Systems, compositions, and methods for converting a cell to a retinal photoreceptor induced neuron (PR-in)

Abstract

Provided herein are recombinant expression systems for converting a cell to a retinal photoreceptor induced neuron (PR-iN) that include (a) a nucleic acid sequence encoding a gene cassette comprising one or more of ASCL1, NEUROG2, OTX2, CRX, LHX4, NRL, NR2E3, NEUROD1, IKZF1, IKZF4, MEF2C, MEF2D, MEIS2, ONECUT1, KLF16, SALL3, ZIC3, POU2F1, POU2F2, RORA, RORB, RXRG, or THRB, and (b) a nucleic acid sequence encoding a porcine teschovirus 2A (p2A) peptide sequence. Additionally, the recombinant expression systems disclosed herein can be used to treat retinal degenerative disorders.

Description

[0001] SYSTEMS, COMPOSITIONS, AND METHODS FOR CONVERTING A CELL TO A RETINAL PHOTORECEPTOR INDUCED NEURON (PR-iN) CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No.

[0002] 63 / 710,852, filed on October 23, 2024. The disclosure of the prior application is considered part of the disclosure of this application and is incorporated herein by reference in its entirety'.

[0003] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with Government support under EY031318 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0005] BACKGROUND

[0006] Photoreceptors (PRs) serve a primary role in visual transduction of light into electrical signals. PRs die in a variety of retinal degenerative disorders, such as age-related macular degeneration, retinitis pigmentosa. Leber congenital amaurosis and since the central nervous system cannot make new photoreceptors, this loss is permanent. In most retinal degenerations, cell loss is primarily in the outer nuclear layer which includes rod and cone photoreceptors. With the loss of post-mitotic neuronal cell populations, an exogenous source of new cells as a replacement for photoreceptor cells is needed.

[0007] SUMMARY

[0008] Provided herein are recombinant expression systems for converting a cell to a retinal photoreceptor induced neuron (PR-iN) that include (a) a nucleic acid sequence encoding a gene cassette comprising one or more of ASCL1, NEUR0G2, 0TX2, CRX, LHX4, NRL, NR2E3, NEURODI, IKZF1, IKZF4, MEF2C, MEF2D, MEIS2, ONECUT1, KLF16, SALL3, ZIC3, POU2F1. POU2F2, RORA, RORB, RXRG, or THRB, and (b) a nucleic acid sequence encoding a porcine teschovirus 2A (p2A) peptide sequence.

[0009] In some embodiments, the gene cassette further comprises an expression control element. In some embodiments, the expression control element comprises (i) a tetracyclineresponsive promoter element (TRE) induced by a reverse tetracycline-controlled transactivator (rtTA), or (ii) a zinc finger-responsive promoter element (ZRE) induced by a synthetic zinc finger transcription regulator (synZiFTR). In some embodiments, the recombinant expression system further comprises a homology arm for a genomic safe harbor site (SHS), an antibiotic selectable marker, a ubiquitous open chromatin element, and a post-transcriptional regulatory element. In some embodiments, the homology arm for SHS genome integration comprises Citrate Lyase Beta-Like (CLYBL), Adeno-Associated Virus integration Site 1 (AAVS1), Region of Gene Insertion 1 (R0GI1). Region of Gene Insertion 2 (ROGI2), L-gulonolactone oxidase (GULOP), or Hippl 1 (Hl 1). In some embodiments, the antibiotic selectable marker is resistant to zeocin, hygromycin, puromycin, or G418 (Geneticin).

[0010] In some embodiments, the recombinant expression system comprises two or more codon-modified p2A peptide sequences. In some embodiments, the two or more codon-modified p2A peptide sequences drive polycistronic expression of the plurality of retinal transcription factors.

[0011] In some embodiments, the cell comprises a fluorescent reporter gene or a reporter tag. In some embodiments, the fluorescent reporter gene comprises a p2A coupled fluorescent protein integrated at a Cone-rod homeobox (CRX) gene, cone arrestin (ARR3) gene, THRB gene, or neural retinal leucine zipper (NRL) gene. In some embodiments, the reporter tag comprises an epitope tag. In some embodiments, the cell comprises a pluripotent stem cell, Muller glia cell, astrocyte, peripheral blood mononuclear cell, urine epithelial cell, or a fibroblast.

[0012] In some embodiments, the gene cassette comprises two or more of ASCLL NEUROG2, OTX2, CRX, LHX4, NRL, NR2E3, NEUR0D1, IKZF1, IKZF4, MEF2C, MEF2D, MEIS2, ONECUT1, KLF16, SALL3, ZIC3, POU2F1, POU2F2, RORA, RORB, RXRG, or THRB. In some embodiments, the gene cassette comprises three or more of ASCL1, NEUR0G2, 0TX2, CRX, LHX4, NRL, NR2E3. NEURODI, IKZF1, IKZF4.

[0013] MEF2C, MEF2D, MEIS2, ONECUT1, KLF16, SALL3, ZIC3, P0U2FL POU2F2, RORA, RORB, RXRG, or THRB.

[0014] Also provided herein are vectors comprising any one of the recombinant expression systems described herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector of an AAV vector. In some embodiments, the vector comprises a class II transposable element (TE).

[0015] Also provided herein are methods of generating a homogeneous population of retinal photoreceptor induced neurons (PR-iNs) that include: obtaining a cell sample; contacting the cell sample with a bone morphogenetic protein (BMP) inhibitor; and administering any one of the recombinant expression systems described herein to the cell sample; thereby generating a homogeneous population of retinal photoreceptor induced neurons (PR-iNs).

