Treatment of eye disorders

Abstract

The present disclosure provides compositions and methods for treating or preventing ischemic optic neuropathy with gene therapy vectors that deliver nucleic acid sequences encoding octamer-binding transcription factor 4 (OCT4), sex-determining region (Y)-box 2 (SOX2), and Krüppel-like factor 4 (KLF4). In particular, non-arteritic anterior ischemic optic neuropathy (NAION) can be treated or prevented.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is an international patent application claiming priority to U.S. Provisional Patent Application No. 63 / 499,864, filed May 3, 2023, and U.S. Provisional Patent Application No. 63 / 478,843, filed January 6, 2023, the entire contents of each of which are incorporated herein by reference in their entirety.

[0002] Incorporation by reference of sequence listing This application contains a Sequence Listing submitted via EFS-Web. The contents of the file named 061189-501001WO_SequenceListing_ST26, created on January 4, 2024, and having a size of 92,025 bytes, are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to gene therapy for ocular disorders that delivers restorative factors to cells, particularly retinal ganglion cells. [Background technology]

[0004] Ischemic optic neuropathy is the most common acute optic neuropathy in patients over the age of 50. Ischemic optic neuropathy is generally classified as anterior (affecting the optic disc) or posterior (rear of the eye) and as arteritic or nonarteritic. Anterior involvement is common in both arteritic and nonarteritic ischemic optic neuropathy.

[0005] Non-arteritic anterior ischemic optic neuropathy (NAION) is the most common form of ischemic optic neuropathy. It is an idiopathic ischemic attack of the optic nerve head, characterized by acute, monocular, painless vision loss accompanied by optic disc swelling. According to the American Academy of Ophthalmology, NAION affects 2.3 to 10.3 people per 100,000 per year, making it the most common cause of acute optic neuropathy in patients over the age of 50. There are approximately 6,000 new cases per year. Men and women are affected approximately equally [www_eyewiki_aao_org / Non-Arteritic_Anterior_Ischemic_Optic_Neuropathy_(NAION)].

[0006] There are no known treatments for NAION that have been proven effective. Although numerous clinical trials have been conducted investigating over 12 different treatments, none have satisfactorily improved visual outcomes for patients with NAION. The present disclosure addresses the need for such treatments. Summary of the Invention

[0007] The present disclosure relates to and provides methods for preventing or treating non-arteritic anterior ischemic optic neuropathy in a subject by administering to the subject a nucleic acid molecule comprising a nucleic acid sequence encoding octamer-binding transcription factor 4 (OCT4), a nucleic acid molecule comprising a nucleic acid sequence encoding sex-determining region (Y)-box 2 (SOX2), and a nucleic acid sequence encoding Krüppel-like factor 4 (KLF4). In some embodiments, the nucleic acid sequences encoding OCT4, SOX2, and KLF4 are on a single nucleic acid molecule. In certain aspects, an adeno-associated virus (AAV) vector comprises nucleic acid molecules encoding OCT4, SOX2, and KLF4. In some embodiments, the nucleic acid molecule does not encode c-Myc and / or another transcription factor, such as Nanog.

[0008] In some embodiments, the method further comprises administering to the subject a nucleic acid molecule comprising a nucleic acid sequence encoding a reverse tetracycline-controlled transactivator (rtTA). According to some embodiments of the method, the AAV vector comprises a nucleic acid molecule encoding a reverse tetracycline-controlled transactivator (rtTA). In some embodiments, the rtTA is rtTA-L, rtTA3, or rtTA4. The AAV vector comprising the nucleic acid molecules encoding OCT4, SOX2, and KLF4, and the AAV vector comprising the nucleic acid molecule encoding a reverse tetracycline-controlled transactivator (rtTA) can be present in a single AAV composition or as separate AAV compositions.

[0009] In some embodiments of the disclosed methods, the nucleic acid molecule comprising a nucleic acid sequence encoding OCT4, a nucleic acid sequence encoding SOX2, and a nucleic acid sequence encoding KLF4 is operably linked to an inducible promoter. In some embodiments, the inducible promoter comprises a tetracycline class antibiotic response element (TRE). In some embodiments, the tetracycline class antibiotic is doxycycline. In some embodiments, the inducible promoter is a TRE2 promoter.

[0010] In some embodiments of the disclosed methods, the nucleic acid molecule comprising a nucleic acid sequence encoding rtTA is operably linked to a CMV promoter.

[0011] In some embodiments, the nucleic acid molecule comprising a nucleic acid sequence encoding OCT4, a nucleic acid sequence encoding SOX2, and a nucleic acid sequence encoding KLF4 is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is serotype 2 (AAV2).

[0012] In some embodiments of the disclosed methods, the nucleic acid molecule comprising a nucleic acid sequence encoding OCT4, a nucleic acid sequence encoding SOX2, and a nucleic acid sequence encoding KLF4 does not comprise a nucleic acid sequence encoding c-Myc.

[0013] In some embodiments of the disclosed methods, the nucleic acid molecule comprising a nucleic acid sequence encoding OCT4, a nucleic acid sequence encoding SOX2, and a nucleic acid sequence encoding KLF4 comprises a nucleic acid sequence encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A peptide.

[0014] In some embodiments of the disclosed methods, the nucleic acid molecules encoding OCT4, SOX2, and KLF4 are flanked by inverted terminal repeats (ITRs), and the distance between the ITRs is 4.7 kb or less.

[0015] In some embodiments, the method further comprises administering to the subject an inducing agent.

[0016] In some embodiments of the disclosed methods, the nucleic acid molecule encoding the reverse tetracycline-controlled transactivator (rtTA) is an AAV vector that does not include nucleic acid molecules encoding OCT4, SOX2, and KLF4. The nucleic acid molecule encoding the reverse tetracycline-controlled transactivator (rtTA) may be an AAV vector comprising SEQ ID NO: 36 or SEQ ID NO: 37.

[0017] In some embodiments of the disclosed methods, the nucleic acid molecules encoding OCT4, SOX2, and KLF4 comprise nucleic acid elements in the following order: a) a first inverted terminal repeat (ITR) sequence, b) a TRE promoter sequence, c) a nucleic acid sequence encoding OCT4, d) a nucleic acid sequence encoding P2A, e) a nucleic acid sequence encoding SOX2, f) a nucleic acid sequence encoding T2A, g) a nucleic acid sequence encoding KLF4, h) a terminator sequence derived from SV-40, and i) a second inverted terminal repeat (ITR) sequence.

[0018] In some embodiments of the disclosed methods, the nucleic acid sequence encoding OCT4 comprises SEQ ID NO: 1. In some embodiments, the nucleic acid sequence encoding SOX2 comprises SEQ ID NO: 3. In some embodiments, KLF4 is a human KLF4 protein. In some embodiments, the nucleic acid sequence encoding KLF4 comprises SEQ ID NO: 5. In some embodiments, the nucleic acid sequence encoding P2A comprises SEQ ID NO: 8. In some embodiments, P2A comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, T2A comprises the amino acid sequence of SEQ ID NO: 11.

[0019] In some embodiments, the nucleic acid sequence encoding T2A is GAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCA (SEQ ID NO: 10).

[0020] In some embodiments, the TRE promoter sequence is SEQ ID NO:7.

[0021] In some embodiments, the terminator sequence from SV-40 is SEQ ID NO:12.

[0022] In some embodiments, the first ITR sequence is SEQ ID NO:16.

[0023] In some embodiments, the nucleic acid molecule encoding OCT4, SOX2, and KLF4 comprises SEQ ID NO:13.

[0024] In some embodiments, the nucleic acid molecule encoding OCT4, SOX2, and KLF4 comprises SEQ ID NO:14.

[0025] In some embodiments, the nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the nucleic acid molecule encoding rtTA are administered sequentially or simultaneously.

[0026] In some embodiments, nucleic acid molecules encoding OCT4, SOX2, and KLF4 are administered intravitreally.

[0027] In some embodiments, the nucleic acid molecule encoding rtTA is administered intravitreally.

[0028] In some embodiments, the nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the nucleic acid molecule encoding rtTA are administered in a ratio of about 1:1.

[0029] In some embodiments, the AAV vector comprising the nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the AAV vector comprising the nucleic acid molecule encoding rtTA are administered in a ratio of about 1:1 (vg:vg).

[0030] In some embodiments, the nucleic acid molecule encoding OCT4, SOX2, and KLF4 is an AAV2-TRE-OSK vector, and the nucleic acid molecule encoding rtTA is pAAV2-CMV-rtTA3VP16 or AAV2-CMV-rtTA4.

[0031] In some embodiments, an effective amount of an AAV2-TRE-OSK vector is about 1 x 10 9 vg / eye ~ approx. 1×10 14 vg / eye range.

[0032] In some embodiments, an effective amount of the pAAV2-CMV-rtTA3VP16 vector is about 1 x 10 9 vg / eye ~ approx. 1×10 14 vg / eye range.

[0033] In some embodiments, an effective amount of an AAV2-CMV-rtTA4 vector is about 1 x 10 9 vg / eye ~ approx. 1×10 14 vg / eye range.

[0034] In some embodiments, nucleic acid molecules encoding OCT4, SOX2, and KLF4 are administered to a subject by left eye (OS) injection, by right eye (OD) injection, or by both eyes (OU) injection.

[0035] In some embodiments, the nucleic acid molecule encoding rtTA is administered to the subject by left eye (OS) injection, by right eye (OD) injection, or by both eyes (OU) injection.

[0036] In some embodiments, the method further comprises administering an effective amount of an antibiotic to the subject. In some embodiments, the antibiotic is administered at least one day before administering the nucleic acid molecule encoding the rtTA. In some embodiments, the antibiotic is administered when the nucleic acid molecule encoding the rtTA is administered. In some embodiments, the antibiotic is administered at least one day after administering the nucleic acid molecule encoding the rtTA.

[0037] In certain aspects, provided herein are methods for the recombinant preparation of AAV, the methods comprising introducing a vector into cells under conditions in which AAV is produced, the vector comprising a) one or more nucleic acid sequences encoding OCT4, SOX2, and KLF4. In some embodiments, the cells comprise a population of HEK293T cells.

[0038] Further provided herein are methods of producing AAV, comprising modifying cells to express one or more plasmids, including one or more AAV2 Rep-Cap plasmids, one or more helper plasmids, and one or more transfer plasmids, wherein the one or more transfer plasmids comprise one or more nucleic acids encoding OCT4, SOX2, and KLF4. In some embodiments, the cells comprise a population of HEK293T cells.

[0039] In certain aspects, provided herein are methods for preventing or treating non-arteritic anterior ischemic optic neuropathy (NAION) in a subject in need thereof, the method comprising administering to the subject a pharmaceutically effective amount of a composition comprising an expression vector comprising a polynucleotide encoding OCT4, SOX2, and KLF4, but not c-Myc. In some embodiments, the polynucleotide does not further encode one or more transcription factors, e.g., Nanog.

[0040] In certain aspects, provided herein are methods for preventing or treating non-arteritic anterior ischemic optic neuropathy (NAION) in a subject in need thereof, the method comprising administering to the subject a composition comprising an expression vector comprising a polynucleotide encoding three transcription factors, the transcription factors consisting of OCT4, SOX2, and KLF4.

[0041] In certain aspects, provided herein are methods for preventing or treating non-arteritic anterior ischemic optic neuropathy (NAION) in a subject in need thereof, the methods comprising administering to the subject a composition comprising an expression vector including polynucleotides encoding four or more transcription factors, the transcription factors including OCT4, SOX2, and KLF4, but excluding c-Myc. In some embodiments, the polynucleotide does not further encode one or more transcription factors, e.g., Nanog.

