid trans-splicing molecule is a pre- RNA trans-splicing molecule (RTM). The design of the trans-splicing molecule permits replacement of the defective or mutated portion of the pre-mRNA exon(s) with a nucleic acid sequence, e.g., the exon(s) having a functional (e.g., normal) sequence without the mutation. The functional sequence can be a wild- type, naturally-occurring sequence or a corrected sequence with some other modification, e.g., codon optimization.

In one embodiment, a trans-splicing molecule is configured to correct one or more mutations located on a 3’ portion of the ABCA4 gene. In one embodiment, a trans-splicing molecule is configured to correct one or more mutations located on a 5’ portion of the ABCA4 gene. The trans-splicing molecules described herein function to repair the defective gene in the target cell of a subject by replacing the defective pre-mRNA gene sequence and removing the defective portion of the target pre-mRNA, yielding a functional ABCA4 gene capable of transcribing a functional gene product in the cell.

Trans-splicing molecules have a binding domain configured to bind a target ABCA4 intron, a splicing domain configured to mediate trans-splicing, and a coding domain having one or more functional ABCA4 exons. In a 5’ trans-splicing molecule, the coding domain, splice site, and binding domain are operatively linked in a 5’-to-3’ direction, such that the trans-splicing molecule is configured to replace the 5’ end of the endogenous gene with the coding domain, which includes a functional ABCA4 exon to replace the mutated ABCA4 exon. Conversely, in a 3’ trans-splicing molecule, the coding domain, splice site, and binding domain are operatively linked in a 3’-to-5’ direction, such that the trans-splicing molecule is configured to replace the 3’ end of the endogenous gene with the coding domain, which includes a functional ABCA4 exon to replace the mutated ABCA4 exon. In some embodiments, the splicing domain resides within an artificial intron, which links the binding domain to the coding domain. The artificial intron may include additional components, such as a spacer. In some embodiments, the trans-splicing molecule or coding domain thereof is up to 4,700 nucleotide bases in length (e.g., from 200 to 300 nucleotide bases in length, from 300 to 400 nucleotide bases in length, from 400 to 500 nucleotide bases in length, from 500 to 600 nucleotide bases in length, from 600 to 700 nucleotide bases in length, from 700 to 800 nucleotide bases in length, form 800 to 900 nucleotide bases in length, from 900 to 1 ,000 nucleotide bases in length, from 1 ,000 to 1 ,500 nucleotide bases in length, from 1 ,500 to 2,000 nucleotide bases in length, from 2,000 to 2,500 nucleotide bases in length, from 2,500 to 3,000 nucleotide bases in length, or from 3,000 to 4,000 nucleotide bases in length, e.g., from 3,100 to 3,800 nucleotide bases in length, from 3,200 to 3,700 nucleotide bases in length, or from 3,300 to 3,500 nucleotide bases in length, e.g., from 3,000 to 3,100 nucleotide bases in length, from 3,100 to 3,200 nucleotide bases in length, from 3,200 to 3,300 nucleotide bases in length, from 3,300 to 3,400 nucleotide bases in length, from 3,400 to 3,500 nucleotide bases in length, from 3,500 to 3,600 nucleotide bases in length, from 3,600 to 3,700 nucleotide bases in length, from 3,700 to 3,800 nucleotide bases in length, from 3,800 to 3,900 nucleotide bases in length, or from 3,900 to 4,000 nucleotide bases in length, e.g., about 2,918 nucleotide bases in length, about 3,328 nucleotide bases in length, about 3,522 nucleotide bases in length, about 3,607 nucleotide bases in length, about 3,632 nucleotide bases in length, about 3,494 nucleotide bases in length, or about 3,300 nucleotide bases in length).

Due to the large size of the ABCA4 gene and the size constraints of AAV delivery, a single transsplicing molecule configured for packaging within an AAV vector may not span all mutations in an ABCA4 gene that may be associated with a disorder and thereby may not correct mutations along the length of the entire ABCA4 gene. Accordingly, the trans-splicing molecules of the invention can be adapted as part of methods described below to correct multiple mutations spanning the entire length of the ABCA4 gene.

An ABCA4 gene targeted by a trans-splicing molecule described herein contains one or multiple mutations that are associated with (e.g., cause, or are correlated with) a disease, such as a Stargardt Disease (e.g., Stargardt Disease 1). An exemplary DNA sequence of a functional (wildtype) human ABCA4 gene is given by the NCBI Reference Sequence: NG_009073. The amino acid sequence of an exemplary protein retinal-specific ATP-binding cassette transporter expressed by ABCA4 is given by Protein Accession No. P78363.

In addition to these published sequences, all corrections later obtained or naturally occurring conservative and non-disease-causing variants sequences that occur in the human or other mammalian population are also included. Additional conservative nucleotide replacements or those causing codon optimizations are also included. The sequences as provided by the database accession numbers may also be used to search for homologous sequences in the same or another mammalian organism.

It is anticipated that the ABCA4 nucleic acid sequences and resulting protein truncates or amino acid fragments may tolerate certain minor modifications at the nucleic acid level to include, for example, modifications to the nucleotide bases which are silent, e.g., preference codons. In other embodiments, nucleic acid base modifications which change the amino acids, e.g., to improve expression of the resulting peptide/protein (for example, codon optimization) are anticipated. Also included as likely modification of fragments are allelic variations, caused by the natural degeneracy of the genetic code.

Also included as modifications of ABCA4 genes are analogs, or modified versions, of the encoded protein fragments provided herein. Typically, such analogs differ from the specifically identified proteins by only one to four codon changes. Conservative replacements are those that take place within a family of amino acids that are related in their side chains and chemical properties. The nucleic acid sequence of a functional ABCA4 gene may be derived from any mammal which natively expresses functional retinal-specific ATP-binding cassette transporter, or homolog thereof. In other embodiments, certain modifications are made to the ABCA4 gene sequence in order to enhance expression in the target cell. Such modifications include codon optimization.