[0016] In some embodiments, the cell sample comprises pluripotent stem cells, Muller glia, astrocytes, peripheral blood mononuclear cells, urine epithelial cells, fibroblasts, or any combination thereof.

[0017] In some embodiments, the method further comprises treating the cell with tetracycline, doxycycline, or grazoprevir. In some embodiments, the recombinant expression system is integrated into the genomic safe harbor site in the genome of the cell by using a Casl2 (Cpfl) or Cas9 CRISPR enzy me. In some embodiments, the BMP inhibitor comprises LDN-193189.

[0018] In some embodiments, the cells of the cell sample comprise a photoreceptor reporter. In some embodiments, the photoreceptor reporter comprises a CRX-p2A-h2b-mRuby3 photoreceptor reporter, a cARR3-p2A-mTagBFP2 reporter, a NRL-p2A-h2b-mGreenLantem reporter, a SIXt>-eGFP / VSX2-tdTomato reporter, a SIX6-eGFP / CRX-mRuby3 reporter, a SIX6-p2A-h2b-GFP / CRX-p2A-h2b-mRuby3 / cARR3-p2A-mTagBFP2 reporter, or aCRX-p2A-h2b-mTagBFP2 / cARR3-p2A-h2b-mScarlet / NRL-p2A-h2b-mGreenLantem reporter.

[0019] Also provided herein are methods of treating a retinal degenerative disorder in a subject in need thereof, the method including (a) administering to the subject a cell previously contacted with any one of the recombinant expression systems or any one of the vectors described herein; or (b) administering to the subject aPR-iN produced by performing any one of the methods described herein. In some embodiments, the retinal degenerative disorder comprises age-related macular degeneration, retinitis pigmentosa, or Leber congenital amaurosis.

[0020] Unless otherwise defined, all 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. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0021] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. BRIEF DESCRIPTION OF DRAWINGS

[0022] FIG. 1 shows an exemplary schematic of a retinal photoreceptor induced neuron (PR-iN) system, and micrographs showing endogenous CRX-h2b-mRuby3 expression indicating photoreceptor identity of stem cell derived neurons.

[0023] FIG. 2 shows an exemplary schematic of photoreceptor fate specification, wherein the schematic shows gene activity leading to photoreceptor (PR) diversity.

[0024] FIGs. 3A-3B show an exemplary schematic of reprogramming strategy and differentiation timeline for PR-iN conversion.

[0025] FIG. 4 shows images of reporters for imaging retinal neurons.

[0026] DETAILED DESCRIPTION

[0027] Retinal photoreceptor neurons are specialized light-sensitive cells in the retina that convert light into neural signals. Retinal photoreceptor degeneration is one of the more dominant etiologies of blindness, caused innately or by other various factors, including retinal dysplasia, retinal degeneration, aged macular degeneration, diabetic retinopathy, retinitis pigmentosa, congenital retinal dystrophy, Leber congenital amaurosis, retinal detachment, glaucoma, optic neuropathy, and trauma. Photoreceptor cell implantation is thought to prevent blindness or recover imperfect eyesight by delaying or restraining retinal degeneration, regenerating degenerated retina, and enhancing retinal functions.

[0028] The disclosure describes a cell-based system that can generate retinal photoreceptor neurons from human pluripotent stem cells (PSCs) and potentially other cell types as well (e.g., Muller glia, astrocytes, peripheral blood mononuclear cells, urine epithelial cells or fibroblasts). More specifically, described herein are methods for converting a cell to a retinal photoreceptor induced neuron (PR-iN) comprising (a) a nucleic acid sequence encoding a gene cassette comprising one or more of ASCL1, NEUR0G2, 0TX2, CRX, LHX4, NRL, NR2E3, NEURODI, IKZF1, IKZF4, MEF2C, MEF2D, MEIS2, ONECUT1, KLF16, SALL3, ZIC3, POU2F1, POU2F2, RORA, RORB, RXRG, or THRB, and (b) a nucleic acid sequence encoding a porcine teschovirus 2A (p2A) peptide sequence. Also provided herein are methods of generating a homogeneous population of retinal photoreceptor induced neurons (PR-iNs) that include: obtaining a cell sample, contacting the cell sample with a bone morphogenetic protein (BMP) inhibitor, and administering any one of the recombinant expression systems described herein to the cell sample. Also described herein are methods of treating a retinal degenerative disorder in a subject in need thereof that include (a) administering to the subject a cell previously contacted with any one of the recombinant expression systems or any one of the vectors described herein, or (b) administering to the subject a PR-iN produced by performing any one of the methods described herein.

[0029] Various non-limiting aspects of these methods are described herein and can be used in any combination without limitation. Additional aspects of various components of the systems or methods described herein are known in the art.

[0030] It must be noted that, as used in the specification and the appended claims, the singular forms '‘a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0031] As used herein, the term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that are within 10% or less of the referred value.

[0032] In some embodiments, the term “expression” refers to the process by which polynucleotides are transcribed (and / or optionally processed, such as by one or more of splicing, capping, editing, etc.) into rnRNA and / or the process by which an mRNA is translated into peptides, polypeptides, or proteins. In some embodiments, for example, if an expressed polynucleotide is or is derived from genomic DNA, expression may include splicing and / or other processing or modification to produce mRNA (e.g.. in a eukaryotic cell). In some embodiments, expression level of a gene may be determined, for example, by measuring amount of rnRNA, or protein encoded thereby, in a sample (e.g., a cell or tissue sample) in which the gene is expressed. In some embodiments, expression level of multiple genes can be determined, e.g., substantially simultaneously, for example to establish an expression profile for a particular sample.