[0042] In some embodiments, the composition does not reprogram a cell, tissue, or organ in the subject to a pluripotent state. In some embodiments, the composition restores at least one cell, tissue, or organ in the subject. In some embodiments, the composition does not induce c-Myc expression in the subject. In some embodiments, the composition does not induce expression of one or more transcription factors (e.g., Nanog) in the subject. In some embodiments, the composition does not induce expression of at least one stem cell marker in the subject. Such at least one stem cell marker may include Esrrb, Nanog, Lin28, TRA-1-60 / TRA-1-81 / TRA-2-54, SSEA1, SSEA4, or any combination thereof. In some embodiments, the composition induces expression of RBPMS, Bm3a, or a combination thereof in the subject. In some embodiments, restoring at least one cell, tissue, or organ comprises increasing repair and / or regeneration in the cell, tissue, or organ. In some embodiments, restoring at least one cell, tissue, or organ comprises restoring epigenetic information in the subject. In some embodiments, restoring at least one cell, tissue, or organ comprises restoring epigenetic information in the cell, tissue, or organ that has been lost due to aging, injury, disease, or any combination thereof. In some embodiments, restoring at least one cell, tissue, or organ comprises restoring the epigenetic state of the cell, tissue, or organ to an epigenetic state that more closely resembles fermentation or terminal differentiation. In some embodiments, restoring at least one cell, tissue, or organ comprises increasing the number of healthy axons in the subject. In some embodiments, restoring at least one cell, tissue, or organ comprises preventing damage to healthy axons in the subject.

[0043] In some embodiments, the polynucleotide comprises DNA, RNA, or a combination thereof. In some embodiments, the DNA comprises plasmid DNA. In some embodiments, the RNA comprises mRNA. In some embodiments, the polynucleotide comprises an inducible promoter, such as the TRE3G promoter, the TRE2 promoter, the P-tight promoter, and a tetracycline response element (TRE).

[0044] In some embodiments, the methods described herein further comprise administering to the subject an inducer to induce expression of OCT4, SOX2, and KLF4 in the subject. In some embodiments, the inducer comprises a tetracycline class antibiotic, such as doxycycline. In some embodiments, the inducer comprises a reverse tetracycline-controlled transactivator (rtTA) or a polynucleotide encoding rtTA. In some embodiments, the polynucleotide encoding rtTA is in an expression vector.

[0045] In some embodiments, the composition and the inducing agent are administered sequentially or simultaneously. In some embodiments, the composition is administered before the administration of the inducing agent. In some embodiments, the composition is administered after the administration of the inducing agent. In some embodiments, the composition is administered simultaneously with the inducing agent.

[0046] In some embodiments, the composition and the inducing agent are administered in a ratio of about 100:1, 50:1, 40:1, 30:1, 25:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:25, 1:30, 1:40, 1:50, or 1:100. In some embodiments, the composition and the inducing agent are administered in a ratio greater than about 100:1. In some embodiments, the composition and the inducing agent are administered in a ratio less than about 1:100. In some embodiments, the composition and the inducing agent are administered in a ratio of about 1:1.

[0047] In some embodiments, the polynucleotide comprises a self-cleaving peptide, such as a 2A peptide.

[0048] In some embodiments, the polynucleotide comprises an inverted terminal repeat (ITR).

[0049] In some embodiments, the expression vector is a viral expression vector selected from lentivirus, retrovirus, adenovirus, alphavirus, vaccinia virus, and adeno-associated virus (AAV) vectors. In some embodiments, the AAV vector is serotype 2 (AAV2).

[0050] In some embodiments, the polynucleotide comprises nucleic acid elements in the following order:

[0051] a. a first inverted terminal repeat (ITR) sequence;

[0052] b. TRE3G promoter sequence,

[0053] c.OCT4 sequence,

[0054] d. P2A cleavage sequence,

[0055] e.SOX2 sequence,

[0056] f. T2A cleavage sequence,

[0057] g.KLF4 sequence,

[0058] h. a terminator sequence derived from SV-40, and

[0059] i. A second inverted terminal repeat (ITR) sequence.

[0060] In some embodiments, i) OCT4 comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2; ii) SOX2 comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:4; and / or iii) KLF4 comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:6.

[0061] In some embodiments, i) OCT4 comprises an amino acid sequence having at least 90% identity to SEQ ID NO:2, ii) SOX2 comprises an amino acid sequence having at least 90% identity to SEQ ID NO:4, and / or iii) KLF4 comprises an amino acid sequence having at least 90% identity to SEQ ID NO:6.

[0062] In some embodiments, i) OCT4 comprises the amino acid sequence of SEQ ID NO:2, ii) SOX2 comprises the amino acid sequence of SEQ ID NO:4, and / or iii) KLF4 comprises the amino acid sequence of SEQ ID NO:6.

[0063] In some embodiments, i) the polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1; ii) the polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:3; and / or iii) the polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:5.

[0064] In some embodiments, i) the polynucleotide comprises a nucleic acid sequence having at least 75% identity to SEQ ID NO:1, ii) the polynucleotide comprises a nucleic acid sequence having at least 75% identity to SEQ ID NO:3, and / or iii) the polynucleotide comprises a nucleic acid sequence having at least 75% identity to SEQ ID NO:5.

[0065] In some embodiments, i) the polynucleotide comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:1, ii) the polynucleotide comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:3, and / or iii) the polynucleotide comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:5.

[0066] In some embodiments, i) the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:1, ii) the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:3, and / or iii) the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:5.

[0067] In some embodiments, the composition is administered systemically. In some embodiments, the composition is administered locally to a tissue or organ. In some embodiments, the composition is administered intravitreally. In some embodiments, the composition is administered to a subject by left eye (OS) injection, right eye (OD) injection, or both eyes (OU) injection.

[0068] In some embodiments, administering the composition improves retinal ganglion cell (RGC) function and / or restores visual function in the subject.

[0069] In some embodiments, the prevention or treatment of NAION is measurable by electroretinogram (pERG). In some embodiments, the prevention or treatment of NAION is measured by electroretinogram (pERG).

[0070] Further provided is a method for treating or preventing ischemic optic neuropathy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a gene therapy vector, the gene therapy vector comprising one or more nucleic acid molecules comprising a nucleic acid sequence encoding octamer-binding transcription factor 4 (OCT4), a nucleic acid sequence encoding sex-determining region (Y)-box 2 (SOX2), and a nucleic acid sequence encoding Krüppel-like factor 4 (KLF4), operably linked to at least one promoter (or operably linked to recombination sites, blunt ligation, or homology sites for genome editing, placing the one or more nucleic acid molecules under the control of the endogenous promoter).

[0071] In some embodiments, the ischemic optic neuropathy is non-arteritic anterior ischemic optic neuropathy (NAION).

[0072] In some embodiments, the ischemic optic neuropathy is arteritic anterior ischemic optic neuropathy (A-AION).

[0073] In some embodiments, the ischemic optic neuropathy is posterior ischemic optic neuropathy (PION).

[0074] In some embodiments, the vector is administered intravitreally.

[0075] In some embodiments, the method of administration delivers one or more nucleic acid molecules to retinal ganglion cells when administered to the eye in vivo.

[0076] In some embodiments, the vector lacks a nucleic acid molecule encoding the Myc proto-oncogene (c-Myc) or Nanog, lacks any nucleic acid molecule encoding a transcription factor, and / or lacks any nucleic acid molecule encoding a reprogramming factor other than octamer-binding transcription factor 4 (OCT4), sex-determining region (Y)-box 2 (SOX2), and Krüppel-like factor 4 (KLF4).

[0077] In some embodiments, the vector is a viral vector.

[0078] In some embodiments, the vector is a liposome or lipid nanoparticle (LNP).

[0079] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector, optionally an oculotropic AAV vector.

[0080] In some embodiments, the AAV vector is an AAV serotype 2 (AAV2) vector or a variant thereof.

[0081] In some embodiments, the AAV vector is AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAVrh74, AAVdj, and AAV.PHP, or a variant thereof, optionally an AAV serotype listed in Table 2 as having specificity for the eye.

[0082] In some embodiments, the one or more nucleic acid molecules is an AAV genome including flanking inverted terminal repeats (ITRs).

[0083] In some embodiments, the vector is an LNP, which comprises an ionizable lipid, a helper lipid, a sterol, and a poly(ethylene glycol)-lipid (PEG-lipid).

[0084] In some embodiments, the promoter is an inducible promoter.

[0085] In some embodiments, the inducible promoter is a tetracycline response element (TRE) promoter, optionally a Tet-On promoter, optionally a TRE3G promoter.

[0086] In some embodiments, the method further comprises administering to the subject, sequentially or simultaneously, in amounts effective to positively regulate a TRE promoter, a second gene therapy vector, and optionally, an adeno-associated virus (AAV), comprising a polynucleotide encoding a reverse tetracycline-controlled transactivator (rtTA).

[0087] In some embodiments, the rtTA is selected from the rtTAs listed in Table 1, and optionally includes: MSRLDKSKIINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLLKQAIELFDRQGAEPAFLFGLEL An rtTA sharing at least 80%, at least 90%, at least 85%, or 100% identity to IICGLEKQLKCESGGPTDALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG (SEQ ID NO: 20), and optionally comprising one or more amino acid substitutions selected from V9I, G12S, F67S, G72V, G72P, and R171K, and optionally comprising amino acid substitutions G12S, F67S, and R171K or amino acid substitutions V9I, G12S, F67S, and R171K.

[0088] In some embodiments, the method further comprises administering an inducer of the tetracycline class, optionally doxycycline or tetracycline.

[0089] In some embodiments, the vector is a multicistronic vector and the one or more nucleic acid molecules is a single nucleic acid molecule comprising open reading frames encoding OCT4, SOX2, and KLF4.

[0090] In some embodiments, the open reading frame does not encode any other proteins.

[0091] In some embodiments, OCT4, SOX2, and KLF4 are linked by a self-cleaving peptide, optionally a 2A-peptide.

[0092] In some embodiments, the open reading frame encodes, in 5' to 3' order, OCT4, SOX2, and KLF4.

[0093] In some embodiments, the methods involve administering to the subject an inducer for a period of time sufficient to restore retinal ganglion cells but not to induce pluripotency of the cells.

[0094] In some embodiments, OCT4 shares at least 80%, at least 90%, at least 85%, or 100% identity to SEQ ID NO:2, SOX2 shares at least 80%, at least 90%, at least 85%, or 100% identity to SEQ ID NO:4, and / or KLF4 shares at least 80%, at least 90%, at least 85%, or 100% identity to SEQ ID NO:6.

[0095] In some embodiments, the method improves retinal ganglion cell (RGC) function and / or restores visual function in a subject.

[0096] In some embodiments, prevention or treatment of NAION is measured by electroretinogram (pERG).

[0097] In another aspect, the disclosure provides a gene therapy vector for use in any of the aforementioned methods.