In some embodiments, the disorder associated with a mutation in ABCA4 is an autosomal recessive disease, for example, Stargardt Disease. In certain instances involving a subject having an autosomal recessive disorder, the subject has a mutation in ABCA4 on both alleles. Compositions comprising trans-splicing molecules can correct the mutations on both alleles, regardless of the location of the mutation within the ABCA4 gene. For instance, for a subject having a mutated ABCA4 exon 1 on a first allele and a mutated ABCA4 exon 30 on a second allele, provided herein is a composition having a 5’ trans-splicing molecule to replace the mutated ABCA4 exon 1 and a 3’ trans-splicing molecule to replace the mutated ABCA4 exon 30. In such embodiments, the two trans-splicing molecules can be co-delivered as part of the same AAV vector or delivered in separate AAV vectors (e.g., in the case in which both trans-splicing molecules exceed the packaging limit of AAV).

Alternatively, in embodiments in which two or more mutations are located on a portion of the ABCA4 gene that can be replaced by the same trans-splicing molecule, a single trans-splicing molecule having a coding region containing a functional ABCA4 exon can replace the one or more exons containing the mutations. Mutations in particular ABCA4 exons are also listed in International Patent Publication No. WO 2017/087900, incorporated herein by reference.

ABCA4 Coding domains

In some embodiments, the coding domain of a 5’ trans-splicing molecule includes all ABCA4 exons (e.g., functional ABCA4 exons) that are 5’ to the target ABCA4 intron. For example, in

embodiments in which a 5’ trans-splicing molecule targets ABCA4 intron 19, the coding domain includes functional ABCA4 exons 1-19. In such embodiments featuring a 5’ trans-splicing molecule having a coding domain including functional ABCA4 exons 1-19, the coding domain is about 2918 bp in length. In embodiments in which a 5’ trans-splicing molecule targets ABCA4 intron 22, the coding domain includes functional ABCA4 exons 1-22. In such embodiments featuring a 5’ trans-splicing molecule having a coding domain including functional ABCA4 exons 1-22, the coding domain is about 3,328 bp in length. In embodiments in which a 5’ trans-splicing molecule targets ABCA4 intron 23, the coding domain includes functional ABCA4 exons 1-23. In such embodiments featuring a 5’ trans-splicing molecule having a coding domain including functional ABCA4 exons 1-23, the coding domain is about 3,522 bp in length. In embodiments in which a 5’ trans-splicing molecule targets ABCA4 intron 24, the coding domain includes functional ABCA4 exons 1-24. In such embodiments featuring a 5’ trans-splicing molecule having a coding domain including functional ABCA4 exons 1-24, the coding domain is about 3,607 bp in length.

The aforementioned embodiments of 5’ ABCA4-targeting trans-splicing molecules are illustrated at the lower left-hand portion of FIG. 1.

In some embodiments, the coding domain of a 3’ trans-splicing molecule includes any one or more of ABCA4 exons 20-50. For example, in embodiments in which a 3’ trans-splicing molecule targets ABCA4 intron 22, the coding domain includes functional ABCA4 exons 23-50. In such embodiments featuring a 3’ trans-splicing molecule having a coding domain including functional ABCA4 exons 23-50, the coding domain is about 3,632 bp in length. In embodiments in which a 3’ trans-splicing molecule targets ABCA4 intron 23, the coding domain includes functional ABCA4 exons 24-50. In such embodiments featuring a 3’ trans-splicing molecule having a coding domain including functional ABCA4 exons 24-50, the coding domain is about 3,494 bp in length. In embodiments in which a 3’ trans-splicing molecule targets ABCA4 intron 24, the coding domain includes functional ABCA4 exons 25-50. In such embodiments featuring a 3’ trans-splicing molecule having a coding domain including functional ABCA4 exons 25-50, the coding domain is about 3,300 bp in length. The aforementioned embodiments of 3’ ABCA4-targeting trans-splicing molecules are illustrated at the upper right-hand portion of FIG. 1.

In some embodiments, the coding domain includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, or 28 functional ABCA4 exons.

In some instances, both mutations occur in the 5’ portion of the target gene, and a 5’ transsplicing molecule is selected to correct both mutations. In one embodiment, the binding domain binds to intron 19, and the coding domain includes functional ABCA4 exons 1-19. In one embodiment, the binding domain binds to intron 22, and the coding domain includes functional ABCA4 exons 1-22. In one embodiment, the binding domain binds to intron 23, and the coding domain includes functional ABCA4 exons 1-23. In one embodiment, the binding domain binds to intron 24, and the coding domain includes functional ABCA4 exons 1-24. Alternatively, in instances in which both mutations occur on the 3’ portion of the target gene, a 3’ trans-splicing molecule is selected to correct both mutations. In one embodiment, the binding domain binds to intron 22, and the coding domain includes functional ABCA4 exons 23-50. In one embodiment, the binding domain binds to intron 23, and the coding domain includes functional ABCA4 exons 24-50. In one embodiment, the binding domain binds to intron 24, and the coding domain includes functional ABCA4 exons 25-50.

As one example, a 3’ pre-mRNA ABCA4 trans-splicing molecule operates as follows: A chimeric mRNA is created through a trans-splicing reaction mediated by the spliceosome between the 5’ splice site of the endogenous target pre-mRNA and the 3’ splice site of the trans-splicing molecule. The transsplicing molecule binds through specific base pairing to a target ABCA4 intron of the endogenous target pre-mRNA and replaces the whole 3’ sequence of the endogenous ABCA4 gene upstream of the targeted intron with the coding domain having a functional ABCA4 exon sequence of the trans-splicing molecule.