[0033] In some embodiments, the term “encode” or “encoding” is used to describe the relationship between a nucleic acid and a polypeptide. For example, an mRNA whose sequence can be translated (e.g., by action of a ribosome) into a polypeptide is said to “encode” that polypeptide. Moreover, a nucleic acid (e.g., DNA or RNA) that, through one or more steps of replication (e.g., transcription, reverse transcription and / or other polymerization) and / or processing (e.g., splicing, capping, editing, etc.) can be used to generate such an mRNA, is also said to “encode” the relevant polypeptide. A nucleic acid strand that encodes a polypeptide is referred to as a “coding” strand; its complement is an “antisense” strand. Systems for Conversion to a Retinal Photoreceptor Induced Neuron (PR-iN) Provided herein are recombinant expression systems for converting a cell to a retinal photoreceptor induced neuron (PR-iN) that include (a) a nucleic acid sequence encoding a gene cassette comprising one or more of ASCL1, NEUR0G2, 0TX2, CRX, LHX4, NRL, NR2E3, NEURODI, IKZF1, IKZF4, MEF2C, MEF2D, MEIS2, ONECUT1, KLF16, SALL3, ZIC3, POU2F1, POU2F2, RORA, RORB, RXRG, or THRB, and (b) a nucleic acid sequence encoding a porcine teschovirus 2A (p2A) peptide sequence. In some embodiments, provided herein are expression vectors (e.g.. a single vector or a plurality of vectors) including any one of the recombinant expression systems described herein. In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral vector (AAV), a lentiviral vector, or an adenoviral vector. In some embodiments, also provided herein are cells comprising any one of the recombinant expression systems or any one of the expression vectors described herein.

[0034] Retinal Photoreceptor Neurons

[0035] The retina is a layer of photoreceptors cells and glial cells within the eye that captures incoming photons and transmits them along neuronal pathways as both electrical and chemical signals for the brain to perceive a visual picture. The retina is located in the posterior segment and forms the innermost boundary among the other major layers of the eye that include the vascular choroid and the fibrous sclera. The retina contains five major classes of retinal neurons: photoreceptors (rods and cones), bipolar cells, ganglion cells, horizontal cells, and amacrine cells.

[0036] Photoreceptors are located in the outer nuclear layer and are responsible for the initial detection and transduction of light into neural signals. As used herein, ‘'retinal photoreceptor neurons” refer to specialized light-sensitive cells in the retina that convert light into neural signals, a process essential for vision. There are two main types: rods for low-light, black-and-white vision and cones for high-acuity, color vision in bright light, wherein the mammalian retina has only one type of rod visual pigment, rhodopsin, and two types of cone opsins that confer dichromatic color vision: S opsin, which has peak sensitivity' in the short wavelength (ultraviolet or blue) region of the spectrum; and M opsin, which has peak sensitivity in the medium-long wavelength (green) region of the spectrum. These neurons work with other retinal neurons like bipolar cells and ganglion cells to send signals to the brain, which interprets them as images.

[0037] Retinal transcription factors have been identified as necessary to generate and maintain diversity in retinal photoreceptor subtypes. In some embodiments, transcription factors playing an important role in photoreceptor development can include, but are not limited to, ASCL1, NEUR0G2, 0TX2, CRX. LHX4, NRL, NR2E3, NEURODI, IKZF1, IKZF4. MEF2C. MEF2D, MEIS2, 0NECUT1, KLF16. SALL3, ZIC3. POU2F1, POU2F2, RORA, RORB, RXRG, or THRB. In some embodiments, transcription factors essential for the development and maintenance of cone photoreceptors can include, but are not limited to, NEUR0G2, 0TX2, CRX, or LHX4. In some embodiments, transcription factors essential for the development and maintenance of rod photoreceptors can include, but are not limited to, NEUR0G2, 0TX2, CRX, or NRL. In some embodiments, transcription factors essential for the development and maintenance of cone photoreceptors can include, but are not limited to, NEURODI, CRX, or NRL.

[0038] Recombinant Expression System

[0039] As used herein, a “recombinant expression system" refers to a system wherein a recombinant DNA is cloned into a vector introduced in a specific expression system (e.g., mammalian, bacteria, yeast, or insect cells) to support the expression of a gene of interest and the production of a recombinant protein. In some embodiments, a recombinant expression system for converting a cell to a retinal photoreceptor induced neuron (PR-iN) includes a nucleic acid sequence encoding a retinal transcription factor. In some embodiments, a recombinant expression system for converting a cell to a retinal photoreceptor induced neuron (PR-iN) includes a nucleic acid sequence encoding one or more retinal transcription factors. In some embodiments, a recombinant expression system for converting a cell to a retinal photoreceptor induced neuron (PR-iN) includes a nucleic acid sequence encoding a gene cassette comprising one or more of ASCL1, NEUR0G2, 0TX2, CRX, LHX4, NRL, NR2E3, NEUR0D1, IKZF1, IKZF4, MEF2C, MEF2D, MEIS2, 0NECUT1, KLF16, SALL3, ZIC3, POU2F1, POU2F2, RORA, RORB, RXRG, or THRB. In some embodiments, a recombinant expression system for converting a cell to a retinal photoreceptor induced neuron (PR-iN) includes a nucleic acid sequence encoding a gene cassette comprising two or more of ASCL1, NEUR0G2, 0TX2, CRX, LHX4, NRL, NR2E3, NEURODI , IKZF1, IKZF4, MEF2C, MEF2D, MEIS2, ONECUT1, KLF16, SALL3, ZIC3, POU2F1, POU2F2, RORA, RORB, RXRG, or THRB. In some embodiments, a recombinant expression system for converting a cell to a retinal photoreceptor induced neuron (PR-iN) includes a nucleic acid sequence encoding a gene cassette comprising three or more of ASCLL NEUR0G2, 0TX2, CRX, LHX4, NRL, NR2E3, NEURODI, IKZF1, IKZF4, MEF2C, MEF2D, MEIS2, ONECUT1, KLF16, SALL3, ZIC3, POU2F1, POU2F2, RORA, RORB, RXRG, or THRB.

