Method to stimulate photorecptor regeneration from glia

Abstract

Compositions, nucleic acid molecules and methods for inducing photoreceptor regeneration and for treating retinal damage, disease, and degeneration in mammalian subjects. Such a nucleic acid molecule comprises a nucleic acid sequence encoding a proneural transcription factor (ASCL1 or NEUROD1) and one or more additional transcription factors selected from OTX2, CRX, and RAX. A method for inducing photoreceptor regeneration in a retina comprises administering to a retina the nucleic acid molecule encoding a proneural transcription factor and one or more additional transcription factors, wherein expression of the nucleic acid molecule stimulates regeneration of photoreceptors from retinal Müller glia (MG).

Description

METHOD TO STIMULATE PHOTORECPTOR REGENERATION FROM GLIA

[0001] This application claims benefit of United States provisional patent application number 63 / 744,729, filed January 13, 2025, the entire contents of which are incorporated by reference into this application.REFERENCE TO A SEQUENCE LISTING

[0002] The content of the XML file of the sequence listing named “UW087_seq”, which is 60 kb in size, created on January 8, 2026, and electronically submitted herewith the application, is incorporated herein by reference in its entirety.BACKGROUND

[0003] Age-related macular degeneration (AMD), characterized by a slow degeneration of the retinal pigment epithelium and photoreceptors cells in the central retina, is one of the leading causes of visual impairment in individuals over 50. Several therapeutic approaches have been developed to delay the progression of the disease and protect the remaining cells, however, in advanced stages, when photoreceptors and more specifically cones have already degenerated, novel therapeutic strategies are necessary to replace the missing cells. Currently, most of the efforts in the field of regenerative medicine for AMD focus on using stem cell transplants to replace these cells, though there are significant barriers to integration of transplanted cells, and to date establishment of synaptic connectivity has been limited. An alternative strategy consists of stimulating the intrinsic regenerative capacity of the retina.

[0004] Although the mammalian retina does not regenerate neurons, some vertebrates such as zebrafish can regenerate their retina after injury. This fascinating process is driven by the Muller glia (MG), which are able to re-enter the cell cycle and reprogram into neurogenic progenitors upon retinal injury. Efforts have been made to better understand the mechanisms underlying the zebrafish's regenerative capacity and to “translate” this to mammals to promote retinal regeneration. A breakthrough in the field was our demonstration that MG can generate new neurons in vivo in the adult mouse retina after the overexpression of the pro-neural transcription factor (TF) ASCL1 (PCT Publication No. W02020223308). The newly generated neurons integrate into the existing circuit and adopt a bipolar-like fate. In addition, a new cocktail of TFs combining Isletl, Pou4f2, and ASCL1 has shown great efficiency in reprograming MG into retinal ganglion cell (RGC)-like cells, thus demonstrating that particular combination of TFscould direct the MG to generate specific retinal cell types. However, none of these previously-identified TF combinations efficiently direct MG to generate new photoreceptors.

[0005] There remains a need to stimulate regeneration of photoreceptor cells in the human retina for the development of new types of regenerative therapies for patients.SUMMARY

[0006] Described herein are compositions, nucleic acid molecules and methods for inducing photoreceptor regeneration and for treating retinal damage, disease, and degeneration in mammalian subjects.

[0007] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding a proneural transcription factor (ASCL1 or NEUROD1) and one or more additional transcription factors selected from OTX2, CRX, and RAX. In some embodiments, the additional transcription factor is OTX2. In some embodiments, the additional transcription factor is CRX. In some embodiments, the additional transcription factor is RAX. In some embodiments, the nucleic acid sequence comprises a promoter sequence In some embodiments, the promoter sequence is a retinal or glia-specific promoter. In some embodiments, the glia-specific promoter is HES1 or RLBPI.

[0008] Also described is a method for inducing photoreceptor regeneration in a retina. In some embodiments, the method comprises administering to a retina the nucleic acid molecule described above, wherein expression of the nucleic acid molecule stimulates regeneration of photoreceptors from retinal Muller glia (MG). In some embodiments, the retina is in a mammalian subject. In some embodiments, the photoreceptors express one or more markers selected from RXRG, Recoverin, OTX2, NR2E3, CRX, OPN1SW, OPN1LW, and ARR3. In some embodiments, the subject is treated for disease, damage or degeneration of the retina.

[0009] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1 and OTX2. In another embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1 and CRX. In another embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1 and RAX. In another embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1, OTX2, CRX, and RAX. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding two, three, or all four of ASCL1, OTX2, CRX, and RAX.

[0010] Also disclosed herein is a method for inducing photoreceptor regeneration in a retina comprising: a) administering to a retina a nucleic acid molecule comprising a nucleic acid sequence encoding an ASCL1 transcription factor and one or more additional transcription factors selected from OTX2, CRX, and RAX. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding two, three, or all four of ASCL1, OTX2, CRX, and RAX. Also disclosed herein is a method for inducing photoreceptor regeneration in a subject comprising: a) administering to a retina of the subject a nucleic acid molecule comprising a nucleic acid sequence encoding an ASCL1 transcription factor and one or more additional transcription factors selected from OTX2, CRX, and RAX. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding two, three, or all four of ASCL1, OTX2, CRX, and RAX. In some embodiments, the administering to the retina is intravitreal or subretinal injection.

[0011] In additional embodiments, the nucleic acid molecules and methods disclosed herein stimulate production of functional photoreceptors from reprogrammed MG. In another embodiment, the number of the MG-derived photoreceptors is increased. In another embodiment, the number of functional photoreceptors is increased by 40%. In another embodiment, the subject is treated for retinal disease, damage or degeneration in the retina. In another embodiment, the subject is an adult. In another embodiment, a vector comprises the nucleic acid molecule. In one embodiment, the vector is a non-viral vector or a viral vector, and the viral vector is an adeno-associated viral (AAV) vector or a lentiviral vector. In an additional embodiment, a promoter sequence is in operable linkage with the nucleic acid encoding ASCL1. In one embodiment, the promoter is a retinal or glia-specific promoter. In one embodiment, administering to the retina is intravitreal or subretinal injection.

[0012] Also, in another embodiment, the nucleic acid molecules disclosed herein comprise a glia-specific promoter sequence. In one embodiment, the glia-specific promoter sequence is a HES1 promoter sequence or a portion thereof. In one embodiment, the glia-specific promoter is a RLBP1 promoter sequence or a portion thereof. In other embodiments, the nucleic acid sequence comprises an IRES or2A self-cleaving sites situated between the sequences encoding the transcription factors, for example, in a multicistronic or polycistronic configuration. In some embodiments, the nucleic acid molecule further comprises a sequence that encodes a reporter. One example of a reporter is green fluorescent protein (GFP).

[0013] In some embodiments, a vector comprises the nucleic acid molecule. In some embodiments, the vector is a non-viral vector or a viral vector. In some embodiments, the viralvector is an adeno-associated viral (AAV) vector or a lentiviral vector. In some embodiments, the nucleic acid molecule further comprises a promoter sequence in operable linkage with the nucleic acid sequence encoding the ASCL1 and one or more additional transcription factors. In some embodiments, the promoter is a retinal progenitor or glia-specific promoter. In some embodiments, the glia-specific promoter is HES1.

[0014] In some embodiments, the ASCL1 and one or more additional transcription factors are expressed as a fusion protein.

[0015] Also provided herein are methods for inducing photoreceptor regeneration comprising administering to a subject a composition as described herein. In some embodiments, the methods are effective to increase the number of cones and / or rods. In some embodiments, the new retinal neurons are bipolar neurons. In some embodiments of the method, the number of photoreceptors increases by at least 40% relative to a baseline level or other reference amount representative of an untreated retina. In some embodiments, the number of retinal neurons increases by 10%, 20%, 25%, 50%, 100%, 150%, 200%, or more.

[0016] The subject in the methods disclosed herein is typically a mammal, such as a human or veterinary subject. In one embodiment, the subject is an adult. The subject, in some embodiments, has a retinal degenerative disease. Examples of such retinal degenerative diseases include, but are not limited to, age-related macular (cone or rod) degeneration (AMD). In some embodiments, the subject has suffered an injury (e.g., solar or laser retinopathy, retinal detachment, or retinal vein occlusion). In some embodiments, the subject is an organoid.