A 3’ trans-splicing molecule includes a binding domain which binds to the target ABCA4 intron 5’ to the mutation or defect, an artificial intron comprising optional spacer and a 3’ splice site, and a coding domain that encodes all exons of the ocular target gene that are 3’ to the binding of the binding domain to the target. A 5’ trans-splicing molecule includes a binding domain binds to the target ABCA4 intron 3’ to the mutation or defect, a 5’ splice site, an optional spacer and a coding domain that encodes all exons of the ocular target gene that are 5’ to the binding of the binding domain to the target.

In some embodiments, the coding domain includes a complementary DNA (cDNA) sequence.

For example, one or more functional ABCA4 exons within the coding domain can be a cDNA sequence.

In some embodiments, the entire coding domain is a cDNA sequence. Additionally or alternatively, all or a portion of the coding domain, or one or more functional ABCA4 exons thereof, can be a naturally- occurring sequence (e.g., a sequence having 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an endogenous ABCA4 exon).

In some embodiments, all or a portion of the coding domain, or one or more functional ABCA4 exons thereof, is a codon-optimized sequence in which a nucleic acid sequence has been modified, e.g., to enhance expression or stability, without resulting in a change in the encoded amino acid. Codon optimization may be performed in a manner such as that described in, e.g., U.S. Patent Nos. 7,561 ,972, 7,561 ,973, and 7,888,1 12, each of which is incorporated herein by reference in its entirety. For delivery via a recombinant AAV, as described herein, in one embodiment, the coding domain can be a nucleic acid sequence of up to 4,000 nucleotide bases in length (e.g., from 3,000 to 4,000 nucleotide bases in length, from 3,100 to 3,800 nucleotide bases in length, from 3,200 to 3,700 nucleotide bases in length, or from 3,300 to 3,500 nucleotide bases in length, e.g., from 3,000 to 3,100 nucleotide bases in length, from 3,100 to 3,200 nucleotide bases in length, from 3,200 to 3,300 nucleotide bases in length, from 3,300 to 3,400 nucleotide bases in length, from 3,400 to 3,500 nucleotide bases in length, from 3,500 to 3,600 nucleotide bases in length, from 3,600 to 3,700 nucleotide bases in length, from 3,700 to 3,800 nucleotide bases in length, from 3,800 to 3,900 nucleotide bases in length, or from 3,900 to 4,000 nucleotide bases in length).

ABCA4 Binding Domains

Trans-splicing molecules of the invention feature a binding domain configured to bind a target ABCA4 intron. In one embodiment, the binding domain is a nucleic acid sequence complementary to a sequence of the target ABCA4 pre-mRNA (e.g., a target ABCA4 intron) to suppress endogenous target cis-splicing while enhancing trans-splicing between the trans-spicing molecule and the target ABCA4 pre- mRNA, e.g., to create a chimeric molecule having a portion of endogenous ABCA4 mRNA and the coding domain having one or more functional ABCA4 exons. In some embodiments, the binding domain is in an antisense orientation to a sequence of the target ABCA4 intron.

A 5’ trans-splicing molecule will generally bind the target ABCA4 intron 3’ to the mutation, while a 3’ trans-splicing molecule will generally bind the target ABCA4 intron 5’ to the mutation. In one embodiment, the binding domain comprises a part of a sequence complementary to the target ABCA4 intron. In one embodiment herein, the binding domain is a nucleic acid sequence complementary to the intron closest to (i.e. , adjacent to) the exon sequence that is being corrected.

In another embodiment, the binding domain is targeted to an intron sequence in close proximity to the 3’ or 5’ splice signals of a target intron. In still another embodiment, a binding domain sequence can bind to the target intron in addition to part of an adjacent exon.

Thus, in some instances, the binding domain binds specifically to the mutated endogenous target pre-mRNA to anchor the coding domain of the trans-splicing molecule to the pre-mRNA to permit transsplicing to occur at the correct position in the target ABCA4 gene. The spliceosome processing machinery of the nucleus may then mediate successful trans-splicing of the corrected exon for the mutated exon causing the disease.

In certain embodiments, the trans-splicing molecules feature binding domains that contain sequences on the target pre-mRNA that bind in more than one place. The binding domain may contain any number of nucleotides necessary to stably bind to the target pre-mRNA to permit trans-splicing to occur with the coding domain. In one embodiment, the binding domains are selected using mFOLD structural analysis for accessible loops (Zuker, Nucleic Acids Res. 2003, 31 (13): 3406-3415).