[0040] In some embodiments, the recombinant expression system further comprises a nucleic acid sequence encoding a porcine teschovirus 2A (p2A) peptide sequence. In some embodiments, the recombinant expression system can include two or more codon-modified p2A peptide sequences. In some embodiments, the two or more p2A peptide sequences drive polycistronic expression of the plurality of retinal transcription factors.

[0041] In some embodiments, a recombinant expression system includes a gene cassette, wherein the gene cassette further comprises an expression control element. As used herein, an expression control element refers to a nucleic acid sequence that regulates the expression of a nucleotide sequence to which it is operably linked. In some embodiments, an expression control element is operably linked to a nucleotide sequence when the expression control sequence controls and regulates the transcription and / or the translation of the nucleotide sequence. Thus, an expression control sequence can include promoters, enhancers, internal ribosome entry' sites (IRES), transcription terminators, a start codon in front of a proteinencoding gene, splicing signals for introns, and stop codons. In some embodiments, an expression control element can include, at a minimum, a sequence whose presence are designed to influence expression, and can also include additional advantageous components. In some embodiments, an expression control element can also include the design of the nucleic acid sequence such that undesirable, potential initiation codons in and out of frame, are removed from the sequence. In some embodiments, an expression control element can be designed to enhance mRNA stability. In some embodiments, an expression control element can modulate a nucleotide sequence to which it is operably linked such that lower expression levels or higher expression levels are achieved. In some embodiments, an expression control element comprises (i) a tetracycline-responsive promoter element (TRE) induced by a reverse tetracycline-controlled transactivator (rtTA), or (ii) a zinc finger-responsive promoter element (ZRE) induced by a synthetic zinc finger transcription regulator (synZiFTR).

[0042] In some embodiments, a recombinant expression system further includes a homology' arm for a genomic safe harbor site (SHS). In some embodiments, the homology' arm for SHS genome integration comprises Citrate Lyase Beta-Like (CLYBL), Adeno-Associated Virus integration Site 1 (AAVS1), Region of Gene Insertion 1 (ROGI1), Region of Gene Insertion 2 (R0GI2), L-gulonolactone oxidase (GULOP). or Hippl 1 (Hl 1).

[0043] In some embodiments, a recombinant expression system further includes an antibiotic selectable marker. In some embodiments, the antibiotic selectable marker is resistant to zeocin, hygromycin, puromycin, or G418 (Geneticin). In some embodiments, a recombinant expression system further includes a ubiquitous open chromatin element. In some embodiments, a recombinant expression system further includes a post-transcriptional regulatory element.

[0044] Cells

[0045] In some embodiments, a recombinant expression system is used for converting a cell to a retinal photoreceptor induced neuron (PR-iN). In some embodiments, the cell comprises a pluripotent stem cell. Muller glia cell, astrocyte, peripheral blood mononuclear cell, urine epithelial cell, or a fibroblast. In some embodiments, a cell is a stem cell. In some embodiments, the stem cell is a pluripotent stem cell, e.g., a stem cell that has the potential to differentiate into any cell existing in organisms derived from ectoblasts, mesoblasts, and endoblasts (i.e., pluripotency) and also have growth capacity. Examples of pluripotent stem cells can include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), somatic stem cells (tissue-specific stem cells), cloned embryo-derived embryonic stem cells obtained by nuclear transfer (ntES cells), germ-line stem cells (GS cells), embryonic germ cells (EG cells), and pluripotent adult progenitor cells (MAPC cells). In some embodiments, the pluripotent stem cell is an induced pluripotent stem cell (iPS). In some embodiments, the pluripotent stem cell is an embryonic stem cell (ES). In some embodiments, the pluripotent stem cell is derived from a mammalian animal.

[0046] In some embodiments, a cell can include a fluorescent reporter gene or a reporter tag. In some embodiments, the fluorescent reporter gene comprises a p2A coupled fluorescent protein integrated at a Cone-rod homeobox (CRX) gene, cone arrestin (ARR3) gene, THRB gene, or neural retinal leucine zipper (NRL) gene. In some embodiments, the reporter tag comprises an epitope tag.

[0047] Nucleic Acids / Vectors

[0048] Also provided herein are nucleic acids that encode any of the recombinant expression systems described herein. In some embodiments, the recombinant expression system for converting a cell to a retinal photoreceptor induced neuron (PR-iN) can include a nucleic acid sequence encoding a gene cassette comprising one or more of ASCL1, NEUR0G2, 0TX2, CRX, LHX4, NRL, NR2E3, NEURODI, IKZF1, IKZF4, MEF2C, MEF2D, MEIS2, ONECUT1, KLF16, SALL3, ZIC3, POU2F1, POU2F2, RORA, RORB, RXRG, or THRB. In some embodiments, the recombinant expression system can further include a second nucleic acid sequence encoding a porcine teschovirus 2A (p2A) peptide sequence. In some embodiments, the recombinant expression system can consist of a single nucleic acid that encodes both (a) a nucleic acid sequence encoding a gene cassette comprising one or more of ASCL1, NEUROG2, OTX2, CRX, LHX4, NRL, NR2E3, NEURODI, IKZF1, IKZF4, MEF2C, MEF2D, MEIS2, ONECUT1, KLF16, SALL3, ZIC3, POU2F1, POU2F2, RORA, RORB. RXRG, or THRB, and (b) a nucleic acid sequence encoding a porcine teschovirus 2A (p2A) peptide sequence.