[0017] An exemplary nucleic acid sequence encoding human ASCL1 (Achaete-scute homolog 1) can be found at NCBI Reference Sequence number NG_008950.1 and also provided herein as SEQ ID NO: 1. In another embodiment disclosed herein is a nucleic acid sequence encoding a human ASCL1 amino acid sequence or portion thereof of UniProtKB / Swiss-Prot: P50553.2, provided herein as SEQ ID NO: 2. ASCL1 homologues, e.g., derived from species such as murine, canine, equine, are included herein, without limitation.

[0018] An exemplary nucleic acid sequence encoding human OTX2 (orthodenticle homeobox 2) can be found at NCBI Reference Sequence number 5015 and also provided herein as SEQ ID NO: 3. In another embodiment disclosed herein is a nucleic acid sequence encoding a human OTX2 amino acid sequence or portion thereof, provided herein as SEQ ID NO: 4. OTX2 homologs, orthologs and / or paralogs, e.g., derived from species such as murine, canine, equine, are included herein, without limitation.

[0019] An exemplary nucleic acid sequence encoding human CRX (cone-rod homeobox) can be found at NCBI Reference Sequence number 1406 and also provided herein as SEQ ID NO: 5. In another embodiment disclosed herein is a nucleic acid sequence encoding a human CRX amino acid sequence or portion thereof, provided herein as SEQ ID NO: 6. CRX homologs, orthologs, and / or paralogs, e.g., derived from species such as murine, canine, equine, are included herein, without limitation.

[0020] An exemplary nucleic acid sequence encoding RAX (retinal homeobox Rx) can be found at NCBI Reference Sequence number NG_013031, and also provided herein as SEQ ID NO: 7. In another embodiment disclosed herein is a nucleic acid sequence encoding a human RAX amino acid sequence or portion thereof, provided herein as SEQ ID NO: 8. RAX homologs, orthologs, and / or paralogs, e.g., derived from species such as murine, canine, equine, are included herein, without limitation.

[0021] Exemplary nucleic acid sequences of the ASCL1, OTX2, CRX, RAX, or other transcription factor for use herein include, without limitation, portions thereof of the corresponding sequences of ASCL1, OTX2, CRX, RAX, for the purposes of configurating multicistronic, bicistronic, and / or tricistronic constructs, plasmids, and / or expression vectors.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 is a schematic illustration of lentiviral constructs.

[0023] FIG. 2 is a set of photomicrographs showing that CRX and OTX2 induce photoreceptor production from mouse Muller glia when over-expressed along with ASCL1 via a lentivirus.

[0024] FIGS. 3A-3B are photomicrographs showing more detail of fetal retinal sphere culture infected with OXT2 and ASCL1 TFs for 7 days. The arrow in 4A shows an example of a cell that is both GFP+ and OTX2+, demonstrating that viral over-expression of OTX2 works. The arrowheads in 4B are examples of GFP+ early photoreceptors, which are GFP+, OTX2+, RXRG+.

[0025] FIG. 4 is a set of photomicrographs showing a fetal retinal sphere culture infected with CRX and ASCL1 TFs for 7 days. The arrowhead points to an example of an early photoreceptor cell that is GFP+ OTX2+ and RXRG+.

[0026] FIG. 5 illustrates photoreceptor regeneration from human Muller glia via the quantitation of effect of expression of RXRG in GFP+ cells. Between 10 -16% of the GFP+ (infected) cellsexpress RXRG after 7 days when using OTX2 and ASCL1 (left bar graph). CRX and ASCL1 were also able to reprogram the Muller glia to generate photoreceptors, though the percentage was closer to 10%. A small subset of the ASCL1 / CRX infected cells generated rod photoreceptors (NR2E3; right bar graph and photomicrographs).

[0027] FIG. 6 illustrates use of a new construct that has the mScarlet reporter in human adult postmortem retinal explants. Over-expression of OTX2 and ASCL1 in Muller glia (using the HES1 promoter) stimulates MG to generate cells with early cone markers (e.g., RXRG). In adult retinas we also used Sodium butyrate.

[0028] FIG. 7 presents a second example of reprogramming MG with ASCL1 and OTX2 in adult human postmortem retina. The MG-derived neurons express early cone markers but not mature cone markers.

[0029] FIG.8 illustrates reprogramming MG with CRX and ASCL1 in adult human postmortem retina. Over-expression of CRX and ASCL1 in Muller glia (using the HES1 promoter) stimulates MG to generate cells with early rod photoreceptor markers (e.g., NR2E3).

[0030] FIG. 9 illustrates another example of reprogramming MG with ASCL1 and CRX in adult human postmortem retina. Butyrate is required for this reprogramming in the adult retina.DETAILED DESCRIPTION

[0031] The invention described herein is based on the discovery that intraocular gene-based delivery and expression of ASCL1 together with one or more of OTX2, CRX, RAX transcription factors in the retina stimulates regeneration of photoreceptors from retinal Muller glia (MG) and reprograms the MG. This addresses the need to stimulate regeneration of photoreceptor cells in the human retina for the development of new types of regenerative therapies for patients.

[0032] Definitions

[0033] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.

[0034] As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude others. As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the recited embodiment. Thus, the term “consisting essentially of as used herein should not be interpreted as equivalent to “comprising.” “Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions disclosed herein. Aspects defined by each of these transition terms are within the scope of the disclosure herein.

[0035] As used herein, “retinal neuron” refers to any of the five types of neurons in the retina: photoreceptors, bipolar cells, ganglion cells, horizontal cells, and amacrine cells. In some particular embodiments, the retinal neurons are bipolar neurons, amacrine, horizontal, and ganglion cells.

[0036] As used herein, “photoreceptors” refers to retinal cells, including their precursors, that transmit signals in response to light. Photoreceptors include rods, which respond to low levels of light, and cones, which respond to higher levels of illumination and specific frequences of light, i.e., color.

[0037] As used herein, the terms “nucleic acid sequence” or “polynucleotide” refers to nucleotides of any length which are deoxynucleotides (i.e. DNAs), or derivatives thereof; ribonucleotides (i.e. RNAs) or derivatives thereof; or peptide nucleic acids (PNAs) or derivatives thereof. The terms include, without limitation, single-stranded, double-stranded, or multistranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, oligonucleotides (oligos), or other natural, synthetic, modified, mutated or non-natural forms of DNA or RNA.

[0038] MicroRNAs, or “miRNAs”, or “miRs”, are short, non-coding RNAs that regulate gene expression by post-transcriptional regulation of target genes.

[0039] “Short hairpin RNAs” or “shRNAs” are synthetic or non-natural RNA molecules. shRNA refers to RNA with a tight hairpin turn used to silence (via RNA interference or RNAi) target gene expression in a cell. An shRNA is typically delivered via an expression vector such as a DNA plasmid or via viral vectors.

[0040] The term “vector” refers to, without limitation, a recombinant genetic construct or plasmid or expression construct or expression vector that retains the ability to infect and transduce non¬ dividing and / or slowly-dividing cells. The vector may be derived from or based on a wild-type virus. Aspects of this disclosure relate to an adeno-associated virus vector, an adenovirus vector, and a lentivirus vector.

[0041] The term “expression control element” as used herein refers to any sequence that regulates the expression of a coding sequence, such as a gene. Exemplary expression controlelements include but are not limited to promoters, enhancers, microRNAs, post-transcriptional regulatory elements, polyadenylation signal sequences, and introns. Expression control elements may be, without limitation, constitutive, inducible, repressible, or tissue-specific. A “promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. In some embodiments, expression control by a promoter is tissue-specific. An “enhancer” is a region of DNA that can be bound by activating proteins to increase the likelihood or frequency of transcription. Non-limiting exemplary enhancers and posttranscriptional regulatory elements include the CMV enhancer and WPRE.