Suitable target binding domains can be from 10 to 500 nucleotides in length. In some embodiments, the binding domain is from 20 to 400 nucleotides in length. In some embodiments, the binding domain is from 50 to 300 nucleotides in length. In some embodiments, the binding domain is from 100 to 200 nucleotides in length. In some embodiments, the binding domain is from 10-20 nucleotides in length (e.g., 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length), 20-30 nucleotides in length (e.g., 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length), 30-40 nucleotides in length (e.g., 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length), 40-50 nucleotides in length (e.g., 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides in length), 50-60 nucleotides in length (e.g., 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides in length), 60-70 nucleotides in length (e.g., 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, or 70 nucleotides in length), 70-80 nucleotides in length (e.g., 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length), 80-90 nucleotides in length (e.g., 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, or 90 nucleotides in length), 90-100 nucleotides in length (e.g., 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length), 100-110 nucleotides in length (e.g., 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, or 110 nucleotides in length), 110-120 nucleotides in length (e.g., 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, or 120 nucleotides in length), 120-130 nucleotides in length (e.g., 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, or 130 nucleotides in length), 130-140 nucleotides in length (e.g., 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, or 140 nucleotides in length), 140-150 nucleotides in length (e.g., 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, or 150 nucleotides in length), 150-160 nucleotides in length (e.g., 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, or 160 nucleotides in length), 160-170 nucleotides in length (e.g., 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, or 170 nucleotides in length), 170-180 nucleotides in length (e.g., 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, or 180 nucleotides in length), 180-190 nucleotides in length (e.g., 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, or 190 nucleotides in length), 190-200 nucleotides in length (e.g., 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, or 200 nucleotides in length), 200-210 nucleotides in length, 210-220 nucleotides in length, 220-230 nucleotides in length, 230-240 nucleotides in length, 240-250 nucleotides in length, 250-260 nucleotides in length, 260-270 nucleotides in length, 270-280 nucleotides in length, 280-290 nucleotides in length, 290-300 nucleotides in length, 300-350 nucleotides in length, 350-400 nucleotides in length, 400-450 nucleotides in length, or 450-500 nucleotides in length. In some embodiments, the binding domain is about 150 nucleotides in length. In another embodiment, the target binding domains may include a nucleic acid sequence up to 750 nucleotides in length. In another embodiment, the target binding domains may include a nucleic acid sequence up to 1000 nucleotides in length. In another embodiment, the target binding domains may include a nucleic acid sequence up to 2000 nucleotides or more in length.

In some embodiments, the specificity of the trans-splicing molecule may be increased by increasing the length of the target binding domain. Other lengths may be used depending upon the lengths of the other components of the trans-splicing molecule.

The binding domain may be from 80% to 100% complementary to the target intron to be able to hybridize stably with the target intron. For example, in some embodiments, the binding domain is 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complimentary to the target intron. The degree of complementarity is selected by one of skill in the art based on the need to keep the trans-splicing molecule and the nucleic acid construct containing the necessary sequences for expression and for inclusion in the rAAV within a 3,000 or up to 4,000 nucleotide base limit. The selection of this sequence and strength of hybridization depends on the complementarity and the length of the nucleic acid.

Any of the aforementioned binding domains may bind to a binding site within intron 19 (SEQ ID NO: 25), intron 22 (SEQ ID NO: 28) intron 23 (SEQ ID NO: 29), or intron 24 (SEQ ID NO: 30). In certain instances of the invention, the trans-splicing molecule is a 5’ trans-splicing molecule and features a binding domain that binds to intron 19 of ABCA4 (SEQ ID NO: 25) and includes a coding domain having functional ABCA4 exons 1-19. In some embodiments, the binding site comprises any one or more of nucleotides 990 to 2,174 of SEQ ID NO: 25 (e.g., any one or more of nucleotides 1 ,670 to 2,174 of SEQ ID NO: 25, any one or more of nucleotides 1 ,810 to 2,000 of SEQ ID NO: 25, any one or more of nucleotides 1 ,870 to 2,000 of SEQ ID NO: 25, or any one or more of nucleotides 1 ,920 to 2,000 of SEQ ID NO: 25).

In some embodiments, the trans-splicing molecule is a 5’ trans-splicing molecule and features a binding domain that binds to intron 22 of ABCA4 (SEQ ID NO: 28) and includes a coding domain having functional ABCA4 exons 1-22. In some embodiments, the binding site comprises any one or more of nucleotides 60 to 570, nucleotides 600 to 800, or nucleotides 900 to 1 ,350 of SEQ ID NO: 28 (e.g., any one or more of nucleotides 70 to 250 of SEQ ID NO: 28).

Alternatively, the trans-splicing molecule can be a 3’ trans-splicing molecule and can feature a binding domain that binds to intron 22 of ABCA4 (SEQ ID NO: 28). This trans-splicing molecules may include a coding domain having functional ABCA4 exons 23-50. In some embodiments, the binding site comprises any one or more of nucleotides 1 to 510 or 880 to 1 ,350 of SEQ ID NO: 28.

In other embodiments, the trans-splicing molecule is a 5’ trans-splicing molecule and features a binding domain that binds to intron 23 of ABCA4 (SEQ ID NO: 29) and includes a coding domain having functional ABCA4 exons 1-23. In some embodiments, the binding site comprises any one or more of nucleotides 80 to 570 or 720 to 1 ,081 of SEQ ID NO: 29.

Alternatively, the trans-splicing molecule can be a 3’ trans-splicing molecule and can feature a binding domain that binds to intron 23 of ABCA4 (SEQ ID NO: 29) and a coding domain having functional ABCA4 exons 24-50. In some embodiments, the binding site comprises any one or more of nucleotides 80 to 1 ,081 of SEQ ID NO: 29 (e.g., any one or more of nucleotides 230 to 1 ,081 of SEQ ID NO: 29, any one or more of nucleotides 250 to 400 of SEQ ID NO: 29, or any one or more of nucleotides 690 to 850 of SEQ ID NO: 29).

In some embodiments, the trans-splicing molecule is a 5’ trans-splicing molecule and features a binding domain that binds to intron 24 of ABCA4 (SEQ ID NO: 30) and includes a coding domain having functional ABCA4 exons 1-24. In some embodiments, the binding site comprises any one or more of nucleotides 600 to 1 ,250 or 1 ,490 to 2,660 of SEQ ID NO: 30 (e.g., any one or more of nucleotides 1 ,000 to 1 ,200 of SEQ ID NO: 30).

In other embodiments, the trans-splicing molecule is a 3’ trans-splicing molecule and features a binding domain that binds to intron 24 of ABCA4 (SEQ ID NO: 30) and includes a coding domain having functional ABCA4 exons 25-50. In some embodiments, the binding site comprises any one or more of nucleotides 1 to 250, nucleotides 300 to 2,100, or nucleotides 2,200 to 2,692 of SEQ ID NO: 30 (e.g., any one or more of nucleotides 360 to 610 of SEQ ID NO: 30 or any one or more of nucleotides 750 to 1 ,110 of SEQ ID NO: 30).