[0049] In some embodiments, a “nucleic acid’’ refers to a compound and / or substance that is, or can be incorporated into, a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is, or can be incorporated into, a polynucleotide chain with a phosphodiester linkage. In some embodiments, nucleic acid can refer to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside), and in some embodiments, nucleic acid can refer to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, a nucleic acid is or comprises ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2’-amino-LNA having a 2'-amino functionalization, and 2’-amino-a-LNA having a 2’-amino functionalization), or a combination thereof. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid residue analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in one or more residues, and in some embodiments, are linked together other than by a phosphodiester. For example, in some embodiments, a nucleic acid includes one or more phosphorothioate and / or phosphoroamidite (e.g., 5'-N-phosphoramidite) linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid includes one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine. 2-aminoadenosine, C 5 -bromouridine, C5 -fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g.. 2'-fluororibose, ribose, 2'-deoxy ribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), enzymatic synthesis in the absence of a complementary template, reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9. 10. 15. 20, 25, 30, 35, 40, 45, 50, 55, 60. 65. 70. 75, 80, 85, 90, 95, 100. 1 10. 120, 130. 140, 150, 160. 170, 180. 190, 20, 225. 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide.

[0050] Also provided herein are vectors that include any of the nucleic acids encoding any of the recombinant expression systems described herein. Also provided herein are vectors that include a nucleic acid sequence encoding a gene cassette comprising one or more of ASCL1, NEUROG2, OTX2, CRX, LHX4, NRL, NR2E3, NEURODI, IKZF1, IKZF4, MEF2C, MEF2D, MEIS2, ONECUT1, KLF16, SALL3, ZIC3, POU2F1, POU2F2, RORA, RORB, RXRG, or THRB. In some embodiments, the gene cassette comprises two or more of ASCL1, NEUROG2, OTX2, CRX, LHX4, NRL, NR2E3. NEURODI, IKZF1. IKZF4.

[0051] MEF2C, MEF2D, MEIS2, ONECUT1, KLF16, SALL3, ZIC3, POU2F1, POU2F2, RORA, RORB, RXRG, or THRB. In some embodiments, the gene cassette comprises three or more of ASCL1, NEUROG2, OTX2, CRX, LHX4, NRL, NR2E3, NEURODI, IKZF1, IKZF4, MEF2C, MEF2D, MEIS2. ONECUT1, KLF16, SALL3, ZIC3, POU2F1. POU2F2, RORA, RORB, RXRG, or THRB. In some embodiments, the vector can further include a nucleic acid encoding a porcine teschovirus 2A (p2A) peptide sequence. In some embodiments, the nucleic acid comprises two or more p2A peptide sequences.

[0052] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is an AAV vector. In some embodiments, the vector comprises a class II transposable element (TE), wherein the class II TE is compatible with transposases comprising piggyBac or Sleeping Beauty transposon systems. Non-limiting examples of vectors can include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vectors (e.g., pSV or pCMV vectors), adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors), and any Gateway® vectors. In some embodiments, a vector can, e.g., include sufficient cis-acting elements for expression: other elements for expression can be supplied by the host mammalian cell or in an in vitro expression system. Skilled practitioners will be capable of selecting suitable vectors and mammalian cells for making any of the recombinant expression systems described herein.

[0053] Method of Generating Retinal Photoreceptor Induced Neurons (PR-iNs)

[0054] Provided herein are methods of generating a homogeneous population of retinal photoreceptor induced neurons (PR-iNs) that include obtaining a cell sample, contacting the cell sample with a bone morphogenetic protein (BMP) inhibitor, and administering any one of the recombinant expression systems or any of the vectors described herein to the cell sample. In some embodiments, the cell sample comprises pluripotent stem cells, Muller glia, astrocytes, peripheral blood mononuclear cells, urine epithelial cells, fibroblasts, or any combination thereof.

[0055] In some embodiments, “delivering”, “gene del i \ ery ‘. “gene transfer”, “transducing” can refer to the introduction of an exogenous polynucleotide into a host cell, irrespective of the method used for the introduction. Such methods include a variety' of well-known techniques such as vector-mediated gene transfer (e.g., viral infection / transfection, or various other protein-based or lipid-based gene delivery7complexes) as well as techniques facilitating the delivery7of “naked” polynucleotides (e.g., electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides). In some embodiments, an introduced polynucleotide may be stably maintained in the host cell. In some embodiments, an introduced polynucleotide may be transiently maintained. Stable maintenance ty pically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.

[0056] In some embodiments, any of the recombinant expressions systems described herein can include (i) a tetracycline-responsive promoter element (TRE) induced by a reverse tetracycline-controlled transactivator (rtTA), or (ii) a zinc finger-responsive promoter element (ZRE) induced by a synthetic zinc finger transcription regulator (synZiFTR). In some embodiments, any of the recombinant expressions systems described herein can include an expression control element that comprises a tetracycline-responsive promoter element (TRE) or a doxycycline (dox)-inducible promoter element. In some embodiments, a method of generating a homogeneous population of retinal photoreceptor induced neurons (PR-iNs) can further include treating the cell with tetracycline, doxycycline, or grazoprevir.

[0057] In some embodiments, a method of generating a homogeneous population of retinal photoreceptor induced neurons (PR-iNs) can further include treating the cell sample with a bone morphogenetic protein (BMP) inhibitor prior to administering the recombinant expression system to the cell.