[0042] The term “multicistronic” or “polycistronic” or “bicistronic” ortricistronic” refers to mRNA with multiple, i.e., double or triple coding areas or exons, and as such will have the capability to express from mRNA two or more, or three or more, or four or more, etc., proteins from a single construct. Multicistronic vectors simultaneously express two or more separate proteins from the same mRNA. The two strategies most widely used for constructing multicistronic configurations are through the use of 1) an IRES or 2) a 2A self-cleaving site. An “IRES” refers to an internal ribosome entry site or portion thereof of viral, prokaryotic, or eukaryotic origin which are used within polycistronic vector constructs. In some embodiments, an IRES is an RNA element that allows fortranslation initiation in a cap-independent manner. The term “self-cleaving peptides” or “sequences encoding self-cleaving peptides” or “2A self-cleaving site” refer to linking sequences which are used within vector constructs to incorporate sites to promote ribosomal skipping and thus to generate two polypeptides from a single promoter, such self-cleaving peptides include without limitation, T2A, and P2A peptides or sequences encoding the self-cleaving peptides.

[0043] The term “substantially complementary,” when used to define either amino acid or nucleic acid sequences, means that a particular sequence, for example, an oligonucleotide sequence, is substantially complementary to the sequence of miR-214 referenced. As such, typically the sequences will be highly complementary to the microRNA “target” sequence, and will have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches throughout the sequence. In many instances, it may be desirable for the sequences to be exact matches, i.e. be completely complementary to the sequence to which the nucleic acid specifically binds, and therefore have zero mismatches along the complementary stretch. As such, highly complementary sequences will typically bind quite specifically to the target sequence region andwill therefore be highly efficient in reducing, and / or even inhibiting the biological activity of the target sequence.

[0044] Substantially complementary nucleic acid sequences will be greater than about 80 percent complementary (or ‘% exact-match’) to the corresponding target sequence to which the nucleic acid specifically binds, and will, more preferably be greater than about 85 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds. In certain aspects, as described above, it will be desirable to have even more substantially complementary nucleic acid sequences for use in the practice of the invention, and in such instances, the nucleic acid sequences will be greater than about 90 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and may in certain embodiments be greater than about 95 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and even up to and including 96%, 97%, 98%, 99%, and even 100% exact match complementary to the target to which the designed nucleic acid specifically binds.

[0045] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of disclosed herein.

[0046] Percent similarity or percent complementary of any of the disclosed sequences may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

[0047] “Nucleotide sequence” refers to a heteropolymer of deoxyribonucleotides, ribonucleotides, or peptide-nucleic acid sequences that may be assembled from smaller fragments, isolated from larger fragments, or chemically synthesized de novo or partially synthesized by combining shorter oligonucleotide linkers, or from a series of oligonucleotides, to provide a sequence which is capable of specifically binding to a target molecule and acting as an antisense construct to alter, reduce, or inhibit the biological activity of the target.

[0048] As used herein, “directed against”, in the context of antisense oligonucleotides, means the antisense oligonucleotide binds to a target miRNA and blocks or suppresses activity of the target.

[0049] As used herein, the terms “protein”, “peptide”, and “polypeptide” refer to amino acid subunits, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In another aspect, the subunit may be linked by other bonds, e.g., ester, ether, etc. As used herein the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids.

[0050] As used herein, the term “recombinant expression system” or “recombinant expression vector” refers to a genetic construct for the expression of certain genetic material formed by recombination.

[0051] The term "effective amount" or "therapeutically effective amount" or "prophylactically effective amount", refer to an amount of an active agent described herein that is effective to provide the desired / intended result and / or biological activity. Thus, for example, in various embodiments, an effective amount of a composition described herein is an amount that is effective to result in regeneration of retinal neurons, and / or to improve or to ameliorate symptoms of and / or to treat retinal degenerative diseases.

[0052] When the disclosure herein relates to a small molecule, polypeptide, protein, polynucleotide, nucleic acid, oligonucleotide, antisense, or miRNA, an equivalent ora biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term “biological equivalent thereof” is intended to be synonymous with “equivalent thereof” when referring to a reference small molecule, polypeptide, protein, polynucleotide, nucleic acid, oligonucleotide, antisense, or miRNA even those reference molecules having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any nucleic acid, polynucleotide, oligonucleotide, antisense, miRNA, polypeptide, or protein mentioned herein also includes equivalents thereof. For example, anequivalent intends at least about 70% homology or identity, or at least 80 % homology or identity and alternatively, or at least about 85 %, or alternatively at least about 90 %, or alternatively at least about 95 %, or alternatively 98 % percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid. Alternatively, when referring to polynucleotides, an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement.

[0053] In some embodiments disclosed herein, the polypeptide and / or polynucleotide sequences are provided herein for use in gene and protein transfer and expression techniques described below. Such sequences provided herein can be used to provide the expression product as well as substantially identical sequences that produce a protein that has the same biological properties. These “biologically equivalent” or “biologically active” or “equivalent” polypeptides are encoded by equivalent polynucleotides as described herein. They may possess at least 60%, or alternatively, at least 65%, or alternatively, at least 70%, or alternatively, at least 75%, or alternatively, at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% or alternatively at least 98%, identical primary amino acid sequence to the reference polypeptide when compared using sequence identity methods run under default conditions. Specific polynucleotide or polypeptide sequences are provided as examples of particular embodiments. Modifications may be made to the amino acid sequences by using alternate amino acids that have similar charge. Additionally, an equivalent polynucleotide is one that hybridizes under stringent conditions to the reference polynucleotide or its complement or in reference to a polypeptide, a polypeptide encoded by a polynucleotide that hybridizes to the reference encoding polynucleotide under stringent conditions or its complementary strand. Alternatively, an equivalent polypeptide or protein is one that is expressed from an equivalent polynucleotide.

[0054] “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0055] Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about 10x SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC. Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about 1x SSC to about 0.1x SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1x SSC, 0.1x SSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes. SSC is 0.15 M NaCI and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.

[0056] As used herein, “treating” or “treatment” of a retinal degenerative disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the compositions, combination therapy, nucleic acid molecules, and methods disclosed herein for inducing neurogenesis from MG and / or generating functional neurons from MG, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms of retinal degeneration, diminishment of extent of a retinal degenerative condition (including a retinal degenerative disease), stabilized ( / .e., not worsening) state of a retinal degenerative condition (including disease), delay or slowing of a retinal degenerative condition (including disease), progression, amelioration or palliation of a retinal degenerative condition (including disease), states of and remission of (whether partial or total) retinal degeneration, whether detectable or undetectable.

[0057] As used herein, the term "isolated” means that a naturally occurring DNA fragment, DNA molecule, coding sequence, or oligonucleotide is removed from its natural environment, or is a synthetic molecule or cloned product. Preferably, the DNA fragment, DNA molecule, coding sequence, or oligonucleotide is purified, i.e., essentially free from any other DNA fragment, DNAmolecule, coding sequence, or oligonucleotide and associated cellular products or other impurities.

[0058] The term “cell” as used herein refers to either a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercially available source. Cells treated, transfected, transformed, or otherwise in contact with compositions and / or nucleic acid molecules disclosed herein, include without limitation, cells of a human, non-human animal, mammal, or non-human mammal, including without limitation, cells of murine, canine, or non-human primate species. Cells treated, transfected, transformed, or otherwise in contact with compositions and / or nucleic acid molecules disclosed herein are, without limitation, retinal cells, Muller glia (MG), and / or retinal neuronal cells such as retinal neurons, bipolar neurons, amacrine cells, horizontal cells, ganglion cells and / or glia. The term “Muller glial” cells “or “Muller glia” or “MG” refer to cells which are found in the vertebrate retina and are support cells for neurons. MG are the most common type of glial cells in the retina. While MG cell bodies are located in the inner nuclear layer of the retina, MG span across the entire retina.

[0059] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects.

[0060] As used herein, “a” or “an” means at least one, unless clearly indicated otherwise.

[0061] As used herein, to “prevent” or “protect against” a condition or disease means to hinder, reduce or delay the onset or progression of the condition or disease.

[0062] The term “encode” as it is applied to nucleic acid sequences refers to a polynucleotide which is said to “encode” a polypeptide, an mRNA, or an effector RNA if, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce the effector RNA, the mRNA, or an mRNA that can for the polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

[0063] As used herein, the term “expression” or “gene expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The expression level of a gene may be determined by measuring the amount of mRNA orprotein in a cell or tissue sample; further, the expression level of multiple genes can be determined to establish an expression profile for a particular sample.