Splicing Domains

The following splicing domains can be used in any of the ABCA4 trans-splicing molecules described herein. The splicing domain can include a splice site, a branch point, and/or a PPT tract to mediate trans-splicing. In some embodiments, a splicing domain has a single splice site, which denotes that the splice site enables trans-splicing, but not cis-splicing, due to the lack of a corresponding splice site. In some embodiments, the splicing domains of the 3’ trans-splicing molecule include a strong conserved branch point or branch site sequence, a polypyrimidine tract (PPT), and a 3’ splice acceptor (AG or YAG) site and/or a 5’ splice donor site. The splicing domains of the 5’ trans-splicing molecule do not contain the branch point or PPT, but comprise a 5’ splice acceptor/or 3’ splice donor splice site.

Splicing domains may be selected by one of skill in the art according to known methods and principles. The splicing domain provides essential consensus motifs that are recognized by the spliceosome. The use of branch point and PPT follows consensus sequences required for performance of the two phosphoryl transfer reaction involved in trans-splicing. In one embodiment a branch point consensus sequence in mammals is YNYURAC (Y=pyrimidine; N=any nucleotide). A polypyrimidine tract is located between the branch point and the splice site acceptor and is important for different branch point utilization and 3’ splice site recognition. Consensus sequences for the 5’ splice donor site and the 3’ splice region used in RNA splicing are well known in the art. In addition, modified consensus sequences that maintain the ability to function as 5’ donor splice sites and 3’ splice regions may be used. Briefly, in one embodiment, the 5’ splice site consensus sequence is the nucleic acid sequence AG/GURAGU (where / indicates the splice site). In another embodiment the endogenous splice sites that correspond to the exon proximal to the splice site can be employed to maintain any splicing regulatory signals.

In one embodiment, a suitable 5’ splice site with spacer is: 5’- GTA AGA GAG CTC GTT GCG ATA TTA T-3’ (SEQ ID NO: 1). In one embodiment, a suitable 5’ splice site is AGGT.

In one embodiment, a suitable 3’ trans-splicing molecule branch site is 5’-TACTAAC-3’. In one embodiment, a suitable 3’ splice site is: 5’- TAC TAA CTG GTA CCT CTT CTT TTT TTT CTG CAG -3’ (SEQ ID NO: 2) or 5’-CAGGT-3’. In one embodiment, a suitable 3’ trans-splicing molecule PPT is: 5’- TGG TAC CTC TTC TTT TTT TTC TG-3’ (SEQ ID NO: 3).

Additional Components or Modifications

In some embodiments of any of the trans-splicing molecules described herein, the splicing domain is included as part of an artificial intron, which may include one or more additional components. For example, a spacer region may be included within an artificial intron to separate the splicing domain from the target binding domain in the trans-splicing molecule. The spacer region may be designed to include features such as (i) stop codons which would function to block translation of any unspliced transsplicing molecule and/or (ii) sequences that enhance trans-splicing to the target pre-mRNA. The spacer may be between 3 to 25 nucleotides or more depending upon the lengths of the other components of the trans-splicing molecule and the rAAV limitations. In one embodiment, a suitable 5’ trans-splicing molecule spacer is AGA TCT CGT TGC GAT ATT AT (SEQ ID NO: 4). In one embodiment, a suitable 3 ' spacer is: 5 '- GAG AAC ATT ATT ATA GCG TTG CTC GAG -3’ (SEQ ID NO: 5).

Still other optional components of the trans-splicing molecules (e.g., as part of artificial introns) include mini introns, and intronic or exonic enhancers (e.g., intronic splice enhancers, e.g., downstream intronic splice enhancers) or silencers that would regulate the trans-splicing.

In another embodiment, the trans-splicing molecule further comprises (e.g., as part of an artificial intron) at least one safety sequence incorporated into the spacer, binding domain, or elsewhere in the trans-splicing molecule to prevent nonspecific trans-splicing. This is a region of the trans-splicing molecule that covers elements of the 3’ and/or 5’ splice site of the trans-splicing molecule by relatively weak complementarity, preventing non-specific trans-splicing. The trans-splicing molecule is designed in such a way that upon hybridization of the binding/targeting portion(s) of the trans-splicing molecule, the 3’ or 5’ splice site is uncovered and becomes fully active. Such safety sequences comprise a

complementary stretch of cis-sequence (or could be a second, separate, strand of nucleic acid) which binds to one or both sides of the trans-splicing molecule branch point, pyrimidine tract, 3’ splice site and/or 5’ splice site (splicing elements), or could bind to parts of the splicing elements themselves. The binding of the safety sequence may be disrupted by the binding of the target binding region of the transsplicing molecule to the target pre-mRNA, thus exposing and activating the splicing elements (making them available to trans-splice into the target pre-mRNA). In another embodiment, the trans-splicing molecule has 3’ UTR sequences or ribozyme sequences added to the 3’ or 5’ end.

In an embodiment, splicing enhancers such as, for example, sequences referred to as exonic splicing enhancers may also be included in the structure of an artificial intron. Additional features can be added to the artificial intron, such as polyadenylation signals to modify RNA expression/stability, or 5’ splice sequences to enhance splicing, additional binding regions, safety-self complementary regions, additional splice sites, or protective groups to modulate the stability of the molecule and prevent degradation. In addition, stop codons may be included in the trans-splicing molecule (e.g., as part of the artificial intron) structure to prevent translation of unspliced trans-splicing molecules. Additional elements, such as a 3’ hairpin structure, circularized RNA, nucleotide base modification, or synthetic analogs can be incorporated into trans-splicing molecules to promote or facilitate nuclear localization and spliceosomal incorporation, and intra-cellular stability.