[0098] Further aspects and embodiments are provided in the detailed description below. [Brief explanation of the drawings]

[0099] [Figure 1] 1 is an exemplary vector map of TRE3G-OSK-SV40pA (SEQ ID NO: 15), an AAV2 vector encoding OSK. [Figure 2]An exemplary vector map of pAAV2-CMV-rtTA3VP16 (SEQ ID NO: 21) is shown, which is a non-limiting example of a vector encoding rtTA (other examples of rtTA include, but are not limited to, rtTA-L and rtTA4). [Figure 3]

[0023] Figure 1 shows an exemplary vector map of pAAV2-CMV-rtTA4 (SEQ ID NO: 28), which is a non-limiting example of a vector encoding rtTA. [Figure 4] FIG. 1 shows a schematic diagram illustrating a non-limiting example of the Tet-ON system for expressing OCT4, SOX2, and KLF4 (OSK) in the presence of tetracycline. [Figure 5] FIG. 1 is a schematic diagram showing the non-human primate (NHP) non-arteritic anterior ischemic optic neuropathy (NAION) study design. [Figure 6] This figure shows the results of pattern electroretinogram (pERG) measurements, demonstrating that induction of non-arteritic anterior ischemic optic neuropathy (NAION) results in a decrease in the pERG signal. pERG uses a contrast-reversed pattern stimulus (checkerboard) to assess macular retinal ganglion cell (RGC) activity. Changes in the pERG waveform indicate RGC dysfunction. [Figure 7] Graph comparing absolute amplitudes from p50 to n95 when treated with vehicle (left bars at each time point) or OSK (controlled by Dox as a Tet-on system, right bars at each time point) before laser treatment (i.e., preventive test). pERG signals were measured on different days and compared under different treatments. [Figure 8] Graph comparing absolute amplitudes from p50 to n95 after laser treatment (i.e., rescue test) when treated with vehicle (left bars at each time point) or OSK (controlled by Dox as a Tet-on system, right bars at each time point). pERG signals were measured on different days and compared under different treatments. [Figure 9]Correlation between early laser damage to the optic nerve head (x-axis, degree of optic nerve head edema measured 8 days after laser) and degree of pattern electroretinogram (pERG, y-axis) defect at 5 weeks is shown. [Figure 10A] Correlation between the degree of early optic nerve head edema and the degree of pERG deficit at 5 weeks in vehicle-treated RGCs is shown (same as Figure 9). [Figure 10B] Figure 1 shows a decreased correlation between the degree of early optic nerve head edema and the degree of pERG deficit at week 5 in OSK-treated NHPs, indicating reduced damage to RGC cells. [Figure 11] Correlation between the initial laser damage to the optic nerve head (x-axis, degree of optic nerve head edema measured 8 days after laser) and the degree of axonal density damage at autopsy (9 weeks) is shown. [Figure 12A] Correlation between the degree of initial optic nerve head edema and the degree of axonal density damage at the end of the study in vehicle-treated NHPs is shown (same as Figure 11). [Figure 12B] We show that the correlation between the degree of initial optic nerve head edema and the degree of axonal density damage at the end of the study in OSK-treated NHPs was reduced, indicating protection from or reversal of axonal damage. DETAILED DESCRIPTION OF THE INVENTION

[0100] The disclosed methods may be more readily understood by reference to the following detailed description in conjunction with the accompanying drawings, which form a part of this disclosure: It is to be understood that the disclosed methods are not limited to the specific methods described and / or illustrated herein, and that the terminology used herein is for the purpose of describing particular embodiments only by way of example and is not intended to limit the methods claimed.

[0101] Unless otherwise specified, any explanation of a possible mechanism, mode of action, or reason for improvement is intended to be exemplary only, and the disclosed methods are not constrained by the accuracy or inaccuracy of any such suggested mechanism, mode of action, or reason for improvement.

[0102] Throughout this specification, descriptions refer to compositions and methods of using the compositions. Where this disclosure describes or claims features or embodiments related to compositions, such features or embodiments are equally applicable to methods of using the compositions. Similarly, where this disclosure describes or claims features or embodiments related to methods of using the compositions, such features or embodiments are equally applicable to the compositions.

[0103] It will be understood that certain features of the disclosed methods that are, for clarity, described herein in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods that are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

[0104] 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. In case of conflict, the present document, including definitions, will control. Exemplary methods and materials are described below; however, methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the disclosed methods. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0105] The terms "comprise," "include," "having," "has," "can," "contain," and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or phrases that do not exclude the possibility of additional acts or structures. The term "comprising" is intended to include examples encompassed by the terms "consisting essentially of" and "consisting of," and similarly, the term "consisting essentially of" is intended to include examples encompassed by the term "consisting of." The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0106] The singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise.

[0107] When numerical ranges are recited herein, each intervening number to the same degree of precision is expressly contemplated. For example, in the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and in the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0108] Some quantitative expressions provided herein are not modified by the term "about." Whether or not the term "about" is explicitly used, it should be understood that all amounts provided are intended to refer to the actual given value, and also to approximations to such given value that can be reasonably inferred by those skilled in the art, including approximations based on experimental and / or measurement conditions for such value.

[0109] As used herein, the term "cell" is meant to include not only an individual cell, but also to refer to the particular tissue or organ from which it is derived.

[0110] The term "gene expression" refers to the degree to which specific genes or all genes in a cell or tissue are transcribed into RNA. In some instances, the RNA is translated into protein by the cell. The epigenome dictates gene expression patterns.

[0111] The terms "condition," "disease," and "disorder" are used interchangeably. As used herein, an "ocular disease" or "ocular condition" is a disease or condition of the eye. An example of an eye disease is non-arteritic anterior ischemic optic neuropathy.

[0112] Any suitable method can be used to measure ocular function, including, but not limited to, visual acuity testing, pattern electroretinogram (pERG), and pathology.

[0113] As used herein, "causes of cellular senescence" include the loss or alteration of epigenetic information. As used herein, the terms "effective amount" and "therapeutically effective amount" refer to an amount of a compound or composition that, when administered to a subject, is effective to at least partially treat a condition from which the subject suffers.

[0114] As used herein, a "functional" or "active" protein is one that retains its biological activity (e.g., can act as a transcription factor or inducer). Conversely, a non-functional or inactive protein is one that is unable to perform one or more of its wild-type functions.

[0115] The term "gene" refers to a nucleic acid fragment that expresses a protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. A "native gene" refers to a gene found in nature with its own regulatory sequences. A "chimeric gene" or "chimeric construct" refers to any gene or construct that is not a native gene, containing regulatory and coding sequences that are not found together in nature. Thus, a chimeric gene or chimeric construct can contain regulatory and coding sequences that are derived from different sources, or regulatory and coding sequences that are derived from the same source but are arranged in a way different from that found in nature. An "endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene that is not normally found in a host organism but is introduced into the host organism by gene transfer. A foreign gene can include a native gene inserted into a non-naturally occurring organism, or a chimeric gene. A "transgene" is a gene that has been introduced into a genome by a transformation procedure.

[0116] "Homologue" or "homology" refers to a specific percent identity (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79% Homologous refers to sequences (e.g., nucleic acid or amino acid sequences) that share at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% percent identity. Homologous sequences include, but are not limited to, paralogous or orthologous sequences. Paralogous sequences result from gene duplication within the genome of a species, while orthologous sequences diverge after a speciation event. Functional homologs retain one or more biological activities of the wild-type protein. In certain embodiments, a functional homolog of OCT4, KLF4, or SOX2 retains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the biological activity (e.g., transcription factor activity) of its wild-type counterpart.

[0117] The term "tissue" refers to any biological tissue (including a group of cells, a body part, or an organ) of a subject, or a portion thereof, including blood and / or lymphatic vessels, to which the compounds, particles, and / or compositions disclosed herein are delivered. The tissue may be abnormal, damaged, or unhealthy tissue that may be in need of treatment. The tissue may also be normal or healthy tissue that is at higher than normal risk of becoming abnormal or unhealthy, and that risk may need to be prevented. In certain embodiments, the tissue is healthy but is considered suboptimal in terms of performance or survival in its current or future state. In certain embodiments, the tissue is the central nervous system. In certain embodiments, the cell or tissue is derived from the eye. In certain embodiments, the tissue is damaged (e.g., due to a congenital defect, injury, accident, or iatrogenic injury), diseased, and / or aged. In certain embodiments, the tissue is a deep tissue accessible with a fiber optic probe.

[0118] The term "tissue repair," with respect to damaged tissue, refers to the restoration of tissue structure, function, or a combination thereof, following tissue injury. Tissue repair includes tissue regeneration, cell proliferation, tissue replacement, and / or rewiring (reprogramming) of existing tissue.

[0119] The term "tissue regeneration" refers to the production of new tissue or cells within a tissue of the same type as the tissue of interest (e.g., the same type as the damaged tissue or cells). In some embodiments, the methods provided herein promote organ regeneration.

[0120] The term "tissue replacement" refers to the production of a different type of tissue compared to the tissue of interest (eg, connective tissue to replace damaged tissue).

[0121] As used herein, the terms "treatment," "treat," and "treating" refer to ameliorating, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder described herein, or one or more symptoms thereof. In certain embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. In other embodiments, treatment may be administered to improve one or more of retinal perfusion, intraocular pressure, retinal layer thickness, RGC survival, retinal electrical response, macular nerve electrical response, optic nerve activation, retinal nerve photoresponse, retinal layer thickness (OCT), retinal RGC cell survival, visual acuity, etc. For example, treatment may be administered to a susceptible individual before the onset of symptoms. Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

[0122] The term "variant" refers to a sequence that contains modifications compared to the wild-type sequence. Non-limiting modifications to amino acid sequences include insertions, deletions, and point mutations. Non-limiting modifications to nucleic acid sequences include frameshift mutations, nucleotide insertions, and nucleotide deletions.

[0123] The term "WPRE" refers to the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). The WPRE can create a tertiary structure in a nucleic acid (e.g., an expression vector) and enhance expression of a transgene (e.g., from a viral vector). In certain embodiments, the WPRE sequence is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100%) identical to SEQ ID NO: 23 or 31.

[0124] gene therapy Gene therapy for eye diseases Ocular diseases encompass a wide range of conditions that can significantly impact vision and overall quality of life. Traditional treatment options aim to alleviate symptoms or slow disease progression, sometimes requiring surgical intervention. These approaches are not always curative, and despite the best available treatments, many patients still experience substantial vision loss. Gene therapy promises to revolutionize ocular treatment and provide new treatments for patients who currently have limited options. The unique properties of the eye, including its immune-privileged state, small size, and compartmentalized structure, facilitate efficient delivery and maintenance of gene therapy components without eliciting excessive immune responses. This allows for localized administration of therapeutic agents, minimizing the risk of systemic exposure. The eye can be evaluated by noninvasive imaging techniques, such as optical coherence tomography, funduscopy, angiography, and new-generation two-photon microscopy, which facilitate real-time monitoring of treatment outcomes and safety. Examples of gene therapy in ocular diseases are described in Ghoraba et al., Clin. Opthalmol. 16:1753-1771 (2022), Choi et al., EXP. Mol. Med. 55(8):1678-1690 (2023), Samiy et al., J Opthalmic Vis res. 9(4):506-509 (2014) and Drag et al., Invest Ophthalmol Vis Sci. 64(7):39 (2023), which are incorporated herein by reference in their entireties.

[0125] In embodiments, the present disclosure provides methods for treating ocular diseases or disorders, including but not limited to those associated with damage to retinal ganglion cells. In particular, the examples provided below demonstrate that the methods described herein can be effective in treating various ocular diseases or disorders associated with eye damage, which are experimentally modeled with laser-induced damage. Without being bound by theory, the examples show that experimental lasers heat blood vessels, such as arteries, in the eye, damaging the blood vessels, which then deprive RGCs and other cells of oxygen, causing ischemic damage to the cells, thus preventing or treating such damage through recovery mechanisms, demonstrating that the described methods can be used to treat various ischemic optic neuropathies, including but not limited to non-arteritic anterior ischemic optic neuropathy.