[0064] As used herein, the term “functional” may be used to modify any molecule, biological, or cellular material to intend that it accomplishes a particular, specified effect.

[0065] As used herein, the term “combined therapy” refers to two or more compositions and / or nucleic acid molecules, delivered in combination, for example and without limitation, sequentially, concurrently, simultaneously, and / or step-wise, in order to achieve a therapeutic effect.

[0066] As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0067] The term “about,” as used herein when referring to a measurable value such as an amount, level or concentration, for example and without limitation, is meant to encompass variations of 20%, 10%, 5%, 1 %, 0.5%, or even 0.1 % of the specified amount, or fold differences in levels of a quantifiable comparison with a standard or control or reference material, such as 1-fold, 2-fold, 3-fold, 4-fold...10-fold, 100-fold, etc. of the specified level of comparison.

[0068] In some embodiments, enhancing expression levels of the two or more transcription factors, endogenous and / or exogenous, refers to an increase in the amount expressed as compared to a control sample or explant levels of endogenous and / or exogenous ASCL1 plus OTX2, and / or CRX, and / or RAX such as, without limitation, untreated, or ASCL1 expression alone. In some embodiments, neurogenesis is increased and / or the production of functional photoreceptors is increased as compared to a control. In some embodiments, expression levels and / or functional neurons are increased about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 1.6 fold, about 1.7 fold, about 1.8 fold, about 1.9 fold, about 2 fold, about 2.5 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 20 fold, about 50 fold, about 100 fold, about 1000 fold, or about 10,000 fold relative to the control.

[0069] The terms “acceptable,” “effective,” or “sufficient” when used to describe the selection of any components, ranges, dose forms, etc. disclosed herein intend that said component, range, dose form, etc. is suitable for the disclosed purpose.

[0070] The term “adeno-associated virus” or “AAV” as used herein refers to a member of the class of viruses associated with this name and belonging to the genus dependoparvovirus, family Parvoviridae. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. At least 11 or 12, sequentially numbered, are disclosed in the prior art. Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 or 12 serotypes, e.g., AAV2, AAV5, and AAV8, or variant serotypes, e.g. AAV-DJ. The AAV structural particle is composed of 60 protein molecules made up of VP1, VP2, and VP3. Each particle contains approximately 5 VP1 proteins, 5 VP2 proteins and 50 VP3 proteins ordered into an icosahedral structure.Compositions / Nucleic acid Molecules

[0071] Provided are compositions and / or nucleic acid molecules for retinal regeneration, the potentiation of retinal regeneration, restoration of vision, and for treatment of retinal degenerative disease, damage, or injury.

[0072] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding two or more transcription factors, namely a proneural bHLH transcription factor, such as Achaete-scute homolog 1 (ASCL1) or Neuronal Differentiation 1 (Neurodi), and at least one of OTX2, CRX, and RAX. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1 and OTX2. In another embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1 and CRX. In another embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1 and RAX. In another embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1, OTX2 and CRX. In another embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1, OTX2 and RAX. In another embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1, CRX and RAX. In another embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1, OTX2, CRX, and RAX.

[0073] An exemplary nucleic acid sequence encoding human ASCL1 can be found at NCBI Reference Sequence number NG_008950.1 (SEQ ID NO: 1). In another embodiment disclosed herein is a nucleic acid sequence encoding a human ASCL1 amino acid sequence or portion thereof of UniProtKB / Swiss-Prot: P50553.2 (SEQ ID NO: 2). ASCL1 homologues, e.g., derived from species such as murine, canine, equine, are included herein, without limitation.

[0074] An exemplary nucleic acid sequence encoding OTX2 can be found at (SEQ ID NO: 3). In another embodiment disclosed herein is a nucleic acid sequence encoding a human OTX2 amino acid sequence or portion thereof of UniProtKB / Swiss-Prot: P50553.2 (SEQ ID NO: 4). OTX2 homologues, e.g., derived from species such as murine, canine, equine, are included herein, without limitation.

[0075] An exemplary nucleic acid sequence encoding CRX can be found at (SEQ ID NO: 5). In another embodiment disclosed herein is a nucleic acid sequence encoding a human CRX amino acid sequence or portion thereof of UniProtKB / Swiss-Prot: P50553.2 (SEQ ID NO: 6). CRX homologues, e.g., derived from species such as murine, canine, equine, are included herein, without limitation.

[0076] An exemplary nucleic acid sequence encoding RAX can be found at (SEQ ID NO: 7). In another embodiment disclosed herein is a nucleic acid sequence encoding a human RAX amino acid sequence or portion thereof of UniProtKB / Swiss-Prot: P50553.2 (SEQ ID NO: 8). RAX homologues, e.g., derived from species such as murine, canine, equine, are included herein, without limitation.

[0077] In one embodiment disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding the ASCL1 and at least one of OTX2, CRX, and RAX in operable linkage with a glia-specific promoter. In some embodiments, the promoter is active in both glia and progenitor cells. In another embodiment, a nucleic acid molecule comprising a nucleic acid sequence encoding the ASCL1 and at least one of OTX2, CRX, and RAX in operable linkage with a HES1 promoter. In some embodiments, the HES1 promoter comprises a sequence modified by introducing nucleotide substitutions and deletions that exhibits improved expression, such as the sequence of SEQ ID NO: 9. In another embodiment, a nucleic acid molecule comprising a nucleic acid sequence encoding the ASCL1 and at least one of OTX2, CRX, and RAX in operable linkage with a human RLBP1 promoter. RLBP1 (Retinaldehyde binding protein 1) is a robust MG tissue specific promoter capable of driving expression of the two or more transcription factors to MG cells at a level sufficient to induce photoreceptor genesis from MG. In one embodiment, a portion of the RLBP1 promoter is used for ASCL1 + OTX2, CRX, and / or RAX expression in MG. In another embodiment, the portion of the RLBP1 promoter in operable linkage with the nucleic acid sequence encoding ASCL1 and OTX2, CRX, and / or RAX is the RLBP1 sequence found in SEQ ID NO: 10. In some embodiments, the promoter that is active in both glia and progenitor cells is LHX2, RAX promoter, HES5, or VSX2.Using promoters active in both cell types allows one to drive the expression of other TFs through the transition from glia to progenitors.

[0078] In one embodiment, the nucleic acid molecule for use in inducing or stimulating photoreceptor genesis from MG comprises a nucleic acid sequence encoding ASCL1 + OTX2, CRX, and / or RAX comprises a native ASCL1 promoter sequence. In another embodiment, the nucleic acid sequence encoding ASCL1 + OTX2, CRX, and / or RAX comprises a non-native ASCL1 promoter such as a retinal specific promoter. In another embodiment, the promoter is a MG-specific promoter such as, for example and without limitation, a HES1 promoter or portion thereof, retinaldehyde binding protein 1 (RLBP1) promoter or portion thereof, glial fibrillary acidic protein (GFAP) promoter, vimentin (VIM) promoter, orCD44, orGLAST promoter. In another embodiment, the promoter is a ubiquitous promoter such as, for example and without limitation, a CMV promoter, CAG promoter, CBh promoter, or miniCMV promoter.

[0079] Such nucleic acid molecules may be delivered by viral or non-viral means. One example of viral delivery is adeno-associated virus (AAV). Other examples include retrovirus, lentivirus, and baculovirus delivery. One example of a non-viral method of miR delivery is cell penetrating peptide (CPP). Polynucleotide constructs may also be modified, such as through chemical modification, to improve their stability and / or suitability for delivery. In some embodiments, the oligonucleotide is modified by locked nucleic acids and / or phosphorothioate linkages. In some embodiments, a delivery system is selected for improved bioavailability, such as PEGylated liposomes, lipidoids, or biodegradable polymers, as examples.Nucleic Acid Molecules and Combined Therapy Compositions

[0080] In some embodiments, the composition further comprises one or more additional potentiating or therapeutic agents, including, for example, reprogramming potentiating agents. In some embodiments, the composition is free of reprogramming potentiating agents.Optionally, a composition comprising one or more small molecule reprogramming potentiating agents can be administered sequentially or concurrently with the nucleic acid molecules disclosed herein. In another embodiment, one or more protein / peptide or miR-based reprogramming potentiators can be incorporated into the nucleic acid molecules disclosed herein. Such one or more reprogramming potentiators are selected from HDACi, STATi, Jak / STATi and RNAi-based ASCL1 activators. See also our previous work in WO2019210320, incorporated herein by reference in its entirety.