[0058] Bone morphogenetic proteins (BMPs) are signaling molecules that are central in a variety of biological processes. BMPs were originally discovered by their ability to induce the formation of bone and cartilage and are also considered to constitute a group of pivotal morphogenetic signals, orchestrating tissue architecture throughout the body. In some embodiments, inhibiting BMP signaling could enhance TF-mediated conversion of a cell to a retinal photoreceptor induced neuron (PR-iN). In some embodiments, converting a cell to a retinal photoreceptor induced neuron (PR-iN) or generating a retinal photoreceptor induced neuron (PR-iN) can be enhanced by blocking BMP signaling with a BMP inhibitor. In some embodiments, converting a cell to a retinal photoreceptor induced neuron (PR-iN) or generating a retinal photoreceptor induced neuron (PR-iN) can be enhanced by blocking BMP signaling with a small molecule inhibitor or a biologic inhibitor of BMP signaling. In some embodiments, converting a cell to a retinal photoreceptor induced neuron (PR-iN) or generating a retinal photoreceptor induced neuron (PR-iN) can be enhanced by blocking BMP signaling with a small molecule inhibitor LDN-193189.

[0059] In some embodiments, for any of the methods described herein, a recombinant expression system can be integrated into the safe harbor site in the genome of a cell by using a gene-editing enzy me. As used herein, a ‘‘gene-editing enzyme” can refer to an agent that can target and bind to a specific sequence in DNA. In some embodiments, a gene-editing enzyme can include a CRISPR-associated enzyme or protein that uses CRISPR sequences as a guide to recognize and cleave specific nucleic acid strands that are complementary to the CRISPR sequence. As used herein, the term “CRISPR” refers to a technique of sequence specific genetic manipulation relying on the clustered regularly interspaced short palindromic repeats pathway, which unlike RNA interference regulates gene expression at a transcriptional level. For example, a gene-editing Cas effector can associate with a CRISPR RNA sequence to bind to and alter DNA or RNA target sequences. In some embodiments, a gene-editing enzyme comprises a Cas9 protein, a Casl3b protein, or a Casl3d protein. In some embodiments, a gene-editing enzyme can be a Cas9 endonuclease that makes a doublestranded break in a target DNA sequence. In some embodiments, a gene-editing enzy me can be a Cas 12a nuclease that also makes a double-stranded break in a target DNA sequence. In some embodiments, a gene-editing enzyme can be a Cas 13 nuclease which targets RNA. In some embodiments, a gene-editing enzyme can be a Cas 12 (Cpfl) enzy me. In some embodiments, a gene-editing enzyme can be a Cas9 enzyme. In some embodiments, a geneediting enzy me can be a class II CRISPR enzy me.

[0060] In some embodiments, the cells of the cell sample can include a photoreceptor reporter. In some embodiments, the cells of the cell sample can include a photoreceptor reporter, wherein the photoreceptor reporter comprises a CRX-p2A-h2b-mRuby3 photoreceptor reporter, a cARR3-p2A-mTagBFP2 reporter, a NRL-p2A-h2b-mGreenLantem reporter, a SIX6-eGFP / VSX2-ldTomalo reporter, a SIX6-eGFP / CRX-mRuby3 reporter, a SIX6-p2A-h2b-GFP / CRX-p2A-h2b-mRuby3 / cARR3-p2A-mTagBFP2 reporter, or aCRX-p2A-h2b-mTagBFP2 / cARR3-p2A-h2b-mScarlet / NRL-p2A-h2b-mGreenLantem reporter.

[0061] Methods of Treatment

[0062] Provided herein are method of treating a retinal degenerative disorder in a subject in need thereof, the method including: (a) administering to the subject a cell previously contacted with any one of the recombinant expression systems or any one of the vectors described herein, or (b) administering to the subject a PR-iN produced by performing any one of the methods described herein.

[0063] As used herein, a “retinal degenerative disorder” refers to an eye disorder that causes progressive damage to the retina, the light-sensitive layer at the back of the eye. Examples of a retinal degenerative disorder can include, but are not limited to, age-related macular degeneration, retinitis pigmentosa, Stargardt disease, rod-cone dystrophy, or Leber congenital amaurosis. In some embodiments, the term “administration"’ refers to the administration of a composition to a subject or system to achieve delivery’ of an agent that is. or is included in. the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, vaginal, vitreal, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality7of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0064] In some embodiments, the term “subject” refers an organism, ty pically a mammal (e.g., a human). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display a particular, or in some embodiments any, symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0065] In some embodiments, the term “treatment” means to ameliorate at least one symptom of a disorder. Generally, the methods of treatment include administering a therapeutically effective amount of composition that reduces at least one symptom of a disorder to a subject who is in need of, or who has been determined to be in need of such treatment. EXAMPLES

[0066] The disclosure is further described in the following examples, which do not limit the scope of the disclosure.

[0067] Example 1 - System for conversion to a retinal photoreceptor induced Neuron (PR-iN) Pluripotent stem cell lines with an integrated CRX-p2A-h2b-mRuby3 specific photoreceptor reporter (PR) were used to convert a pluripotent stem cell to a retinal photoreceptor induced neuron (PR-iN), wherein stem cell to PR conversion was enhanced using LDN-193189, a small molecule inhibitor of BMP (FIG. 1). To distinguish between rod and cones, cell reporters for cones (cARR3-p2A-mTagBFP2) and rods (NRL-p2A-h2b-mGreenLantem) were additionally built. Two stem cell lines were made for visualizing rod / cone cells and include triple color reporters for (1) SIX6-p2A-h2b-GFP (for general retina) / CRX-p2A-h2b-mRuby3(for all PRs) / cARR3-p2A-mTagBFP2(for all cones) and (2) CRX-p2A-h2b-mTagBFP2 / cARR3-p2A-h2b-mScarlet / NRL-p2A-h2b-mGreenLantem (for all rods).