[0126] eye disease NAION Non-arteritic anterior ischemic optic neuropathy (NAION) comprises 95% of all anterior ischemic optic neuropathies (AION) and is the most common cause of acute optic neuropathy in people aged 50 years or older, affecting approximately 2–10 per 100,000 people (approximately 1500–6000 new cases per year in the United States). Currently, there is no generally accepted treatment or secondary prevention for NAION, although steroids have traditionally been used in some patients.

[0127] NAION is a condition characterized by impaired blood flow to the small blood vessels supplying the anterior portion of the optic nerve. Vision loss in NAION is painless, rapid, and usually permanent. Risk factors for NAION include atherosclerosis (which impairs blood flow in the blood vessels supplying the optic nerve) and a "dense" optic nerve. Optic nerves with small or absent optic cups, also known as "discs at risk," pass through a "dense" sclera as they enter the eye. This dense passage through the sclera is thought to put additional pressure on the small blood vessels supplying the optic nerve. This process ultimately leads to a loss of adequate blood flow to the optic nerve, resulting in ischemic optic neuropathy. Attempts to treat NAION have included radial optic neurotomy to relieve mechanical pressure on the optic nerve and its supporting vasculature. This procedure carries all the risks of intraocular surgery and is difficult to perform. Collateral damage to these structures is not uncommon. In some embodiments, the ischemic optic neuropathy treated or prevented is non-arteritic anterior ischemic optic neuropathy (NAION).

[0128] A-AION Arteritic arteritis-induced optic neuropathy (A-AION) is an eye condition caused by inflammation of the arteries supplying blood to the optic nerve. A-AION accounts for 5-10% of AION cases. This inflammation results from a condition known as giant cell arteritis (GCA) or temporal arteritis, which causes inflammation of medium-sized and large arteries. GCA is potentially fatal, and if not promptly diagnosed and treated, it can damage the entire optic nerve head, leading to permanent and widespread vision loss. A-AION is three times more common in women than men and most commonly affects people 55 years of age or older. In an embodiment, the ischemic optic neuropathy being treated or prevented is arteritic anterior ischemic optic neuropathy (A-AION).

[0129] PION Posterior ischemic optic neuropathy (PION) is a potentially fatal condition characterized by acute, painless vision loss in one or both eyes. PION can be classified into three types: arteritic PION, which is caused by giant cell arteritis; non-arteritic PION; and perioperative PION. PION is caused by reduced blood flow and oxygenation to the intraorbital optic nerve. This can occur due to several risk factors: reduced arterial perfusion pressure due to hypotension caused by volumetric blood loss; increased peripheral vascular resistance causing reduced downstream blood flow; increased peripheral venous pressure due to orbital edema; increased intraocular pressure; or reduced blood oxygen-carrying capacity. In some embodiments, the ischemic optic neuropathy being treated or prevented is posterior ischemic optic neuropathy (PION).

[0130] physiological measurements To assess the effectiveness of the therapeutic and prophylactic methods described herein, various physiological measurements can be applied. These include:

[0131] retinal ganglion cells Retinal ganglion cells (RGCs) are a type of neuron located near the inner surface (ganglion cell layer) of the retina of the eye. They receive visual information from photoreceptors via two types of intermediate neurons, bipolar cells and amacrine cells. Retinal ganglion cells collectively transmit image-forming and non-image-forming visual information from the retina to several regions of the thalamus, hypothalamus, and midbrain.

[0132] Although RGCs differ significantly in their size, connectivity, and response to visual stimuli, they all share the crucial property of having long axons that span the brain. These axons form the optic nerve, optic chiasm, and optic tract. A small number of RGCs contribute little or nothing to vision but are themselves photosensitive. Their axons form the retinohypothalamic tract and contribute to circadian rhythms and the pupillary light reflex, i.e., changes in pupil size. RGC types include midget RGCs, parasol RGCs, small stratified RGCs, large bistratified RGCs, smooth unstratified RGCs, recursive unstratified / bistratified RGCs, stubborn RGCs, large sparse RGCs, and melanopsin-containing intrinsically photosensitive RGCs. RGC degeneration underlies several conditions that cause significant visual problems, including glaucoma, hereditary optic neuropathies, ischemic optic neuropathies, and demyelinating diseases.

[0133] In embodiments, treating or preventing an eye disease or eye disorder may include treating or preventing damage to RGCs, as assessed by ocular histopathology or other known methods for assessing RGC viability and function. In embodiments, the methods described herein prevent a decrease in axon density or number (disease prevention) or result in an increase in axon density or number (disease treatment).

[0134] Pattern electroretinogram The pattern electroretinogram (pERG) provides an objective measure of central retinal function. The pERG contains two major components: a positive (P50) at approximately 50 ms and a larger negative (N95) at approximately 95 ms. The P50 component is affected by macular dysfunction, accompanied by a decrease in the N95 component. The pERG complements the Ganzfeld ERG in the evaluation of patients with retinal disease. In contrast, the ganglion cell origin of the N95 component allows for electrophysiological assessment of ganglion cell function in both primary disease and dysfunction secondary to optic nerve disease, where selective loss of the N95 component can be observed. Both macular dysfunction and optic nerve disease can produce abnormalities in visual evoked potentials (VEPs), and therefore the pERG facilitates more meaningful interpretation of VEPs. Retinal electrical activity related to RGC function can be assessed by pERG.

[0135] In embodiments, treating or preventing eye diseases or eye disorders may include treating or preventing damage to RGCs, which is evaluated by pERG. In embodiments, the methods described herein prevent a decrease in p50 amplitude (prevention of disease) or result in an increase in p50 amplitude (treatment of disease). In embodiments, the methods described herein prevent a decrease in p50-n95 amplitude (prevention of disease) or result in an increase in p50-n95 amplitude (treatment of disease).

[0136] Vision test A visual acuity test is an eye test that determines how well a subject perceives details of letters or symbols from a specific distance. Visual acuity refers to a subject's ability to distinguish shapes and details in what they see. Visual acuity is only one component of a subject's overall vision. Other components include color vision, peripheral vision, and depth perception. Examples of visual acuity tests include the Snellen chart, dynamic visual acuity testing, pinhole visual acuity testing, Cardiff visual acuity test, best corrected visual acuity (BCVA) using the Electronic Visual Acuity (EVA) and E-ETDRS algorithms, and random E charts. Visual acuity tests can be used to diagnose common conditions that affect vision, including myopia, hyperopia, astigmatism, presbyopia, and color blindness.

[0137] In some embodiments, the methods described herein prevent a decrease in vision (prevention of a disease) or result in an increase in vision (treatment of a disease).

[0138] Slit-lamp examination A slit lamp examination is an examination that allows for the visual examination and evaluation of all parts of a subject's eye. A slit lamp examination is performed with a slit lamp microscope, which is equipped with a bright light that can be used to examine and evaluate various parts of a subject's eye. The slit lamp light can be adjusted to see into and through the layers of a subject's eye. A slit lamp examination allows for the examination and evaluation of the overall health of a subject's eye and can diagnose any problems or symptoms. During a slit lamp examination, the cornea, sclera, conjunctiva, pupil, iris, lens, retina, and optic nerve can be examined. A slit lamp examination can be used to screen for eye conditions including cataracts, glaucoma, dry eye, scarred corneas, corneal diseases, macular degeneration, and retinitis pigmentosa.

[0139] nucleic acid The present disclosure provides a nucleic acid molecule comprising a nucleic acid sequence encoding OCT4, a nucleic acid sequence encoding SOX2, a nucleic acid sequence encoding KLF4, or any combination thereof, in the absence of an exogenous nucleic acid sequence encoding c-Myc. The nucleic acid molecule may be a vector, including, for example, an expression vector. In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding OCT4. In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding SOX2. In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding KLF4. In certain embodiments, the nucleic acid molecule comprises any two of a nucleic acid sequence encoding OCT4, a nucleic acid sequence encoding SOX2, and a nucleic acid sequence encoding KLF4. In certain embodiments, the nucleic acid molecule comprises a first nucleic acid sequence encoding OCT4, a second nucleic acid sequence encoding SOX2, and a third nucleic acid sequence encoding KLF4. In certain embodiments, the OCT4 comprises an amino acid sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 2. In certain embodiments, the nucleic acid sequence encoding OCT4 is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:1. In certain embodiments, SOX2 comprises an amino acid sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:4. In certain embodiments, the nucleic acid sequence encoding SOX2 is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:3. In certain embodiments, KLF4 comprises an amino acid sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:6. In certain embodiments, the nucleic acid sequence encoding KLF4 is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) to SEQ ID NO: 5. In certain embodiments, OCT4, SOX2, KLF4, or any combination thereof, is a human protein.In certain embodiments, OCT4, SOX2, KLF4, or any combination thereof, is a non-human protein, e.g., a protein derived from one or more mammals, including one or more primates (e.g., cynomolgus monkeys, rhesus monkeys). When two or more of OCT4, SOX2, and KLF4 are present on a single nucleic acid molecule, they can be in any order. The terms "first," "second," and "third" do not necessarily refer to the order of genes on a nucleic acid molecule.

[0140] The terms "nucleic acid," "polynucleotide," "nucleotide sequence," "nucleic acid molecule," "nucleic acid sequence," and "oligonucleotide" refer to a series of nucleotide bases (also called "nucleotides") in DNA and RNA, and mean any chain of two or more nucleotides. These terms include double- or single-stranded genomic and cDNA, RNA, and any synthetic and genetically engineered polynucleotide. This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA, and RNA-RNA hybrids.

[0141] The nucleic acids described herein can be synthesized by standard methods known in the art, for example, by using an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, etc.). Such DNA sequences can be incorporated into a wide variety of vectors incorporating an appropriate RNA polymerase promoter, such as the T7 or SP6 polymerase promoter. The vector can be introduced in vivo so that it is taken up by a cell and directs transcription of the nucleic acid molecule. Such a vector can remain episomal, as long as it is transcribed, or can be integrated into a chromosome. Such vectors can be constructed by standard recombinant DNA technology methods known in the art. Vectors can be plasmid, viral, or other vectors used for replication and expression in mammalian cells. Expression of the sequence can be by any promoter known in the art that acts in mammalian, preferably human, cells. Such promoters can be inducible or constitutive. Any type of plasmid, cosmid, yeast artificial chromosome, or viral vector can be used to prepare recombinant DNA constructs that can be administered to a subject.

[0142] Nucleic acid molecules may contain natural regulatory (expression control) sequences or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5" and 3" non-coding regions, etc. A "recombinant nucleic acid molecule" or "engineered nucleic acid molecule" is a nucleic acid molecule that has undergone a molecular biological manipulation, i.e., a nucleic acid molecule that does not occur in nature or that has been genetically engineered. Furthermore, the terms "recombinant DNA molecule" or "engineered nucleic acid" refer to a nucleic acid sequence that does not occur in nature or that can be made by the artificial combination of two otherwise separate segments of nucleic acid sequence, i.e., by joining together pieces of DNA that are not normally contiguous. "Recombinantly produced" means an artificial combination, often accomplished by chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques using restriction enzymes, ligases, and similar recombinant techniques described, e.g., by Sambrook et al., Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, NY; (1989), or Ausubel et al., Current Protocols in Molecular Biology, Current Protocols (1989), and DNA Cloning: A Practical Approach, Volumes I and II (ed. D.N. Glover) IREL Press, Oxford, (1985), each of which is incorporated herein by reference.