[0081] In one embodiment, the HDAC signaling pathway inhibitor is selected from the group consisting of peptidomimetics, small molecule inhibitors, oligonucleotides, peptides and proteins. Representative examples of small molecule HDACi include, but are not limited to, trichostatin A (TSA), Istodax™ also known as (Pro) / romidepsin, Beleodaq™, also known as (Pro) / belinostat, Farydak™, also known as (Pro) / panobinostat, and Zolinza™, also known as (Pro) / vorinostat, Quisinostat, Abexinostat, Givinostat, Resminostat, Phenylbutyrate, Valproic Acid, Depsipeptide, Entinostat, Mocetinostat, and Tubastatin A. Exemplary HDACi peptides are, without limitation, 16cyc-HxA, 16lin-HxA and 16KA (See WO2023192994).

[0082] In one embodiment, the STAT signaling pathway inhibitor or Jak / STATI is selected from the group consisting of natural compounds, peptidomimetics, peptides, proteins, small molecules and oligonucleotides. Examples of endogenous STAT pathway inhibitors, include, but are not limited to, suppressor of cytokine signaling (SOCS) proteins, phosphatases, and protein inhibitor of activated STAT (PIAS) proteins. Such endogenous inhibitors provide a basis for therapeutic molecules and compounds for STAT inhibition. In one embodiment, protein or peptide inhibitors of STAT include, for example and without limitation, Socsl, Socs2, Socs3, Socs4, Socs5, Socs6, Socs7, CIS, and / or XpYL (See WO2023192994). In one embodiment, a peptidomimetic inhibitor of STAT is ISS610. In another embodiment, small molecule inhibitors of STAT and / or Jak / STAT include, without limitation, STA-21, LLL3, S31-201, Stattic, OPB-31121, OPB-51602, SH-4-54, Tofactinib, Ruxolitinib, Baricitinib, Oclacitinib, AZD1480, and Dasatinib. In one embodiment, the STAT signaling pathway inhibitor is an inhibitor of STAT3. An exemplary STAT3 inhibitor includes, but is not limited to, SH-4-54. See also Fagard et al. JAKSTAT. 2013 Jan 1; 2(1): e22882. In another embodiment, STAT and / or Jak / STAT inhibitors include natural compounds such as Butein and Capsaicin.

[0083] In some embodiments, the inhibitor, mimic, activator, orantagomir is an oligonucleotide or a nucleotide sequence. The invention thus provides nucleotide constructs for use in the compositions or combined therapy or nucleic acid molecules and methods described herein.

[0084] The reprogramming potentiating agents, in some embodiments, are selected from one or more STAT signaling pathway inhibitors; and one or more ASCL1 activators such as, without limitation, miR-25 and / or miR-124; and one or more let-7 family inhibitors. Exemplary sequences for such agents include, without limitation, those provided in WO2023192994.

[0085] Optionally, provided herein is a composition comprising any one or more of the combined therapy of RNAi-based ASCL1 activators and / or HDACi + STATi, and / or a nucleic acid sequence encoding the two or more proneural bHLH transcription factors or a vectorcomprising the nucleic acid sequences disclosed herein, and a carrier. In some embodiments, the carrier is a pharmaceutically acceptable carrier.

[0086] Viral Vectors

[0087] In some embodiments, the vector disclosed herein is a viral vector, or pair of viral vectors. In some embodiments, the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, or a lentiviral vector. In some embodiments, the vector is a retroviral vector, an adenoviral / retroviral chimera vector, a herpes simplex viral I or II vector, a parvoviral vector, a reticuloendotheliosis viral vector, a polioviral vector, a papillomaviral vector, a vaccinia viral vector, or any hybrid or chimeric vector incorporating favorable aspects of two or more viral vectors. In some embodiments, the vector further comprises one or more expression control elements operably linked to the polynucleotide. In some embodiments, the vector further comprises one or more selectable markers.

[0088] In some embodiments, the vector disclosed herein is an AAV vector with low toxicity. In some embodiments, the AAV vector does not incorporate into the host genome, thereby having a low probability of causing insertional mutagenesis. In some embodiments, the AAV vector can encode a range of total polynucleotides from 4.5 kb to 4.75 kb. In some embodiments, exemplary AAV vectors that may be used in any of the herein described compositions, systems, methods, and kits can include an AAV1 vector, a modified AAV1 vector, an AAV2 vector, a modified AAV2 vector, an AAV3 vector, a modified AAV3 vector, an AAV4 vector, a modified AAV4 vector, an AAV5 vector, a modified AAV5 vector, an AAV6 vector, a modified AAV6 vector, an AAV7 vector, a modified AAV7 vector, an AAV8 vector, an AAV9 vector, an AAV.rhIO vector, a modified AAV.rhIO vector, an AAV.rh32 / 33 vector, a modified AAV.rh32 / 33 vector, an AAV.rh43 vector, a modified AAV.rh43 vector, an AAV.rh64R1 vector, and a modified AAV.rh64R1 vector and any combinations or equivalents thereof.

[0089] In some embodiments, the vector disclosed herein is a lentiviral vector. In one embodiment, the lentiviral vector is an integrase-competent lentiviral vector (ICLV). In some embodiments, the lentiviral vector can refer to the transgene plasmid vector as well as the transgene plasmid vector in conjunction with related plasmids (e.g., a packaging plasmid, a rev expressing plasmid, an envelope plasmid) as well as a lentiviral-based particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism. Lentiviral vectors are well-known in the art. In some embodiments, exemplary lentiviral vectors that may be used in relation to any of the herein described compositions, nucleic acid molecules and / or methods, and can include a human immunodeficiency virus (HIV) 1 vector, a modifiedhuman immunodeficiency virus (HIV) 1 vector, a human immunodeficiency virus (HIV) 2 vector, a modified human immunodeficiency virus (HIV) 2 vector, a sooty mangabey simian immunodeficiency virus (SIVSM) vector, a modified sooty mangabey simian immunodeficiency virus (SIVSM) vector, a African green monkey simian immunodeficiency virus (SIVAGM) vector, a modified African green monkey simian immunodeficiency virus (SIVAGM) vector, a equine infectious anemia virus (EIAV) vector, a modified equine infectious anemia virus (EIAV) vector, a feline immunodeficiency virus (FIV) vector, a modified feline immunodeficiency virus (FIV) vector, a Visna / maedi virus (VNV / VMV) vector, a modified Visna / maedi virus (VNV / VMV) vector, a caprine arthritis-encephalitis virus (CAEV) vector, a modified caprine arthritis-encephalitis virus (CAEV) vector, a bovine immunodeficiency virus (BIV), ora modified bovine immunodeficiency virus (BIV).

[0090] In some embodiments of the compositions and / or nucleic acid molecules and / or methods of the disclosure, a vector of the disclosure is a viral vector. In some embodiments, the viral vector comprises a sequence isolated or derived from a retrovirus. In some embodiments, the viral vector comprises a sequence isolated or derived from a lentivirus. In some embodiments, the viral vector comprises a sequence isolated or derived from an adenovirus. In some embodiments, the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV). In some embodiments, the viral vector is replication incompetent. In some embodiments, the viral vector is isolated or recombinant. In some embodiments, the viral vector is self-complementary.

[0091] In some embodiments of the compositions and / or nucleic acid molecules and / or methods of the disclosure, the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV). In some embodiments, the viral vector comprises an inverted terminal repeat sequence or a capsid sequence that is isolated or derived from an AAV of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or the vector and / or components are derived from a synthetic AAV serotype, such as, without limitation, Anc80 AAV (an ancestor of AAV 1, 2, 6, 8 and 9). In some embodiments, the viral vector is replication incompetent. In some embodiments, the viral vector is isolated or recombinant (rAAV). In some embodiments, the viral vector is self-complementary (scAAV).

[0092] In some embodiments of the compositions and methods of the disclosure, a vector of the disclosure is a non-viral vector. In some embodiments, the vector comprises or consists of a nanoparticle, a micelle, a liposome or lipoplex, a polymersome, a polyplex or a dendrimer.