[0068] Specifically, an inducible gene cassette was integrated into a safe harbor docking site in human cells, wherein the gene cassette expresses 3-4 retinal transcription factors after the addition of doxycycline. As shown in FIG.2, transcription factors play an important role in photoreceptor development and can include, but are not limited to, ASCL1, NEUR0G2, 0TX2, CRX, LHX4, NRL, NR2E3, NEURODI, IKZF1, IKZF4, MEF2C, MEF2D, MEIS2, 0NECUT1, KLF16, SALL3, ZIC3, POU2F1, POU2F2, RORA, RORB, RXRG, or THRB.

[0069] Example 2 - Reprogramming strategy and differentiation timeline for PR-iN conversion

[0070] PR-iNs were generated by a biphasic reprogramming strategy wherein Tetracycline (dox) and Grazoprevir (gzv) inducible gene cassettes were integrated at two different safe harbor sites for differentiation phases 1 and 2 respectively. Constitutively expressed rtTA integrated at the first locus enables dox activation of a TRE driven gene cassette which induces polycistronic expression of three transcription factors (TFs) and results inactivation of an endogenous CRX reporter. Subsequently, ubiquitously expressed SynZiFTR integrated at the second locus enables gzv activation of a ZRE driven gene cassette which induces polycistronic expression of three transcription factors (TFs) and results in activation of an endogenous photoreceptor subtype specific (for e.g., ARR3 / NRL) reporter (FIG.3A). PR-iN conversion was enabled by dox and LDN administration to result in neurite outgrowth and CRX expression in the first phase. In the second phase, PR fate was specified by addition of gzv and maturity was ascertained via PR subtype specific reporters.

[0071] Differentiation timeline across three conditions highlights the biphasic and temporally controlled nature of directed differentiation (FIG.3B).

[0072] Example 3 - Direct conversion of photoreceptor induced neurons (PR-iNs)

[0073] Briefly, pluripotent stem cells (PSCs) maintained in B8 media under 5%02 / 10%C02 were pretreated overnight with 100 nM LDN-193189 (Sigma #SML0559) and 1 pg / ml doxycycline (Sigma #D5207) to activate TRE controlled TF expression cassettes. Cells were dissociated into a single cell suspension with Accutase (Sigma #A6964) for 12 min at 37 °C. quenched with B8T with 2 pM Thiazovivin (B8T; Cayman #14245), pelleted at 80 x g for 5 min, resuspended in neural induction medium (NIM) with Thiazovivin and 1-2 pg / ml of dox and passed through a Falcon 35 pm cell strainer (Falcon #352235). NIM consists of DMEM / F12 (Gibco #11330032), N2 supplement (Gibco #17502048), lx non-essential amino acids (NEAA; Gibco#l 1140050) and GlutaMAX (Gibco #35050061) all from Life technologies. Cells were plated at ~25,000cells / well in TC-treated 12-well plates (7000 cells / cmA2) additionally coated with 0.2 mg / ml poly-L-omithine (PLO; Sigma #P3655, rinsed 3x in sterile cell culture grade ater and coated a final time with 1 %GFR Matrigel (Coming #354230). After 2 days, cells were fed by the addition of 1 ml of fresh NIM plus dox and lx CultureOne supplement (Gibco #A3320201) and every other day thereafter with a one-third medium exchange. For cultures beyond 6 days, cells were transitioned to BrainPhys (StemCell Technologies#05790) containing B27 supplement (Gibco #17504044), 1 pM Grazoprevir (Medchem Express HY-15298), 500 nM All Trans Retinoic Acid (ATRA; Sigma #R2625), ImM Taurine (Sigma #T8691), and 10 mM nicotinamide (NIC; Sigma #72340)

[0074] [Table 1] Phase 1 and 2 PR enriched transcription factors

[0075] Phase 1 Transcription Factors Phase 2 Transcription Factors ASCL1 NRL

[0076] NEUROG2 LHX4

[0077] NEURODI RORB CRX NR2E3

[0078] OTX2 THRB

[0079] RXRG

[0080]

[0081] IKZF1 IKZF4

[0082] MEF2C

[0083] MEF2D

[0084] ONECUT1

[0085] MEIS 2

[0086] KLF16

[0087] SALL3

[0088] ZIC3

[0089] P0U2F1

[0090]

[0091] POU2F2

[0092] Example 5 - Reporters for imaging retinal neurons

[0093] A D45 cryoembedded SIX6-eGFP / VSX2-tdTomato dual reporter was imaged by confocal microscopy (FIG. 4 panels A-C). A live D65 SIX6-eGFP / CRX-mRuby3 dual reporter was imaged in an organoid (FIG. 4 panels D-E). Cell counting was performed in living samples by automated counting in MetaXpress to tag CRX-Ruby+ nuclei (FIG.4 panel F). Cone Arrestin (cARR3)-mTagBFP2 and CRX-p2A-mRuby3 co-label photoreceptors at DI 73 were imaged (FIG. 4 panel G-I).

Claims

WHAT IS CLAIMED IS:

1. A recombinant expression system for converting a cell to a retinal photoreceptor induced neuron (PR-iN) comprising:(a) a nucleic acid sequence encoding a gene cassette comprising one or more of ASCL1, NEUR0G2, 0TX2:CRX, LHX4, NRL, NR2E3, NEURODI, IKZF1, IKZF4, MEF2C, MEF2D, MEIS2, ONECUT1, KLF16, SALL3, ZIC3, POU2F1, POU2F2, RORA, RORB. RXRG, or THRB; and(b) a nucleic acid sequence encoding a porcine teschovirus 2A (p2A) peptide sequence.