In some embodiments, binding of a trans-splicing molecule to the target pre-mRNA is mediated by complementarity (i.e. based on base-pairing characteristics of nucleic acids), triple helix formation, or protein-nucleic acid interaction (as described in documents cited herein). In one embodiment, the nucleic acid trans-splicing molecule includes DNA, RNA, or DNA/RNA hybrid molecules, wherein the DNA or RNA is either single or double stranded. Also included herein are RNAs or DNAs, which can hybridize to one of the aforementioned RNAs or DNAs, preferably under stringent conditions, for example, at 60°C in 2.5x SSC buffer and several washes at 37°C at a lower buffer concentration, for example, 0.5x SSC buffer. These nucleic acids can encode proteins exhibiting lipid phosphate phosphatase activity and/or association with plasma membranes. When trans-splicing molecules are synthesized in vitro, such transsplicing molecules can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization to the target mRNA, transport into the cell, stability in the cells to enzymatic cleavage, etc. For example, modification of a trans-splicing molecule to reduce the overall charge can enhance the cellular uptake of the molecule. In addition, modifications can be made to reduce susceptibility to nuclease or chemical degradation. The nucleic acid molecules may be synthesized in such a way as to be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

Various other well-known modifications to the nucleic acid molecules can be introduced as a means of increasing intracellular stability and half-life (see also above for oligonucleotides). Possible modifications are known to the art. Modifications, which may be made to the structure of synthetic transsplicing molecules include backbone modifications. EXAMPLES

These Examples describe development of ABCA4 cell lines, which can be used, e.g., for testing therapies (e.g., trans-splicing molecules) that restore ABCA4 expression in patients suffering from diseases associated with mutations in ABCA4 (e.g., Stargardt Disease).

Example 1. HEK293T cells

Human kidney-derived HEK293T cell lines expressing ABCA4 were generated. The ABCA4 gene is only known to be expressed in living photoreceptors in the retina, and full-length ABCA4 pre- mRNA and protein are not generally detectable in cultured cells in vitro. Therefore, to test trans-splicing strategies for ABCA4, cells were engineered to express ABCA4 from its native genomic locus on chromosome 1 (1 p22.1). Two strategies were pursued. In the first case, stable cell lines were derived to express site-specific (upstream of the ABCA4 transcriptional start site) DNA-binding TALENs that were fused to the VP64 viral trans-activator. In the second case, a constitutive eukaryotic promoter was directly inserted (using CRISPR/Cas9) into the genomic locus immediately upstream of the ABCA4 transcriptional start site. The results in both cases were stable cell lines that robustly expressed ABCA4 pre-mRNA and protein.

TALEN cell lines

TALENs targeted to specific domains upstream of the ABCA4 transcriptional start site were designed and fused to a VP-64 trans-activator sequence (FIG. 1). This combination of three TALENs were transfected into HEK293T cells and stable single cell clones were derived. Two clones were shown to direct expression of ABCA4 protein (FIG. 2).

CAG promoter cell lines

The general strategy for deriving CAG promoter cell lines is outlined in FIGS. 3-5. Site-specific guides (FIG. 4A) were designed to insert the CAG promoter and a puromycin selectable marker using homology arms (FIG. 4B). Primers used to validate CAG insertion are shown in FIG. 4C. Puromycin resistant cells were cloned and analyzed by PCR for the desired insertion. Several clonal lines were selected for further analyses. RNA and protein expression for two lines (B6 and C3) are shown in FIGS. 6A and 6B. Both lines clearly contained the promoter insertion, as demonstrated by RNA and protein analyses.

ABCA4 knockout cell lines

Once stable ABCA4 expression was established in cultured cells, knock-outs of ABCA4 expression were generated for testing of ABCA4 trans-splicing molecules designed to restore ABCA4 protein expression. In general, guide RNA and Cas9 protein were co-transfected in B6 cells (CAG- promotor knocked into ABCA4 locus and mediating ABCA4 expression). After nine days, a second transfection with guide RNA and Cas9 protein was performed. The basic design targeting exons 3 and 4 is shown in FIG. 7. Single cells were plated by limiting dilution and once grown, evaluated for protein expression of ABCA4 by Western blot. FIG. 8 shows the RNA and protein profiles of single cell clones derived after treatment with CRISPR/Cas9, as depicted in FIG. 7. There were varying degrees of RNA and protein ablation. Clones 17+06 and 17+21 were chosen because they exhibited complete ABCA4 protein knockout. Mutation analyses (FIGS. 9A-9B and 10) confirmed that exons 3 and 4 were targeted and interrupted.

Example 2. ARPE-19 cells

To confirm that the results observed in Example 1 were not an artifact of the genetic background or transcriptome of the HEK293T cell line, the genetic engineering techniques described in Example 1 were applied to the human retinal pigment epithelia cell line ARPE-19 to induce expression of ABCA4. This process generated the clone ASC_A12, which expressed ABCA4 (FIG. 1 1).

Knock-out engineering as described in Example 1 was performed to generate protein-deficient sub-clones of ARPE-19 cells. ARPE-19 clone 5I was selected for characterization and downstream evaluation. The 5I genotype [NM_00023.3 c.264delAinsTG; c.265_266delGA] is distinct from HEK293T clone 17+06.

Restoration of protein expression was achieved in both 17+06 and 5I clones following transduction of AAV encoding a 5’ trans-splicing molecule targeting ABCA4 (intron 22), but not when a non-targeted trans-splicing molecule was transduced into these cells. FIG. 12.

Together, these results demonstrate that the genetic engineering techniques for trans-splicing molecule-based rescue are applicable across multiple cell types.

The following additional numerated paragraphs further define some embodiments of the invention described herein.