[0093] In some embodiments, expression vector or viral vector disclosed herein is used to transfect, transform, or come in contact with a cell which is a eukaryotic cell. In some embodiments, the cell is an animal cell. In some embodiments, the cells is a zebrafish cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a bovine, murine, feline, equine, porcine, canine, simian, or human cell. In particular embodiments, the cell is a retinal neuron or MG of an animal or mammal.

[0094] In some embodiments, a cell is a packaging cell or a producer cell for production of a viral particle.

[0095] In some embodiments, provided herein are viral particles comprising, consisting of, or consisting essentially of a vector comprising, consisting of, or consisting essentially of a polynucleotide sequence encoding an ASCL1 protein in combination with another transcription factor.

[0096] In general, methods of packaging genetic material such as RNA or DNA into one or more vectors is well known in the art. For example, the genetic material may be packaged using a packaging vector and cell lines and introduced via traditional recombinant methods.

[0097] In some embodiments, the packaging vector may include, but is not limited to retroviral vector, lentiviral vector, adenoviral vector, and adeno-associated viral vector. The packaging vector contains elements and sequences that facilitate the delivery of genetic materials into cells. For example, the retroviral constructs are packaging plasmids comprising at least one retroviral helper DNA sequence derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required to package a replication incompetent retroviral vector, and for producing virion proteins capable of packaging the replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus. The retroviral DNA sequence lacks the region encoding the native enhancer and / or promoter of the viral 5’ LTR of the virus, and lacks both the psi function sequence responsible for packaging helper genome and the 3’ LTR, but encodes a foreign polyadenylation site, for example the SV40 polyadenylation site, and a foreign enhancer and / or promoter which directs efficient transcription in a cell type where virus production is desired. The retrovirus is a leukemia virus such as a Moloney Murine Leukemia Virus (MMLV), the Human Immunodeficiency Virus (HIV), or the Gibbon Ape Leukemia virus (GALV). The foreign enhancer and promoter may be the human cytomegalovirus (HCMV) immediate early (IE) enhancer and promoter, the enhancer and promoter (U3 region) of the Moloney Murine Sarcoma Virus (MMSV), the U3 region of RousSarcoma Virus (RSV), the U3 region of Spleen Focus Forming Virus (SFFV), or the HCMV IE enhancer joined to the native Moloney Murine Leukemia Virus (MMLV) promoter.

[0098] The retroviral packaging plasmid may consist of two retroviral helper DNA sequences encoded by plasmid-based expression vectors, for example where a first helper sequence contains a cDNA encoding the gag and pol proteins of ecotropic MMLV or GALV and a second helper sequence contains a cDNA encoding the env protein. The Env gene, which determines the host range, may be derived from the genes encoding xenotropic, amphotropic, ecotropic, polytropic (mink focus forming) or 10A1 murine leukemia virus env proteins, or the Gibbon Ape Leukemia Virus (GALV env protein, the Human Immunodeficiency Virus env (gp160) protein, the Vesicular Stomatitus Virus (VSV) G protein, the Human T cell leukemia (HTLV) type I and II env gene products, chimeric envelope gene derived from combinations of one or more of the above env genes or chimeric envelope genes encoding the cytoplasmic and transmembrane of the above env gene products and a monoclonal antibody directed against a specific surface molecule on a desired target cell. Similar vector-based systems may employ other vectors such as sleeping beauty vectors or transposon elements.

[0099] The resulting packaged expression systems may then be introduced via an appropriate route of administration, discussed in detail with respect to the method aspects disclosed herein.

[0100] Pharmaceutical compositions

[0101] Pharmaceutical compositions disclosed herein include one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the disclosure may be formulated for intraocular administration.

[0102] Cells

[0103] In some embodiments of the compositions and / or nucleic acid molecules and / or methods of the disclosure, a cell of the disclosure is a retinal cell, such as a Muller glial (MG) cell, or a rod or cone photoreceptor cell. In some embodiments, the cell is a neuronal cell. In some embodiments, a neuronal cell of the disclosure is a neuron of the retina. In some embodiments, a neuron cell of the disclosure is a neuron of an optic nerve. In some embodiments, a neuron cell of the disclosure is a neuroglial ora glial cell. In someembodiments, a cell is a bipolar neuron, a horizontal cell, a ganglion cell, or an amacrine cell. In some embodiments, a cell of the disclosure is an astrocyte. In some embodiments, cells of the disclosure are macroglia or microglia or glia.

[0104] In some embodiments of the compositions and methods of the disclosure, a cell of the disclosure is a cultured cell.

[0105] In some embodiments of the disclosure, a cell is in vivo, in vitro, ex vivo, or in situ. In some embodiments, the cells are modified ex vivo and transplanted into and / or administered to the retina of a subject in need thereof.

[0106] In some embodiments, a cell of the disclosure is autologous or allogeneic and used for transplantation.

[0107] In some embodiments, a cell of the disclosure is a stem cell-derived or an embryonic stem cell-derived retinal cell. In some embodiments, the cell is derived from an induced pluripotent stem cell (iPS cell)-dervied retinal cell.

[0108] Methods

[0109] Described herein are methods for inducing photoreceptor regeneration in a subject. Also provided are methods for enhancing retinal regeneration, improving retinal neurogenesis, potentiating retinal regeneration, restoring vision, and treating retinal degenerative disease, damage, injury, or blindness.

[0110] In one embodiment is a method for inducing photoreceptor regeneration in a subject comprising: a) administering to a retina of the subject the nucleic acid molecules and / or compositions disclosed herein. In one embodiment, a method for inducing retinal regeneration in a subject comprises: a) administering to a retina of the subject a nucleic acid molecule comprising a nucleic acid sequence encoding two or more transcription factors, wherein expression of the transcription factors stimulates regeneration of photoreceptors from retinal Muller glia (MG). In particular, in one embodiment of the method disclosed herein, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1 and OTX2, CRX, and / or RAX. In another embodiment of the method disclosed herein, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1 and OTX2. In another embodiment of the method disclosed herein, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1 and CRX. In another embodiment of the method disclosed herein, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1 and RAX. In another embodiment of the method disclosed herein, the nucleic acid molecule comprises a nucleic acidsequence encoding ASCL1, OTX2, and CRX. In another embodiment of the method disclosed herein, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1, OTX2 and RAX. In another embodiment of the method disclosed herein, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1, CRX, and RAX. In another embodiment of the method disclosed herein, the nucleic acid molecule comprises a nucleic acid sequence encoding ASCL1, OTX2, CRX, and RAX.

[0111] In one embodiment of the method disclosed herein, the nucleic acid molecule comprising a nucleic acid sequence encoding the two or more transcription factors is in operable linkage with a glia-specific promoter. A representative example of the glia-specific promoter is a HES1 promoter or portion thereof. In another particular embodiment of the method disclosed herein, a nucleic acid molecule comprising a nucleic acid sequence encoding the two or more transcription factors (e.g., ASCL1 plus OTX2, CRX, and / or RAX) is in operable linkage with a human RLBP1 promoter.

[0112] Also provided herein are methods for inducing photoreceptor regeneration comprising administering to a subject a composition as described herein. In some embodiments, the methods are effective to increase the number of Muller glial-derived photoreceptors, to induce Muller glial (MG) cells to enter the mitotic cell cycle, and / or to generate new photoreceptor cells. In some embodiments of the method, the number of photoreceptors increases by at least 25% relative to a baseline level or other reference amount representative of an untreated retina. In other embodiments, the number of photoreceptors increases by at least 40%. In some embodiments, the number of photoreceptors increases by 5%, 10%, 20%, 50%, 100%, 150%, 200%, or more.

[0113] Optionally, methods disclosed herein may utilize combined therapy compositions comprising one or more, or two or more, small molecule reprogramming potentiating agents. The agents can be administered sequentially or concurrently with the nucleic acid molecules disclosed herein. In another embodiment, one or more protein / peptide or miR-based reprogramming potentiators can be incorporated into the nucleic acid molecules used in the methods disclosed herein. Such one or more reprogramming potentiators are selected from HDACi, STATi, Jak / STATi and RNAi-based ASCL1 activators. See also our previous work in W02019210320, incorporated herein by reference in its entirety. In some embodiments, the method is performed in the absence of such reprogramming potentiators.