[0143] Such manipulations may be performed to replace a codon with a redundant codon that encodes the same or a conservative amino acid, typically while introducing or removing a sequence recognition site. Alternatively, the manipulations may be performed to join nucleic acid segments of desired functions together to generate a single gene entity containing a desired combination of functions not found in nature. Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site-specific targets, such as promoters, DNA replication sites, regulatory sequences, control sequences, open reading frames, or other useful features, may be incorporated by design.

[0144] As used herein, a "terminator" or "terminator sequence" is a nucleic acid (or nucleic acids) sequence that terminates transcription. Terminators may be unidirectional or bidirectional. Terminators are composed of DNA sequences involved in the specific termination of an RNA transcript by an RNA polymerase. Terminator sequences prevent transcriptional activation of downstream nucleic acid sequences by an upstream promoter. Thus, in certain embodiments, terminators that terminate the production of an RNA transcript are contemplated.

[0145] The most commonly used type of terminator is a forward terminator. When placed downstream of a nucleic acid sequence that is normally transcribed, a forward transcription terminator will interrupt transcription. In some embodiments, a bidirectional transcription terminator may be used, which normally terminates transcription on both the forward and reverse strands. In some embodiments, a reverse transcription terminator may be used, which normally terminates transcription only on the reverse strand.

[0146] Non-limiting examples of mammalian terminator sequences include the bovine growth hormone terminator and viral termination sequences, such as the SV40 terminator, spy, yejM, secG-leuU, thrLABC, rrnB Tl, hisLGDCBHAFI, metZWV, rrnC, xapR, aspA, and arcA terminators. In certain embodiments, the terminator sequence is SV40 and comprises a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:12.

[0147] In certain embodiments, a nucleic acid molecule of the present disclosure comprises a terminator sequence derived from SV40. In certain embodiments, the SV40-derived sequence is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 12 or 30.

[0148] In certain embodiments, the nucleic acid molecules of the present disclosure contain a separator sequence, which can be useful for generating two separate amino acid sequences from a single transcript. The separator sequence can encode a self-cleaving peptide (e.g., a 2A peptide comprising a 2A peptide sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 8 or 10). In certain embodiments, the separator sequence is an internal ribosome entry site (IRES).

[0149] OSK "OCT4," which may also be referred to as octamer-binding transcription factor 4, OCT3, OCT3 / 4, POU5F1, or POU class 5 homeobox 1, is a transcription factor involved in embryonic development and cell fate determination. Like other OCT transcription factors, OCT4 is characterized by a bipartite DNA-binding domain called the POU domain. As used herein, OCT4 transcription factors, homologs, or variants thereof may be derived from any species, including humans. In certain embodiments, a nucleic acid (e.g., an engineered nucleic acid) encoding human OCT4 is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a nucleic acid (e.g., an engineered nucleic acid) set forth in NCBI RefSeq under accession numbers NM_002701, NM_203289, NM_001173531, NM_001285986, or NM_001285987. In certain embodiments, a nucleic acid (e.g., an engineered nucleic acid) encoding OCT4 comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to the nucleic acid (e.g., engineered nucleic acid) sequence provided as SEQ ID NO: 1. SEQ ID NO: 1 is a non-limiting example of a nucleotide sequence encoding OCT4 from Mus musculus. In certain embodiments, a nucleic acid (e.g., an engineered nucleic acid) encoding human OCT4 comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to the nucleic acid (e.g., engineered nucleic acid) sequence provided as SEQ ID NO: 40. SEQ ID NO: 40 is a non-limiting example of a nucleotide sequence encoding human OCT4. Non-limiting examples of OCT4 variants encompassed herein include POU5F1, transcript variant 1, POU5F1, transcript variant 2, POU5F1, transcript variant 3, POU5F1, transcript variant 4, and POU5F1 transcript variant 5.In certain embodiments, the amino acid sequence encoding human OCT4 is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a nucleic acid (e.g., an engineered nucleic acid) set forth in NCBI RefSeq under accession numbers NP_001167002.1, NP_001272915.1, NP_001272916.1, NP_002692.2, or NP_976034.4. In certain embodiments, OCT4 comprises an amino acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 2. SEQ ID NO: 2 is a non-limiting example of an amino acid sequence encoding OCT4 from Mus musculus. In certain embodiments, OCT4 comprises an amino acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 41. SEQ ID NO: 41 is a non-limiting example of an amino acid sequence encoding human OCT4. Other OCT4 transcription factors (e.g., from other species) are known, and nucleic acids (e.g., engineered nucleic acids) encoding OCT4 transcription factors can be found in public databases, including GenBank. For detailed descriptions of the crystal structure and structure-function analysis of OCT4, see Remenyi et al., Genes Dev. 17(16):2048-2059 (2003), Yesudhas et al., PLos One 11(1):e0147240 (2016), and Michel et al., Science 368(6498):1460-1465 (2020); each of which is incorporated by reference in its entirety. Yesudhas et al. identified key residues and hydrogen bonds in OCT4 that potentially facilitate protein-protein and protein-DNA interactions.

[0150] "SRY-box 2" or "SOX2" is a member of the SRY-related HMG-box (SOX) family of transcription factors. SOX2 is involved in promoting embryonic development. Members of the SOX (SRY-related HMG-box) family of transcription factors are characterized by a high mobility group 5 (HMG)-box DNA sequence. This HMG box is a DNA-binding domain that is highly conserved across eukaryotic species. As used herein, a SOX2 transcription factor, homolog, or variant thereof can be derived from any species, including humans. In certain embodiments, a nucleic acid (e.g., an engineered nucleic acid) encoding SOX2 comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a nucleic acid (e.g., an engineered nucleic acid) set forth in NCBI RefSeq under accession number NM_011443.4. In certain embodiments, a nucleic acid (e.g., an engineered nucleic acid) encoding human SOX2 comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a nucleic acid (e.g., an engineered nucleic acid) set forth in NCBI RefSeq under accession number NM_003106.4. In certain embodiments, SOX2 comprises a nucleic acid (e.g., an engineered nucleic acid) sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:3 or SEQ ID NO:42. SEQ ID NO:3 is a non-limiting example of a nucleotide sequence encoding SOX2 from Mus musculus. SEQ ID NO:42 is a non-limiting example of a nucleotide sequence encoding human SOX2. In certain embodiments, the nucleic acid (e.g., an engineered nucleic acid) encoding human SOX2 comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to the amino acid sequence set forth in NCBI RefSeq under accession number NP_003097.1.In some examples, SOX2 comprises an amino acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 4. In some examples, SOX2 comprises an amino acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 43. SEQ ID NO: 4 is a non-limiting example of an amino acid sequence encoding SOX2 from Mus musculus. SEQ ID NO: 43 is a non-limiting example of an amino acid sequence encoding human SOX2. For detailed descriptions of the crystal structure and structure-function analysis of SOX2, see Dodonova et al., Nature 580(7805):669-672 (2020), Holmes et al., Nat Commun 11:1805 (2020), and Yesudhas et al., PLos One 11(1):e0147240 (2016), which are incorporated by reference in their entireties. Yesudhas et al. identified key residues and hydrogen bonds that potentially facilitate protein-protein and protein-DNA interactions in SOX2.

[0151] "KLF4," which may also be referred to as Krüppel-like factor 4, EZF, or GKLF, is a zinc finger transcription factor. KLF4 is involved in regulating differentiation and proliferation and can interact with coactivators, including members of the p300-CBP coactivator family. As used herein, a KLF4 transcription factor, homolog (e.g., functional homolog), or variant thereof can be derived from any species, including humans. In certain embodiments, a nucleic acid (e.g., an engineered nucleic acid) encoding human KLF4 comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a nucleic acid (e.g., an engineered nucleic acid) described in the NCBI RefSeq database under accession number NM_004235.5 or NM_001314052.1. Non-limiting examples of KLF4 variants include Krüppel-like factor 4 transcript variant 1 and Krüppel-like factor 4 transcript variant 2. In certain embodiments, KLF4 comprises a nucleic acid (e.g., an engineered nucleic acid) sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 5 or SEQ ID NO: 44. SEQ ID NO: 5 is a non-limiting example of a nucleotide sequence encoding KLF4 from Mus musculus. SEQ ID NO: 44 is a non-limiting example of a nucleotide sequence encoding human KLF4. In certain embodiments, KLF4 comprises an amino acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to NP_001300981.1 or NP_004226.3. In certain embodiments, KLF4 comprises an amino acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 6. In certain embodiments, KLF4 comprises an amino acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 45.SEQ ID NO: 6 is a non-limiting example of an amino acid sequence encoding KLF4 from Mus musculus. SEQ ID NO: 45 is a non-limiting example of an amino acid sequence encoding human KLF4. For detailed descriptions of the crystal structure and structure-function analysis of KLF4, see Borisova et al., iScience 25(1):103525 (2021), Schu et al., Cell Mol Life Sci. 68(18):3121-3131 (2011), and Liu et al., Nucleic Acids Res. 42(8):4859-4867 (2014); each of which is incorporated by reference in its entirety. Borisova et al. identified amino acid substitutions in the KLF4 zinc finger domain that enhance protein function.

[0152] The term "c-Myc" or "Myc" refers to a nuclear phosphoprotein involved in cell cycle progression. c-Myc can form a heterodimer with the transcription factor MAX, and the heterodimer can bind to an E-box sequence on a nucleic acid (e.g., an engineered nucleic acid) to regulate transcription of a target gene. In certain embodiments, a nucleotide sequence encoding c-Myc comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a sequence set forth in the NCBI RefSeq database under accession number NM_001354870.1 or NM_002467.5. In certain embodiments, the amino acid sequence encoding c-Myc comprises a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to NP002458.2 or NP001341799.1. In certain embodiments, the method comprises inducing expression of OCT4, KLF4, SOX2, or any combination thereof, without inducing c-Myc expression or activating c-Myc. Failure to induce c-Myc expression may refer to the absence of substantial induction of c-Myc expression above endogenous levels of c-Myc expression in a cell, tissue, subject, or any combination thereof. Lack of substantial induction of c-Myc expression compared to endogenous levels of c-Myc expression in a cell, tissue, subject, or any combination thereof can refer to increasing c-Myc expression by less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or any value therebetween, compared to endogenous levels of c-Myc expression in a cell, tissue, subject, or any combination thereof. Failure to activate c-Myc expression can refer to no substantial activity of c-Myc (e.g., activity) in a cell, tissue, subject, or any combination thereof above endogenous c-Myc activity.No substantial induction of c-Myc activity compared to endogenous c-Myc activity in a cell, tissue, subject, or any combination thereof may refer to an increase in c-Myc activity to less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or any value therebetween, compared to endogenous c-Myc activity in a cell, tissue, subject, or any combination thereof.

[0153] Constitutive promoter The term "promoter" refers to a regulatory region of a nucleic acid sequence that controls the initiation and rate of transcription of the remainder of the nucleic acid sequence. A promoter may also contain subregions to which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. A promoter may be constitutive, inducible, activatable, repressible, tissue-specific, or any combination thereof. A promoter drives the expression or drives the transcription of a nucleic acid sequence that it regulates. As used herein, a promoter is considered to be "operably linked" when it is in the correct functional location and orientation relative to the nucleic acid sequence that it regulates, so as to control ("drive") the initiation of transcription of that sequence, the expression of that sequence, or a combination thereof.

[0154] A promoter can promote ubiquitous or tissue-specific expression of an operably linked nucleic acid sequence from any species, including humans. In some embodiments, the promoter is a eukaryotic promoter. Non-limiting examples of eukaryotic promoters include TDH3, PGK1, PKC1, TDH2, PYK1, TPI1, AT1, CMV, EF1 alpha, SV40, PGK1 (human or mouse), Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIIa, GAL1, GAL10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, and U6, as are well known to those of skill in the art (see, for example, the Addgene website: blog.addgene.org / plasmids-101-the-promoter-region).