1. A cell modified to express ABCA4, wherein the cell comprises an ABCA4 expression modification that increases expression of ABCA4 from a native ABCA4 locus.

2. The cell of paragraph 1 , wherein the cell comprises a detectable level of pre-mRNA comprising an ABCA4 intron.

3. The cell of paragraph 1 or 2, wherein the ABCA4 expression modification is a TALEN.

4. The cell of paragraph 3, wherein the TALEN is fused to a viral trans-activator.

5. The cell of paragraph 3 or 4, wherein the TALEN is expressed upstream of the ABCA4 transcriptional start site.

6. The cell of paragraph 4 or 5, wherein the viral trans-activator is VP16 or VP64.

7. The cell of any one of paragraphs 3-6, wherein the TALEN comprises a DNA binding domain that specifically binds to a target sequence in a 5^: UTR of the ABCA4 locus.

8. The cell of paragraph 7, wherein the target sequence is located within SEQ ID NO: 56.

9. The cell of paragraph 8, wherein the target sequence is located within any one of SEQ ID NOs: 57-59.

10. The cell of any one of paragraphs 1 -9, wherein the native ABCA4 locus further comprises an endogenous promoter.

11. The cell of paragraph 10, wherein the chimeric protein drives expression of ABCA4 from the endogenous promoter at its native genomic locus.

12. The cell of paragraph 1 , wherein the expression modification is an exogenous eukaryotic promoter. 13. The cell of paragraph 12, wherein the exogenous eukaryotic promoter is a CAG promoter.

14. The cell of paragraph 13, wherein the CAG promoter comprises:

(a) a cytomegalovirus (CMV) early enhancer element,

(b) a promoter, first exon, and first intron of chicken beta-actin gene, and

(c) a splice acceptor of a rabbit beta-globin gene.

15. The cell of paragraph 13 or 14, wherein the CAG promoter is located upstream of the ABCA4 gene.

16. The cell of any one of paragraphs 13-15, wherein the CAG promoter drives expression of the ABCA4 gene.

17. The cell of any one of paragraphs 13-16, wherein the cell further comprises a mouse PGK promoter.

18. The cell of paragraph 17, wherein the mouse PGK promoter is located upstream of the CAG promoter.

19. The cell of any one of paragraphs 1-18, wherein the cell is a eukaryotic cell.

20. The cell of paragraph 19, wherein the eukaryotic cell is an HEK293T cell.

21. The cell of paragraph 19, wherein the cell is a retinal derived cell.

22. The cell of paragraph 21 , wherein the retinal derived cell is an AKPE-19 cell.

23. Thecell of any one of paragraphs 1-22, further comprising an ABGA4 disruption modification that decreases expression of functional ABCA4 from a native ABCA4 locus.

24. The cell of paragraph 23, wherein the ABCA4 disruption modification causes a disruption in one or more ABCA4 exons.

25. The cell of paragraph 24, wherein the ABCA4 disruption modification causes a disruption in two ABCA4 exons.

26. The cell of paragraph 25, wherein the ABCA4 disruption modification causes a disruption in ABCA4 exons 3 and 4.

27. The cell of any one of paragraphs 23-26, wherein the ABCA4 disruption modification is a CRISPR/Cas9-mediated ablation.

28. A method of generating a cell of any one of paragraphs 1-27, the method comprising inserting an ABCA4 expression modification in the native genome of a host cell, wherein the ABCA4 expression modification increases expression of ABCA4 from a native ABCA4 locus.

29. A method of testing a candidate agent for treatment of an ABCA4-associated disorder, the method comprising:

(a) providing a candidate agent for testing;

(b) contacting the candidate agent with the cell of any one of paragraphs 24-27 for a time and condition suitable for change in genetic and/or protein expression in the cell;

(c) measuring expression of ABCA4 by the cell after step (b); and

(d) based on the measurement of step (c), determining whether the candidate agent restores ABCA4 expression.

30. The method of paragraph 29, wherein the candidate agent is an ABCA4-restoring agent.

31. The method of paragraph 30, wherein the ABCA4-restoring agent is an ABCA4 trans-splicing molecule. 32. The method of paragraph 30 or 31 , wherein step (d) comprises comparing the ABCA4 expression by the cell after step (b) with ABCA4 expression by the cell before step (b).

33. The method of paragraph 32, wherein step (d) comprises determining that the candidate agent restores ABCA4 expression based on at least a 1.5-fold restoration of ABCA4 expression by the cell after step (b) compared to ABCA4 expression by the cell before step (b).

34. The method of paragraph 30 or 31 , wherein step (d) comprises comparing the ABCA4 expression by the cell after step (b) with ABCA4 expression by a control cell contacted with a reference agent.

35. The method of paragraph 34, wherein step (d) comprises determining that the candidate agent restores ABCA4 expression based on at least a two-fold restoration of ABCA4 expression by the cell after step (b) compared to ABCA4 expression by the control cell.

36. The method of paragraph 34 or 35, wherein the candidate agent is a candidate ABCA4 transsplicing molecule and the reference agent is a reference ABCA4 trans-splicing molecule.

37. The method of any one of paragraphs 30-36, wherein the ABCA4-restoring agent is for treatment of an ABCA4-associated disorder.

38. The method of paragraph 37, wherein the ABCA4-associated disorder is selected from the group consisting of Stargardt macular dystrophy (STGD), fundus flavimaculatus, cone-rod dystrophy, retinitis pigmentosa, and age-related macular degeneration.

39. The method of any one of paragraphs 30-38, further comprising, administering the ABCA4- restoring agent to a subject.