[0114] The subject is typically a mammal, such as a human or veterinary subject. In one embodiment, the subject is an adult. The subject, in some embodiments, has a retinaldegenerative disease. Examples of such retinal degenerative diseases include, but are not limited to, age-related macular (cone or rod) degeneration (AMD). In some embodiments, the subject has suffered an injury (e.g., solar or laser retinopathy).

[0115] Administration and Dosage

[0116] The compositions and / or nucleic acid molecules disclosed herein are administered in any suitable manner, often with pharmaceutically acceptable carriers. Suitable methods of administering compositions, compounds, molecules, nucleic acids, and vectors in the context of the present invention to a subject’s eye or retina are available, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. For treatment of the retina, intraocular injection, such as, for example and without limitation, intravitreal injection and subretinal injection are the most common routes of delivery to the retina. In some embodiments, however, periocular, suprachoroidal, systemic, or topical administration is more suitable for efficacy and safety of delivery.

[0117] The dose administered to a patient, in the context of the disclosure herein, should be sufficient to result in a beneficial therapeutic response in the patient over time, or to inhibit disease progression. Thus, the composition is administered to a subject in an amount sufficient to elicit an effective response and / or to alleviate, reduce, cure or at least partially arrest symptoms and / or complications from the retinal disease or injury. An amount adequate to accomplish this is defined as a "therapeutically effective dose."

[0118] Routes, order and / or frequency of administration of the therapeutic compositions disclosed herein, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. In general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and / or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome in treated patients as compared to non-treated patients.EXAMPLES

[0119] The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.

[0120] Example 1: Photoreceptor-like cells regenerated from Muller glial cells

[0121] Age-related macular degeneration (AMD), characterized by a slow degeneration of the retinal pigment epithelium and photoreceptors cells in the central retina, is one of the leading causes of visual impairment in individuals over 50 (1 ). Several therapeutic approaches have been developed to delay the progression of the disease and protect the remaining cells. In advanced stages, however, when photoreceptors and, more specifically, cones, have already degenerated, novel therapeutic strategies are necessary to replace the missing cells. Currently, most of the efforts in the field of regenerative medicine for AMD focus on using stem cell transplants to replace these cells, though there are significant barriers to integration of transplanted cells, and to date establishment of synaptic connectivity has been limited. An alternative strategy consists of stimulating the intrinsic regenerative capacity of the retina.

[0122] Although the mammalian retina does not regenerate neurons, some vertebrates such as zebrafish can regenerate their retina after injury (2). This fascinating process is driven by the Muller glia (MG), which are able to re-enter the cell cycle and reprogram into neurogenic progenitors upon retinal injury. Efforts have been made to better understand the mechanisms underlying the zebrafish’s regenerative capacity and to “translate” this to mammals to promote retinal regeneration. A breakthrough in the field from the inventors’ previous work was the demonstration that MG can generate new neurons in vivo in the adult mouse retina after the overexpression of the pro-neural transcription factor (TF) ASCL1 (3). The newly generated neurons integrate into the existing circuit and adopt a bipolar-like fate. In addition, a new cocktail of TFs combining Isletl, Pou4f2, and ASCL.1 has shown great efficiency in reprograming MG into retinal ganglion cell (RGC)-like cells (4,5), thus demonstrating that particular combination of TFs could direct the MG to generate specific retinal cell types. However, we had not yet found a specific TF combination that efficiently directs MG to generate new photoreceptors.

[0123] As prior studies were done in mice, we recently tested the same strategy on MG dissociated cultures from human fetal retinas and human retinal organoids. Using a lentiviral mediated system to overexpress ASCL1, we demonstrated that MG cultures can be reprogrammed into neurons (6). Although these results are promising, the MG cultures are limited: the MG-derived neurons in dissociated cultures do not maintain their normal morphology and remain immature. Following this initial study on human MG reprograming in dissociated cultures, we next tested this strategy in a more intact retinal environment: Retinospheres, an in vitro culture system of fetal retinal tissues, that can grow and mature in culture for many months and, unlike retinal organoids, their lamination is preserved overtime. We modified our lentiviralconstruct to integrate a glial specific promoter instead of CMV to drive the expression of ASCL1 and track the MG derived neurons. Specifically, we tested different promoters and determined that HES1 is highly specific to glial cells (7). One major advantage of this approach is the maintenance of the expression of ASCL1, even as cells reprogrammed from glia to progenitor cells, since HES1 is also expressed in progenitor cells during fetal development. With this new approach we find that 7- and 14-days post infection with ASCL1, approximately 20% of the GFP+ MG-derived cells expressed the OTX2 neuronal marker. Furthermore, 14 days after ASCL1 overexpression, GFP+ cells also expressed the bipolar cell marker PCP2, thus showing that ASCL1 stimulates bipolar cell regeneration in 3D retinospheres. Recently, we found that combining ASCL1 with ATOH1 induces MG to generate OTX2+ and HuC / D+ neurons in human fetal MG thus demonstrating combinations of TFs can expand the diversity of MG-derived neurons just like in mouse. Taken together, these results provide a rationale fortesting additional TFs to potentially unlock still inaccessible cell fates, such as photoreceptors.

[0124] New method to stimulate photoreceptors from Muller glia

[0125] By studying the development of photoreceptors, we and others have identified many TFs required for photoreceptor genesis (8-10). More specifically, our recent Multiome data generated on young fetal retinal tissues (D59 and D76) allowed us to identify additional candidate genes for early human photoreceptor development. We generated lentiviral vectors carrying over 10 of these potential “cone-inducing” transcription factors, along with the ASCL1 proneural gene and tested these individually for their ability to stimulate cone photoreceptor genesis from mouse and human Muller Glia. Figure 1 shows examples of three of these vectors, using the HES1 promoter to drive expression specifically in glial cells.

[0126] Mouse (Figure 2). As noted above, we have previously shown that Muller glia can be grown from mice as dissociated cell cultures and that proneural transcription factors, like ASCL1 and Atohl can induce the glial cells to acquire a retinal progenitor-like state and generate new neurons to replace those lost from injury or degenerative diseases. To date, we have been successful at regenerating bipolar neurons and ganglion cells, two classes of inner retinal neurons, but we have not been able to induce the MG to produce photoreceptors. In this experiment, we isolated Muller glia from young mice (P12) and grew them in dissociated cultures. We then infected the cells (DO) with a lentivirus that induces expression of either OTX2+ASCL1, CRX+ASCL1 or RAX+ASCL1. Both of these transcription factor combinations induced the Muller glia to make cells that express markers of photoreceptors (RXRG andRecoverin) with high efficiency after 8 days (D8). We find that adding Crx to ASCL1 is more effective than Otx2 at inducing photoreceptors in mouse Muller glia.

[0127] Human (Figure 3). We have also tested the expression of ASCL1 in human MG and previously found that this can stimulate neurogenesis in fetal and adult human MG. However, the over-expression of ASCL1 alone or in combination with other proneural factors, ATOH1 or NEUROD1 stimulates neurogenesis of inner retinal neurons, but not photoreceptors. We now find that over-expression of the combination of either OTX2 + ASCL1 or CRX + ASCL1 in human MG also stimulates photoreceptor production from the cells. Figure 3 shows examples of infection of fetal retinal explants with these combinations and some of the infected cells express markers of photoreceptors (OTX2, RXRG, ARR3) and the GFP lineage tracer. These factors were selectively expressed in MG using the HES1 promoter, and so the cells labeled with photoreceptor markers and GFP were generated by MG.

[0128] After screening over 10 factors fortheir ability to stimulate cone genesis from Muller glia in human and mice, we have discovered that three factors have this ability. The combination of either OTX2, CRX or RAX with the proneural factor ASCL1 enable the Muller glia to generate new rod and cone photoreceptors. These results may be applicable to restore vision in patients that have lost eyesight from cone or rod degeneration (AMD), or injury (e.g., solar or laser retinopathy).

[0129] References

[0130] 1. Rein DB, et al. JAMA Ophthalmol. 2022 Dec 15; 140(12): 1202-8.