[0155] Non-limiting examples of ubiquitous promoters include tetracycline-responsive promoters (under relevant conditions), CMV (e.g., SEQ ID NO: 17), chicken β-actin (CBA), short CMV early enhancer / chicken β-actin / short β-globulin intron, human synapsin, EF1 alpha, SV40 promoter, PGK1, Ubc, CAG, human β-actin gene promoter, RSV promoter, EFS promoter, and promoters containing an upstream activating sequence (UAS). In certain embodiments, the promoter is a mammalian promoter. Examples of constitutive promoters tested in RGCs and RGC axons are described in Nieuwenhuis et al., Gene Ther. 30:503-519 (2023), the entire contents of which are incorporated herein by reference.

[0156] Non-limiting examples of constitutive promoters include CP1, CMV, EF1 alpha, SV40, PGK1, Ubc, human beta actin, beta tubulin, CAG, Ac5, Rosa26 promoter, COL1A1 promoter, polyhedrin, TEF1, GDS, CaM3 5S, Ubi, H1, U6, red opsin promoter (red promoter), rhodopsin promoter (rho promoter), cone arrestin promoter (car promoter), and rhodopsin kinase promoter (rk promoter). In some examples, the constitutive promoter is the Rosa26 promoter. In some examples, the constitutive promoter is the COL1A1 promoter. Tissue-specific promoters can be used to drive expression of engineered nucleic acids, including, for example, nucleic acids encoding rtTA, tTA, OCT4, KLF4, SOX2, or any combination thereof. In some embodiments, tissue-specific promoters are used to drive expression of rtTA or rTA. In some embodiments, tissue-specific promoters are used to drive expression of OCT4, KLF4, and SOX2. In some embodiments, the SV40 promoter is used to drive expression of OCT4, KLF4, and SOX2.

[0157] In certain embodiments, a nucleic acid molecule of the present disclosure comprises one or more constitutive promoters, such as CP1, CMV, EF1 alpha, SV40, PGK1, Ubc, human beta-actin, CAG, Ac5, polyhedrin, TEF1, GDS, CaM3 5S, Ubi, H1, and / or U6 promoters. The constitutive promoter may be operably linked to a nucleic acid sequence encoding OCT4, KLF4, SOX2, an inducer, or a combination thereof. In some embodiments, the nucleic acid molecule comprises one constitutive promoter. In some embodiments, the nucleic acid molecule comprises two or more constitutive promoters.

[0158] Inducible promoters An "inducible promoter" is a promoter that is characterized by initiating or enhancing transcriptional activity when in the presence of, affected by, or contacted by an inducing agent. The inducing agent can be an endogenous or, usually, exogenous condition, compound, drug, or protein that contacts a nucleic acid engineered in such a way that it is active in inducing transcriptional activity from the inducible promoter. In certain embodiments, the inducing agent is a tetracycline-sensitive protein (e.g., tTA or rtTA, a TetR family regulator).

[0159] Inducible promoters for use in accordance with the present disclosure include any inducible promoter described herein or known to one of skill in the art. Examples of inducible promoters include, but are not limited to, chemically / biochemically regulated and physically regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, including tetracycline repressor proteins (TetR or TetRKRAB), tetracycline operator sequences (tetO) and tetracycline transactivator fusion proteins (tTA), and tetracycline operator sequences (tetO) and reverse tetracycline transactivator fusion proteins (rtTA)). These include steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptor, and promoters from the steroid / retinoid / thyroid 25 receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (a protein that binds and sequesters metal ions) genes from yeast, mouse, and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene, or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters), pH-regulated promoters, and light-regulated promoters. Non-limiting examples of inducible systems using light-regulated promoters are provided in Wang et al., Nat. Methods. 2012 Feb 12;9(3):266-9.

[0160] In the field of genetic engineering, precise control of gene expression is a useful tool for studying, manipulating, and controlling developmental and other physiological processes. Gene expression is a complex biological process that involves several specific protein-protein interactions. Tightly controlled inducible gene expression systems, or "gene switches," are useful for a variety of applications, such as gene therapy, large-scale production of proteins in cells, cell-based high-throughput screening assays, functional genomics, and trait control in transgenic plants and animals. Inducible promoters are useful because they can turn on or off the expression of genes operably linked to them at specific developmental stages, specific tissues, or specific therapeutic stages of an organism. Examples of inducible promoters and on / off gene expression systems include alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenesis-regulated promoters, temperature-regulated promoters, light-regulated promoters, dihydrofolate reductase (DHFR) protein destabilization domains, riboswitches, and hormone-activated promoters. Many other systems have been described, and those skilled in the art can readily select one. Specific examples of inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, the ecdysone insect promoter, the tetracycline-repressible system, the tetracycline-inducible system, the RU486-inducible system, and the rapamycin-inducible system. Other types of inducible promoters that may be useful in this context are those regulated by specific physiological conditions, such as temperature, acute phase, specific differentiation states of cells, or only in replicating cells. Any type of inducible promoter that is tightly regulated and specific to a particular target ocular cell type may be used.Exemplary inducible promoters for use in ocular treatments are described in U.S. Patent No. 10,383,922, Buck et al., Int. J. Mol Sci. 21, 12:4197 (2020), and Lipinski et al., Adv. Exp. Med. Biol. 1185:79-83 (2019), which are incorporated herein by reference in their entireties. Exemplary inducible promoters for use in mammalian cells are described in U.S. Patent No. 20200283778, Kallunke et al., Cells 8:796 (2019), Doshi et al., Crit. Rev. Biotechnol. 40, 8:1131-1150 (2020), and Siddiqui et al., Current Opinion in Biotechnology 78:102823 (2022), which are incorporated herein by reference in their entireties.

[0161] Further non-limiting examples of inducible promoters include mifepristone-responsive promoters (e.g., the GAL4-Elb promoter) and coumermycin-responsive promoters. See, e.g., Zhao et al., Hum Gene Ther. 2003 Nov 20;14(17):1619-29.

[0162] In some embodiments, the nucleic acid molecule of the present disclosure comprises an inducible promoter. In some embodiments, the nucleic acid molecule has one inducible promoter. In such examples, expression of OCT4, SOX2, and KLF4 is under the control of the same inducible promoter. In some embodiments, the nucleic acid molecule has two or more inducible promoters. Inducible promoters may include tetracycline response elements (TREs) (e.g., the TRE3G promoter, the TRE2 promoter, or the P-tite promoter), mifepristone-responsive promoters (e.g., the GAL4-Elb promoter), or coumermycin-responsive promoters. As an example, a TRE (e.g., TRE3G) promoter may comprise a nucleic acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 7. See, e.g., U.S. Published Application No. 2021-0403923A and International Publication No. WO2020 / 069339.

[0163] In certain embodiments, the inducing agent is capable of inducing expression of the first (e.g., OCT4), second (e.g., SOX2), third (e.g., KLF4) nucleic acid, or any combination thereof, from an inducible promoter in the presence of tetracycline (e.g., doxycycline). In certain embodiments, the inducing agent is a reverse tetracycline-controlled transactivator (rtTA) (e.g., M2-rtTA, rtTA3, or rtTA4). rtTA variants are described in Urlinger et al., Proc. Natl. Acad. Sci. USA 97(14):7693-8 (2000), Das et al., J Biol Chem. 279(18):18776-18782 (2004), U.S. Published Application Nos. 2021-0403923A, US 7541446B2, US 5650298A, US 8383364B2, Zhou et al., Gene Ther. 13:1382-90 (2006), and International Publication No. WO 2020 / 069339, entitled MUTANT REVERSE TETRACYCLINE TRANSACTIVATORS FOR EXPRESSION OF GENES, each of which is incorporated herein by reference in its entirety. In certain embodiments, the rtTA is rtTA3, which comprises an amino acid sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 20. In certain embodiments, the rtTA3 is encoded by a nucleic acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 19. In certain embodiments, the rtTA is rtTA4, which comprises an amino acid sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 27. In certain embodiments, rtTA4 is encoded by a nucleic acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:26.

[0164] In certain embodiments, the inducing agent is capable of inducing expression of a first nucleic acid (e.g., OCT4), a second nucleic acid (e.g., SOX2), a third nucleic acid (e.g., KLF4), or any combination thereof, from an inducible promoter in the absence of tetracycline (e.g., doxycycline).

[0165] In certain embodiments, the inducing agent is a tetracycline-controlled transactivator (tTA).

[0166] tissue-specific promoters Non-limiting examples of tissue-specific promoters include eye-specific promoters. Non-limiting examples of eye-specific promoters include the human GRK1 (rhodopsin kinase) promoter, the human CRX (cone homeobox transcription factor) promoter, human synapsin, mouse phosphoglycerate kinase, neurofilament light polypeptide (NEFL), neurofilament heavy polypeptide (NEFH), doublecortin (DCX), phosphodiesterase 6H (PDE6H), Purkinje cell protein 2 (PCP2), gamma-synuclein promoter, interphotoreceptor binding protein (IRBP), short promoter variants of glial fibrillary acidic protein (GFAP), monocyte chemoattractant protein-1 (Mcp1), short promoter variants of mouse cone arrestin (mCAR), short promoter variants of human neurofilament heavy polypeptide, and the human NRL promoter (neuroretinal leucine zipper transcription factor enhancer upstream of the human TK terminal promoter), all of which are described in Nieuwenhuis et al., Gene Ther. 30:503-519 (2023) and Kani et al., Invest Ophthalmol Vis Sci. 48(9):3954-3961 (2007), which are incorporated herein by reference in their entireties.Non-limiting examples of RGC-specific promoters include those derived from neurofilament heavy chain (NEFH), neurofilament light polypeptide (NEFL), neurofilament medium chain (NEFM), vinivin-like 1 (VSNL1), SPARC-like 1 (SPARCL1), solute carrier family 17 member 6 (SLC17A6), thymosin beta 10 (TMSB10), annexin A2 (ANXA2), stathmin 2 (STMN2), peripherin (PRPH1), cartilage acidic protein 1 (CRTAC1), RNA-binding protein-mRNA processing factors (RBPMS), Ras-related protein Rab-13 (RAB13), ATPase Na+ / K+ transport subunit beta 1 (ATP1B1), fatty acid binding protein 3 (FABP3), mouse γ-synuclein, and human gamma-synuclein genes, all of which are described in detail in Hanlon et al. al., Front. Neurosci., 11:521 (2017) and Ward et al., Sci Rep 10:16515 (2020), Simpson et al., Hum. Gene Ther., 30(3):257-272 (2019), Wang et al., J. Neurosci. 40(20):3896-3914 (2020), and Chaffiol et al., Mol. Ther. 25(11):2546-256- (2017), which are incorporated herein by reference in their entireties.

[0167] In some embodiments, the promoters of the present disclosure are suitable for use in AAV vectors. See, e.g., U.S. Patent Application Publication No. 2018 / 0155789, which is incorporated herein by reference in its entirety for this purpose.

[0168] rtTA system "Tetracycline" refers to antibiotic compounds of the tetracycline class, including, but not limited to, tetracycline, chlortetracycline, oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, doxycycline, tigecycline, eravacycline, sarecycline, and omadacycline. Regarding the use of tetracycline derivatives to induce rtTa activity, see Gossen et al., Science 268:1766-9 (1995), which is incorporated herein by reference in its entirety.