40. The method of paragraph 39, wherein the subject has an ABCA4-assoicated disorder.

41. The method of paragraph 40, wherein the ABCA4-associated disorder is selected from the group consisting of Stargardt macular dystrophy (STGD), fundus flavimaculatus, cone-rod dystrophy, retinitis pigmentosa, and age-related macular degeneration.

42. The method of any one of paragraphs 39-41 , wherein the subject is a human.

Claims

CLAIMS What is claimed is:

1 . A cell modified to express ABCA4, wherein the cell comprises an ABCA4 expression modification that increases expression of ABCA4 from a native ABCA4 locus.

2. The cell of claim 1 , wherein the cell comprises a detectable level of pre-mRNA comprising an ABCA4 intron.

3. The cell of claim 1 , wherein the ABCA4 expression modification is a TALEN.

4. The cell of claim 3, wherein the TALEN is fused to a viral trans-activator.

5. The cell of claim 3, wherein the TALEN is expressed upstream of the ABCA4 transcriptional start site.

8. The cell of claim 4, wherein the viral trans-activator is VP16 or VP84.

7. The cell of claim 3, wherein the TALEN comprises a DNA binding domain that specifically binds to a target sequence in a 5’ UTR of the ABCA4 locus

8. The cell of claim 7, wherein the target sequence is located within SEQ ID NO: 56.

9. The cell of claim 8, wherein the target sequence is located within SEQ ID NO: 57.

10. The cell of claim 1 , wherein the native ABCA4 locus further comprises an endogenous promoter.

1 1 . The cell of claim 10, wherein the chimeric protein drives expression of ABCA4 from the endogenous promoter at its native genomic locus.

12. The cell of claim 1 , wherein the expression modification is an exogenous eukaryotic promoter.

13. The cell of claim 12, wherein the exogenous eukaryotic promoter is a GAG promoter.

14. The cell of claim 13, wherein the CAG promoter comprises:

(a) a cytomegalovirus (CMV) early enhancer element,

(b) a promoter, first exon, and first intron of chicken beta-actin gene, and

(c) a splice acceptor of a rabbit beta-globin gene.

15. The cell of claim 13, wherein the CAG promoter is located upstream of the ABCA4 gene.

16. The cell of claim 13, wherein the CAG promoter drives expression of the ABCA4 gene.

17. The cell of claim 13, wherein the cell further comprises a mouse PGK promoter.

18. The cell of claim 17, wherein the mouse PGK promoter is located upstream of the CAG promoter.

19. The cell of claim 1 , wherein the cell is a eukaryotic cell.

20. The cell of claim 19, wherein the eukaryotic cell is an HEK293T cell.

21. The cell of claim 19, wherein the cell is a retinal derived cell.

22. The cell of claim 21 , wherein the retinal derived cell is an ARPE-19 ceil.

23. The cell of claim 1 , further comprising an ABCA4 disruption modification that decreases expression of functional ABCA4 from a native ABCA4 locus.

24. The cell of claim 23, wherein the ABCA4 disruption modification causes a disruption in one or more ABCA4 exons.

25. The cell of claim 24, wherein the ABCA4 disruption modification causes a disruption in two ABCA4 exons.

26. The cell of claim 25, wherein the ABCA4 disruption modification causes a disruption in ABCA4 exons 3 and 4.

27. The cell of claim 23, wherein the ABCA4 disruption modification is a CRISPR/Cas9-mediated ablation.

28. A method of generating a cell of claim 1 , the method comprising inserting an ABCA4 expression modification in the native genome of a host cell, wherein the ABCA4 expression modification increases expression of ABCA4 from a native ABCA4 locus.

(a) providing a candidate agent for testing;

(b) contacting the candidate agent with the cell of any one of claims 24-27 for a time and condition suitable for change in genetic and/or protein expression in the cell;

(c) measuring expression of ABCA4 by the cell after step (b); and (d) based on the measurement of step (c), determining whether the candidate agent restores ABCA4 expression.

30. The method of claim 29, wherein the candidate agent is an ABCA4-restoring agent.

31. The method of claim 30, wherein the ABCA4-restoring agent is an ABCA4 trans-splicing molecule.

32. The method of claim 30, wherein step (d) comprises comparing the ABCA4 expression by the cell after step (b) with ABCA4 expression by the cell before step (b).

33. The method of claim 32, wherein step (d) comprises determining that the candidate agent restores ABCA4 expression based on at least a 1.5-fold restoration of ABCA4 expression by the cell after step (b) compared to ABCA4 expression by the cell before step (b).

34. The method of claim 30, wherein step (d) comprises comparing the ABCA4 expression by the cell after step (b) with ABCA4 expression by a control cell contacted with a reference agent.

35. The method of claim 34, wherein step (d) comprises determining that the candidate agent restores ABCA4 expression based on at least a two-fold restoration of ABCA4 expression by the cell after step (b) compared to ABCA4 expression by the control cell.

36. The method of claim 34, wherein the candidate agent is a candidate ABCA4 trans-splicing molecule and the reference agent is a reference ABCA4 trans-splicing molecule.

37. The method of claim 30, wherein the ABCA4-restoring agent is for treatment of an ABCA4- associated disorder.

38. The method of claim 37, wherein the ABCA4-associated disorder is selected from the group consisting of Stargardt macular dystrophy (STGD), fundus flavimaculatus, cone-rod dystrophy, retinitis pigmentosa, and age-related macular degeneration.

39. The method of claim 30, further comprising, administering the ABCA4-restoring agent to a subject.

40. The method of claim 39, wherein the subject has an ABCA4-assoicated disorder.

41. The method of claim 40, wherein the ABCA4-associated disorder is selected from the group consisting of Stargardt macular dystrophy (STGD), fundus flavimaculatus, cone-rod dystrophy, retinitis pigmentosa, and age-related macular degeneration.

42. The method of claim 39, wherein the subject is a human.