[0131] 2. Goldman D. Nature Reviews Neuroscience. 2014(15): 431-42.

[0132] 3. Jorstad NL, et al. Nature. 2017;548(7665):103-7.

[0133] 4. Todd L, et al. Sci Adv 8, eabq7219(2022). DOI:10.1126 / sciadv.abq7219

[0134] 5. Todd L, et al. Cell Rep. 2021;37(3):109857. doi.org / 10.1016 / j.celrep.2021.109857

[0135] 6. Wohlschlegel J, et al. Stem Cell Reports. 2023 Dec 12; 18(12):2400-2417.

[0136] 7. Wohlschlegel J, et al. bioRxiv. 2024 Jan 1;2024.09.24.614778.

[0137] 8. Sridhar A, et al. Cell Rep. 2020;30(5):1644-1659.e4.

[0138] 9. Finkbeiner C, et al. Cell Rep. 2022;38(4).

[0139] 10. Lu Y, et al. Dev Cell. 2020;53(4):473-491,e9.Example 2: Not all genes tested were able to reprogram Muller glial cells into photoreceptor cells

[0140] The studies described in Example 1 were also performed using other candidate genes, many of which were not successful in achieving the reprogramming of Muller glial cells into photoreceptor cells in human or mouse cells. Listed below are the genes tested that do not reprogram Muller glial cells into photoreceptor cells. Tested were genes that are expressed during early photoreceptor development. The genes were cloned into lentiviral vectors along with ASCL1 and the vectors were used to infect fetal 3D retinal cultures. After 7 days in vitro, new photoreceptor production from the MG was assessed with immunofluorescence for photoreceptor genes, such as RXRgamma.

[0141] List of genes that do not reprogram MG into photoreceptor cells in human or mouse cellsPRDM1 ZHX1MYBL1 RUNX1THRB CUX2HMX1 EYA2ONECUT1 MEIS2ZEB1 ZNF608POU2F1 RORBPAX6 / HMGB3 K. LF7ZFP281 MYBSOX11 OLIG2NEUROG2 LIN28MYTL1 FOXN4RFX3\SOX2 EBNA1

[0142] Example 3: HES1 promoter drives efficient expression

[0143] Exemplary sources for the promoter sequence can be found in HES1 (SEQ ID NO: 9), RLBP1 (SEQ ID NO: 10), orGLAST (also known as SL1A3; SEQ ID NOs: 11-13; see NCBI Reference Sequence Number NM_004172). We have found that the human RLBP1 promoter works very well to drive expression in Muller glia (mouse, human or NHP), but, it is 2.8 kB andtoo cumbersome for 2-3 reprogramming TFs. Thus we began working with a much smaller promoter: HES1. HES1 promoter provides a good alternative to RLBP1 promoter. HES1 promoter is only 337 bp, it is expressed in adult Muller glia, and in retinal progenitors. In addition, HES1 is increased in expression after ASCL1 infection. HES1 promoter drives very good expression specifically in Muller glia in human retinal organoids. GFP is expressed in HES1 and Sox9 positive cells seven days after the infection with a HES1-GFP construct. The construct is specific at 7 days. HES1-promoter also directs GFP expression in MG in adult NHP dissociated cultures.

[0144] This small and specific promoter offers the potential for a single AAV virus to drive reprogramming TFs in Muller glia. The HES1 promoter shows specificity. The related HES5 offers another alternative.

[0145] Example 4: Further data showing photoreceptor regeneration

[0146] Fetal retinal sphere cultures were infected with CRX and ASCL1 TFs for 7 days. FIG. 5 presents fluorescent photomicrographs demonstrating successful expression and photoreceptor regeneration. The arrowhead in FIG. 5 points to an example of an early photoreceptor cell that is GFP+ OTX2+ and RXRG+.

[0147] FIG. 6 illustrates photoreceptor regeneration from human Muller glia via the quantitation of effect of expression of RXRG in GFP+ cells. Between 10 -16% of the GFP+ (infected) cells express RXRG after 7 days when using OTX2 and ASCL1 (left bar graph). CRX and ASCL1 were also able to reprogram the Muller glia to generate photoreceptors, though the percentage was closer to 10%. A small subset of the ASCL1 / CRX infected cells generated rod photoreceptors (NR2E3; right bar graph and photomicrographs).

[0148] A new construct that has the mScarlet reporter was tested in human adult postmortem retinal explants (FIG. 7). Over-expression of OTX2 and ASCL1 in Muller glia (using the HES1 promoter) stimulates MG to generate cells with early cone markers (e.g., RXRG). In adult retinas we also used Sodium butyrate at 1 mM (final concentration). In some embodiments, small molecule inhibitors of histone deacetylase (HDAC) are added to improve reprogramming efficiency. Such HDAC inhibitors (HDACi) are not always needed in mouse, but it has proven to be helpful in the adult. The HDACi can be applied systemically, or through an intravitreal injection. Examples of HDACi include, but are not limited to, sodium butyrate, Trichostatin A (TSA), Vorinostat (SAHA), Romidepsin (FK228), Belinostat (PXD101), and Valproic Acid.

[0149] FIG. 8 presents a second example of reprogramming MG with ASCL1 and OTX2 in adult human postmortem retina. The MG-derived neurons express early cone markers but not mature cone markers.

[0150] FIG. 9 illustrates reprogramming MG with CRX and ASCL1 in adult human postmortem retina. Over-expression of CRX and ASCL1 in Muller glia (using the HES1 promoter) stimulates MG to generate cells with early rod photoreceptor markers (e.g., NR2E3).

[0151] FIG. 10 illustrates another example of reprogramming MG with ASCL1 and CRX in adult human postmortem retina. Sodium butryate was used as an adjunct for reprogramming in the adult retina.

[0152] Thus, in fetal retinal spheres, OTX2+ASCL1 and CRX+ASCL lead to expression of an early cone marker. In adult postmortem retina, with sodium butyrate, OTX2+ASCL1 leads to expression of an early cone marker and CRX+ASCL1 leads to expression of a rod marker.

[0153] Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention pertains. Also incorporated by reference herein in its entirety is provisional application number 62 / 840,264, filed April 29, 2019.

[0154] From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

What is claimed is:

1. A nucleic acid molecule comprising a nucleic acid sequence encoding an ASCL1 transcription factor and one or more additional transcription factors selected from OTX2, CRX, and RAX.

2. The nucleic acid molecule of claim 1, wherein the additional transcription factor is OTX2.

3. The nucleic acid molecule of claim 1, wherein the additional transcription factor is CRX.

4. The nucleic acid molecule of claim 1, wherein the additional transcription factor is RAX.

5. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence comprises a promoter sequence.

6. The nucleic acid molecule of claim 5, wherein the promoter sequence is a retinal progenitor or glia-specific promoter.

7. The nucleic acid molecule of claim 6, wherein the glia-specific promoter is HES1 or RLBP1.

8. A method for inducing photoreceptor regeneration in a retina comprising: administering to a retina the nucleic acid molecule of claim 1, wherein expression of the nucleic acid molecule stimulates regeneration of photoreceptors from retinal Muller glia (MG).

9. The method of claim 8, wherein the retina is in a mammalian subject.

10. The method of claim 9, wherein the photoreceptors express one or more markers selected from RXRG, Recoverin, OTX2, and ARR3.

11. The method of claim 9, wherein the subject is treated for disease, damage or degeneration of the retina.

12. The method of claim 9, wherein the subject is an adult.

13. The method of claim 8, wherein a vector comprises the nucleic acid molecule.

14. The method of claim 13, wherein the vector is a non-viral vector or a viral vector.

15. The method of claim 14, wherein the viral vector is an adeno-associated viral (AAV) vector or a lentiviral vector.

16. The method of claim 8, wherein the nucleic acid molecule further comprises a promoter sequence in operable linkage with the nucleic acid sequence encoding the ASCL1 and one or more additional transcription factors.

17. The method of claim 16, wherein the promoter is a retinal or glia-specific promoter.

18. The method of claim 17, wherein the glia-specific promoter is HES1.

19. The method of claim 9, wherein the administering to the retina is intravitreal or subretinal injection.

20. The method of claim 8, wherein the ASCL1 and one or more additional transcription factors are expressed as a fusion protein.

Images