[0169] In certain embodiments, the inducible promoter comprises a tetracycline (Tet) response element. For example, the inducible promoter may be a TRE3G promoter (e.g., a TRE3G promoter comprising a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 7). As an example, a TRE (e.g., TRE2) promoter may comprise a nucleic acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 7.

[0170] As used herein, a "reverse tetracycline transactivator" ("rtTA") is an inducing agent that binds to a TRE promoter (e.g., a TRE3G, TRE2, or P-tight promoter) in the presence of tetracycline (e.g., doxycycline) and can drive expression of a transgene operably linked to the TRE promoter. rtTAs generally comprise a mutant tetracycline repressor DNA-binding protein (TetR) and a transactivation domain (see, e.g., Gossen et al., Science. 1995 Jun 23;268(5218):1766-9 and any of the transactivation domains listed therein). The mutant TetR domain, when bound to tetracycline, is capable of binding to a TRE promoter. See, e.g., U.S. Published Application Nos. 2021-0403923A, US7541446B2, US5650298A, US8383364B2, Zhou et al., Gene Ther. 13:1382-90 (2006), Das et al., Curr Gene Ther. 16(3):156-167 (2016), and International Publication No. WO2020 / 069339, entitled "MUTANT REVERSE TETRACYCLINE TRANSACTIVATORS FOR EXPRESSION OF GENES," each of which is incorporated by reference herein in its entirety.

[0171] As used herein, a "Tet-Off" system is a type of inducible system that can suppress expression of a specific transgene in the presence of tetracycline (e.g., doxycycline (DOX)). Conversely, a Tet-Off system can induce expression of a specific transgene in the absence of tetracycline (e.g., doxycycline, DOX). In certain embodiments, a Tet-Off system comprises a tetracycline-responsive promoter and a tetracycline-controlled transactivator (tTA) operably linked to a transgene (e.g., encoding OCT4, KLF4, SOX2, or any combination thereof). The transgenes with the tetracycline-responsive promoter (e.g., TRE3G, P-tite, or TRE2) and the tetracycline-controlled transactivator may be encoded on the same vector or on separate vectors. See, for example, U.S. Published Application No. 2021-0403923A and International Publication No. WO2020 / 069339, entitled MUTANT REVERSE TETRACYCLINE TRANSACTIVATORS FOR EXPRESSION OF GENES, each of which is incorporated by reference herein in its entirety.

[0172] As used herein, a "Tet-On" system is a type of inducible system that can induce expression of a particular transgene in the presence of tetracycline (e.g., doxycycline (DOX)). In certain embodiments, the Tet-On system comprises a tetracycline-responsive promoter and a reverse tetracycline-controlled transactivator (rtTA) operably linked to a transgene (e.g., encoding OCT4, KLF4, SOX2, or any combination thereof). For example, the rtTA can be any rtTA system listed in Table 1 or a variant thereof. In certain embodiments, the nucleic acid encoding rtTA3 comprises a sequence at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%) identical to SEQ ID NO:19. In certain embodiments, rtTA3 comprises an amino acid sequence at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100%) identical to SEQ ID NO: 20. In certain embodiments, a nucleic acid encoding rtTA4 comprises a sequence at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100%) identical to SEQ ID NO: 26. In certain embodiments, rtTA4 comprises an amino acid sequence at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100%) identical to SEQ ID NO: 27. The tetracycline-responsive promoter (e.g., a TRE-containing promoter, including TRE3G, P-tite, and TRE2) and the expression cassette encoding the reverse tetracycline-controlled transactivator may be encoded on the same vector or on separate vectors. See, e.g., U.S. Published Application No. 2021-0403923A and International Publication No. WO2020 / 069339.

[0173] As used herein, a "TRE promoter" refers to a promoter comprising a tetracycline response element (TRE). As used herein, a TRE comprises at least one (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) Tet-O sequence. In some embodiments, a TRE promoter further comprises a minimal promoter located downstream of the tet-O sequence. A minimal promoter is a promoter that comprises minimal promoter elements (e.g., a TATA box and a transcription start site) but is inactive in the absence of an upstream enhancer (e.g., a sequence comprising a Tet-O). In one example, a minimal promoter may be a minimal CMV promoter comprising a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 17 or 24. For example, the TRE promoter may be a TRE3G promoter (e.g., a TRE3G promoter comprising a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 7). [Table 1]

[0174] Multicistronic vectors A "multicistronic vector" is a vector encoding two or more amino acid sequences (e.g., a vector encoding OCT4 and KLF4, OCT4 and SOX2, KLF4 and SOX2, or OCT4, SOX2, and KLF4 (OSK)). Multicistronic vectors allow for the expression of multiple amino acid sequences from a nucleic acid sequence. The nucleic acid sequences encoding each transcription factor (e.g., OCT4, KLF4, or SOX2) may be linked or separated so that they produce an unlinked protein. For example, an internal ribosome entry site (IRES) or polypeptide cleavage signal may be positioned between the nucleic acid sequences encoding each transcription factor in the vector. Exemplary polypeptide cleavage signals include 2A peptides (e.g., T2A, P2A, E2A, and F2A). The T2A peptide may comprise a sequence at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) to SEQ ID NO: 10. The P2A peptide can comprise a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:8.

[0175] In some embodiments, the expression vector of the present disclosure is a multicistronic expression vector.

[0176] In certain embodiments, the nucleic acid molecule is a viral vector (e.g., a lentivirus, retrovirus, or adeno-associated virus (AAV) vector). The AAV vector of the present disclosure generally comprises inverted terminal repeats (ITRs) flanking the transgene of interest (e.g., a nucleic acid sequence encoding OCT4, SOX2, KLF4, an inducer, or a combination thereof). In some embodiments, the distance between two inverted terminal repeats is less than 5.0 kilobases (kb) (e.g., less than 4.9 kb, less than 4.8 kb, less than 4.7 kb, less than 4.6 kb, less than 4.5 kb, less than 4.4 kb, less than 4.3 kb, less than 4.2 kb, less than 4.1 kb, less than 4 kb, less than 3.5 kb, less than 3 kb, less than 2.5 kb, less than 2 kb, less than 1.5 kb, less than 1 kb, or less than 0.5 kb).

[0177] In certain embodiments, a nucleic acid molecule of the present disclosure (e.g., an expression vector encoding OCT4, KLF4, SOX2, an inducer, or a combination thereof) may further comprise a nucleic acid sequence encoding a selection agent (e.g., an antibiotic, including blasticidin, geneticin, hygromycin B, mycophenolic acid, puromycin, zeocin, actinomycin D, ampicillin, carbenicillin, kanamycin, and neomycin) and / or a detectable marker (e.g., GFP, RFP, luciferase, CFP, mCherry, DsRed2FP, mKate, biotin, a FLAG tag, an HA tag, a His tag, a Myc tag, a V5 tag, etc.).

[0178] In some embodiments, the expression vector encoding OCT4, KLF4, and SOX2 comprises the sequence set forth in SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In some embodiments, the expression vector encoding OCT4, KLF4, and SOX2 comprises the elements shown in Figure 1, or a combination thereof. The expression vector may be a viral vector. The viral vector may be an adeno-associated viral (AAV) vector, a retroviral vector, a lentiviral vector, a herpes viral vector, or the like.

[0179] Delivery Vehicle viral vectors A "recombinant virus" is a virus (e.g., a lentivirus, adenovirus, retrovirus, herpesvirus, alphavirus, vaccinia virus, or adeno-associated virus (AAV)) that has been isolated from its natural environment (e.g., from a host cell, tissue, or subject) or that is artificially produced.

[0180] Currently, multiple serotypes of adeno-associated virus (AAV) have been identified, including 12 human serotypes and over 100 serotypes from non-human primates (Howarth et al., 2010, Cell Biol Toxicol 26:1-10). Among these serotypes, human serotype 2 was the first AAV to be developed as a gene transfer vector. Other AAV serotypes currently in use include, but are not limited to, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAVrh74, AAVdj, and AAV.PHP, as well as variants thereof. Additionally, AAV serotypes for use in treating ocular diseases are described in Ghoraba et al., Clin. Opthalmol. 16:1753-1771 (2022), Bordet et al., Drug Discovery Today 24, 8:1685-1693 (2019), and Dalara et al., Sci. Transl. Med. 5, 189 (2013), which are incorporated herein by reference in their entireties. Exemplary AAV serotypes used in ocular treatments are described in U.S. Patent Nos. 9,567,376, 10,383,922, and 10,426,844, which are incorporated herein by reference in their entireties. Additionally, non-naturally engineered variants and chimeric AAVs may also be useful. In particular, the capsid protein may be a variant containing one or more amino acid substitutions that enhance transduction efficiency.

[0181] "AAV" or "adeno-associated virus" is a non-enveloped virus capable of carrying and delivering nucleic acids (e.g., engineered nucleic acids encoding OCT4, KLF4, SOX2, or any combination thereof) and belongs to the genus Dependoparvovirus. In some instances, AAV can deliver nucleic acids encoding inducers. Generally, AAV does not integrate into the genome. The tissue-specific targeting ability of AAV is often determined by the AAV capsid serotype (e.g., see Table 2 below for examples of AAV serotypes and their usefulness in tissue-specific delivery). Non-limiting serotypes of AAV include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and variants thereof. In certain embodiments, the AAV serotype is a variant of AAV9 (e.g., AAV PHP.b). [Table 2]

[0182] As used herein, the term "AAV vector" refers to a nucleic acid comprising AAV inverted terminal repeats (ITRs) flanking an expression cassette (e.g., an expression cassette comprising nucleic acids encoding OCT4, KLF4, and SOX2, each alone or in combination, or an expression cassette encoding rtTA or tTA). An AAV vector may further comprise a promoter sequence.

[0183] "Inverted terminal repeats" or "ITRs" are nucleic acid sequences that are reverse complementary to each other. Generally, in AAV vectors, ITRs are found on either side of a cassette (e.g., an expression cassette containing a nucleic acid encoding OCT4, KLF4, SOX2, or any combination thereof). For example, the ITRs flanking an OSK cassette may include SEQ ID NOs: 16 and 32. Similarly, in some examples, the pAAV2-CMV-rtTA3VP16 vector disclosed herein may include ITRs containing SEQ ID NOs: 22 and 33, and the AAV2-CMV-rtTA4 vector disclosed herein may include ITRs containing SEQ ID NOs: 29 and 34. In some examples, the cassette encodes an inducing agent. AAV ITRs include ITRs from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV variants thereof.

[0184] In one aspect, the present disclosure provides a recombinant virus. The recombinant virus may include one or more lentiviruses, adenoviruses, retroviruses, herpesviruses, alphaviruses, vaccinia viruses, or adeno-associated viruses (AAVs), including any of the expression vectors described herein. The use of recombinant lentiviruses to treat ocular diseases is described in Arsenijevic Y. et al., Pharmaceuticals 14(8);1605(2022), Miyazaki M. et al., Hum. Gen Ther., 22(5):559-565(2011), Ralph GS et al., Clin. Sci. (London) 110(1);37-46(2006), and Balaggan KS. et al., Gene Ther. 19(2):145-53(2012), each of which is incorporated herein by reference in its entirety. In certain embodiments, the recombinant virus encodes a transcription factor selected from OCT4, KLF4, SOX2, and any combination thereof. In certain embodiments, the recombinant virus encodes two or more transcription factors selected from the group consisting of OCT4, KLF4, and SOX2. In certain embodiments, the recombinant virus encodes OCT4 and SOX2, OCT4 and KLF4, or SOX2 and KLF4. In certain embodiments, the recombinant virus encodes OCT4, KLF4, and SOX2. In certain embodiments, the recombinant virus encodes four or more transcription factors, for example, OCT4, SOX2, KLF4, and another transcription factor.