2. The recombinant expression system of claim 1, wherein the gene cassette further comprises an expression control element.

3. The recombinant expression system of claim 2, wherein the expression control element comprises (i) a tetracycline-responsive promoter element (TRE) induced by a reverse tetracycline-controlled trans activator (rtTA), or (ii) a zinc finger-responsive promoter element (ZRE) induced by a synthetic zinc finger transcription regulator (synZiFTR).

4. The recombinant expression system of any one of claims 1-3, further comprising a homology arm for a genomic safe harbor site (SHS), an antibiotic selectable marker, a ubiquitous open chromatin element, and a post-transcriptional regulatory element.

5. The recombinant expression system of claim 4, wherein the homology arm for SHS genome integration comprises Citrate Lyase Beta-Like (CLYBL). Adeno-Associated Virus integration Site 1 (AAVS1), Region of Gene Insertion 1 (R0GI1), Region of Gene Insertion 2 (R0GI2), L-gulonolactone oxidase (GULOP), or Hippl 1 (Hll).

6. The recombinant expression system of claim 4, wherein the antibiotic selectable marker is resistant to zeocin, hygromycin, puromycin, or G418 (Geneticin).

7. The recombinant expression system of any one of claim 1-6, comprising two or more codon-modified p2A peptide sequences.

8. The recombinant expression system of claim 7, wherein the two or more codon-modified p2A peptide sequences drive polycistronic expression of the plurality of retinal transcription factors.

9. The recombinant expression system of any one of claims 1-8, wherein the cell comprises a fluorescent reporter gene or a reporter tag.

10. The recombinant expression system of claim 9, wherein the fluorescent reporter gene comprises a p2A coupled fluorescent protein integrated at a Cone-rod homeobox (CRX) gene, cone arrestin (ARR3) gene, THRB gene, or neural retinal leucine zipper (NRL) gene.

11. The recombinant expression system of claim 9, wherein the reporter tag comprises an epitope tag.

12. The recombinant expression system of any one of claims 1-11, wherein the cell comprises a pluripotent stem cell, Muller glia cell, astrocyte, peripheral blood mononuclear cell, urine epithelial cell, or a fibroblast.

13. The recombinant expression system of any one of claims 1-12, wherein the gene cassette comprises two or more of ASCL1, NEUROG2. OTX2. CRX, LHX4, NRL, NR2E3. NEURODI, IKZF1, IKZF4, MEF2C, MEF2D, MEIS2, ONECUT1, KLF16, SALL3, ZIC3, POU2F1, POU2F2, RORA, RORB, RXRG, or THRB.

14. The recombinant expression system of any one of claims 1-13, wherein the gene cassette comprises three or more of ASCL1, NEUROG2, OTX2, CRX, LHX4, NRL, NR2E3, NEURODI, IKZF1, IKZF4, MEF2C, MEF2D, MEIS2, ONECUT1, KLF16, SALL3, ZIC3, POU2F1, POU2F2, RORA, RORB, RXRG, or THRB.

15. A vector comprising the recombinant expression system of any one of claims 1-14.

16. The vector of claim 15, wherein the vector is a viral vector.

17. The vector of claim 16, wherein the viral vector is a lentiviral vector of an AAV vector.

18. The vector of claim 15, wherein the vector comprises a class II transposable element (TE).

19. A method of generating a homogeneous population of retinal photoreceptor induced neurons (PR-iNs), the method comprising:obtaining a cell sample;contacting the cell sample with a bone morphogenetic protein (BMP) inhibitor; and administering the recombinant expression system of any one of claims 1-14 to the cell sample;thereby generating a homogeneous population of retinal photoreceptor induced neurons (PR-iNs).

20. The method of claim 19, wherein the cell sample comprises pluripotent stem cells, Muller glia, astrocytes, peripheral blood mononuclear cells, urine epithelial cells, fibroblasts, or any combination thereof.

21. The method of claim 19 or 20, further comprising treating the cell with tetracycline, doxycycline, or grazoprevir.

22. The method of any one of claims 19-21 , wherein the recombinant expression system is integrated into the genomic safe harbor site in the genome of the cell by using a Casl2 (Cpfl) or Cas9 CRISPR enzy me.

23. The method of any of claims 19-22, wherein the BMP inhibitor comprises LDN-193189.

24. The method of any of claims 19-23, wherein the cells of the cell sample comprise a photoreceptor reporter.

25. The method of claim 24, wherein the photoreceptor reporter comprises a CRX-p2A-h2b-mRuby3 photoreceptor reporter, a cARR3-p2A-mTagBFP2 reporter, a NRL-p2A-h2b-mGreenLantem reporter, a SIX6-eGFP / VSX2-tdTomato reporter, a SIX6-eGFP / CRX-mRuby3 reporter, a SIX6-p2A-h2b-GFP / CRX-p2A-h2b-mRuby3 / cARR3-p2A-mTagBFP2reporter, or a CRX-p2A-h2b-mTagBFP2 / cARR3-p2A-h2b-mScarlet / NRL-p2A-h2b-mGreenLantem reporter.

26. A method of treating a retinal degenerative disorder in a subject in need thereof, the method comprising:(a) administering to the subject a cell previously contacted with a recombinant expression system of any one of claims 1-14 or a vector of any one of claims 15-18; or (b) administering to the subject a PR-iN produced by performing any one of the methods of claims 19-25.

27. The method of claim 26, wherein the retinal degenerative disorder comprises age-related macular degeneration, retinitis pigmentosa, or Leber congenital amaurosis.

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