[0185] Lipid nanoparticles (LNPs) Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used to introduce the compositions of the present disclosure into suitable host cells. In particular, any nucleic acid (e.g., engineered nucleic acid) capable of inducing OCT4, KLF4, and / or SOX2 expression (e.g., expression vector), any engineered protein, any chemical agent that activates (e.g., induces expression of) OCT4, KLF4, and / or SOX2, any antibody that activates (e.g., induces expression of) OCT4, KLF4, and / or SOX2, engineered cells, and / or any recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) can be encapsulated in a lipid particle, liposome, vesicle, nanosphere, nanoparticle, or the like. In some embodiments, any of the nucleic acids (e.g., engineered nucleic acids) (e.g., expression vectors) capable of inducing expression of OCT4, KLF4, SOX2, or any combination thereof, any of the engineered proteins, any of the chemical agents that activate (e.g., induce expression of) OCT4, KLF4, SOX2, or any combination thereof, any of the antibodies that activate (e.g., induce expression of) OCT4, KLF4, SOX2, or any combination thereof, any of the engineered cells, and / or any of the recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) can be encapsulated in lipid particles, liposomes, vesicles, nanospheres, nanoparticles, etc. Inducing agents (e.g., nucleic acids encoding the inducing agents, or proteins encoding the inducing agents, and / or recombinant viruses encoding the inducing agents) and / or chemical agents that can modulate the activity of the inducing agents can be encapsulated in lipid particles, liposomes, vesicles, nanospheres, nanoparticles, etc.

[0186] In some embodiments, nucleic acids, engineered proteins, chemicals, antibodies, and / or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) are formulated in lipid nanoparticles. See, e.g., Cullis and Hope Mol Ther. 2017 Jul. 5;25(7):1467-1475. In some embodiments, the lipid nanoparticles contain one or more membrane fusion proteins, which deliver plasmids directly to the cytoplasm or factors OCT4, KLF4, SOX2, or any combination thereof, which may be fused directly to a targeting protein, with or without nanoparticle encapsulation. In some embodiments, the lipid nanoparticles are Fusogenix lipid nanoparticles. In some embodiments, the lipid particles are "wrapped liposomes" (WL). See, e.g., Yamauchi et al., Biochim Biophys Acta. 2006 January;1758(1):90-7. In some embodiments, the lipid nanoparticle is a PEGylated liposome (e.g., DOXIL®) (e.g., Allen & Hansen, Biochim Biophys Acta. 1991 Jul. 1; 1066(1):29-36), 1,2-dioleoyl-sn-glycerol-3 phosphatidylethanolamine (DOPE), the neutral helper lipid phosphatidylethanolamine (PE), or a combination thereof (e.g., Farhood et al., Biochim Biophys Acta. 1995 May 4; 1235(2):289-95, Zhou & Huang, Biochim Biophys Acta. 1994 Jan. 19; 1189(2):195-203).In some embodiments, the lipid nanoparticles or fusion proteins comprise molecules or proteins that mimic the method used by viruses for intracellular delivery of macromolecules, for example, using vesicular stomatitis virus protein (VSV G), phage coat protein and / or shGALA, and / or fusion-associated small transmembrane (FAST) proteins, such as those found in avian reoviruses (ARV), Nelson Bay reovirus (NBV), and baboon reoviruses (BBV), aquareovirus reovirus (AQV), and reptilian reoviruses (RRV), and / or various pH-sensitive peptides such as bombesin targeting peptides (e.g., Kohashi et al., Bioconjug Chem. 2009 May 20;20(5):953-9). See, e.g., Peisajovich et al., Eur J Biochem. 2002 September;269(17):4342-50; Sakurai et al., 2011). See also Nesbitt, Targeted Intracellular Therapeutic Delivery Using Liposomes Formulated with Multifunctional FAST proteins, Western University Thesis, 2012.

[0187] In some embodiments, nucleic acids (e.g., RNA or DNA containing plasmids) encoding OCT4, KLF4, SOX2, or combinations thereof are encapsulated in Fusogenix lipid nanoparticles. In some embodiments, nucleic acids encoding inducers (e.g., rtTA or tTA) are encapsulated in Fusogenix lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise a viral membrane protein. Without being bound by theory, the lipid nanoparticles may be non-toxic because they comprise a membrane fusion protein that is not a viral membrane fusion protein. Non-limiting examples of membrane fusion proteins include those disclosed in U.S. Pat. Nos. 7,851,595 and 8,252,901, International Application Publication No. WO 2012 / 040825, and International Application Publication No. WO 2002 / 044206.

[0188] In some embodiments, compositions of the present disclosure (e.g., comprising nucleic acids encoding OCT4, KLF4, SOX2, or combinations thereof) are delivered non-virally. Methods for non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid complexes, naked nucleic acid (e.g., RNA or DNA), artificial virions, and enhanced uptake of nucleic acids (e.g., RNA or DNA).

[0189] In some embodiments, the delivery vehicle targets the cargo. For example, any of the nucleic acids, engineered proteins, chemicals, antibodies, and / or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) described herein can be delivered via nanoparticles that deliver the cargo to specific tissues or cell types. For example, nanoparticles coated with galactose polymers are known to release cargo in senescent cells as a result of endogenous beta-galactosidase activity. See, e.g., Lozano-Torres et al., J Am Chem Soc. 2017 Jul. 5;139(26):8808-8811.

[0190] In some embodiments, delivery of nucleic acids not on a viral vector includes administration of naked nucleic acids, electroporation, the use of nanoparticles, and / or the use of liposomes. As a non-limiting example, the engineered nucleic acids of the present disclosure (e.g., RNA, including mRNA, or DNA) can be formulated in nanoparticles for delivery. See, e.g., Dong et al., Nano Lett. 2016 Feb. 10; 16(2):842-8. In some embodiments, the nanoparticles comprise acetylated galactose. See, e.g., Lozano-Torres et al., J Am Chem Soc. 2017 Jul. 5; 139(26):8808-8811. In some embodiments, the engineered nucleic acids (e.g., RNA, including mRNA, or DNA) are electroporated or transfected into cells. In certain embodiments, the engineered nucleic acids are delivered as naked nucleic acids (e.g., naked DNA or naked RNA).

[0191] In some embodiments, the engineered nucleic acid formulated in nanoparticles for delivery is not an AAV vector. Suitable vector backbones for formulation in nanoparticles include, but are not limited to, NANOPLASMID® vectors and NTC "8" series mammalian expression vectors. Non-limiting examples of vector backbones for formulation in nanoparticles include NTC9385R and NTC8685. Without being bound by theory, NTC "8" series mammalian expression vectors may be useful because they are generally eliminated by cells within a few weeks. NTC "8" series mammalian expression vectors contain a CMV promoter, which may be operably linked to sequences encoding OCT4, KLF4, SOX2, or combinations thereof. Without being bound by theory, NANOPLASMID® vectors may be less immunogenic than other vectors and may express at higher levels and for longer periods of time, which may be useful for long-term expression of operably linked nucleic acids. In some embodiments, NANOPLASMID® vectors may be useful for long-term expression of OCT4, KLF4, SOX2, or combinations thereof.

[0192] In some embodiments, proteins encoding OCT4, SOX2, and / or KLF4 and / or inducers are formulated within nanoparticles (e.g., for nuclear delivery). In some embodiments, proteins encoding OCT4, SOX2, KLF4, or any combination thereof (e.g., OCT4 and SOX2, KLF4 and SOX2, OCT4 and KLF4, or KLF4, SOX2, and OCT4) are formulated within nanoparticles (e.g., for nuclear delivery). In certain embodiments, the nanoparticles further comprise proteins encoding inducers. For example, chitosan [poly(N-acetylglucosamine)] is a biodegradable polysaccharide that can be used to formulate nanoparticles by several methods. In some embodiments, chitosan polymer nanoparticles are loaded with proteins encoding OCT4, SOX2, and / or KLF4 and / or inducers and delivered to the nucleus of a cell. See, for example, Tammam et al., Oncotarget. 2016 Jun. 21;7(25):37728-37739.

[0193] Delivery route As used herein, the terms "administer," "administering," or "administration" refer to the introduction of any of the compositions described herein into a subject; Any of the nucleic acids capable of inducing OCT4, KLF4, and / or SOX2 expression, any of the nucleic acids capable of inducing expression of one or more transcription factors selected from the group consisting of OCT4, KLF4, SOX2, and any combination thereof, any of the engineered proteins described herein, any of the chemical agents that activate (e.g., induce expression of) OCT4, KLF4, and / or SOX2, any of the chemical agents that activate (e.g., induce expression of) one or more transcription factors selected from OCT4, KLF4, SOX2, and any combination thereof, any of the antibodies that activate (e.g., induce expression of) OCT4, KLF4, and / or SOX2, and any combination thereof, and / or any of the recombinant viruses described herein (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV), alone or in combination, are administered to any cell, tissue, organ, and / or subject. In some embodiments, a nucleic acid encoding an inducer, an engineered protein encoding an inducer, a chemical agent capable of modulating (e.g., activating or inhibiting) the activity of an inducer, and / or a recombinant virus encoding an inducer is also administered to a cell, tissue, organ, and / or subject.Any of the compositions described herein, including any of the nucleic acids capable of inducing expression of one or more transcription factors selected from OCT4, KLF4, SOX2, and any combination thereof; any of the chemical agents that activate OCT4, KLF4, and / or SOX2 (e.g., induce the expression of tetracycline); any of the engineered proteins encoding OCT4, SOX2, KLF4, or any combination thereof; any of the chemical agents that activate OCT4, KLF4, SOX2, or any combination thereof (e.g., induce the expression of tetracycline); any of the antibodies that activate (e.g., induce the expression of) OCT4, KLF4, and / or SOX2; and / or any of the recombinant viruses described herein (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV), may be administered intravitreally, intraocularly, subconjunctivally, or subretinaly, alone or in combination. In other embodiments, administration can be intravenous, intradermal, intraarterial, intralesional, intratumoral, intracranial, intraarticular, intraprostatic, intrapleural, intranasal, intravitreal, intravaginal, intrarectal, topically, intratumoral, intramuscular, intraperitoneal, subcutaneous, subconjunctival, intravesicular, mucosally, intrapericardially, intraumbilical, intraocular, oral, topical, locally, systemically, by injection, infusion, continuous infusion, localized perfusion directly into target cells, by catheter, cream, lipid composition (e.g., liposomes), or other method, or any combination of the above, as would be known to one of skill in the art (see, e.g., Remington's Pharmaceutical Sciences (1990), incorporated herein by reference). In some embodiments, compositions comprising a nucleic acid encoding an inducer, an engineered protein encoding an inducer, a chemical agent capable of modulating (e.g., activating or inhibiting) the activity of an inducer, and / or a recombinant virus encoding an inducer are also administered to cells, tissues, organs, and / or subjects using any suitable method, such as intravitreally, intraocularly, subconjunctivally, or subretinally.

[0194] How to Treat NAION Further provided herein are methods for treating non-arteritic anterior ischemic optic neuropathy (NAION) in a subject in need thereof, comprising administering to the subject one or more agents for upregulating OCT4, SOX2, KLF4, and / or a combination of one or more thereof. The one or more agents do not upregulate c-Myc. The one or more agents for upregulating OCT4, SOX2, KLF4 (OSK) may include one or more means for inducing expression of OSK, including DNA, RNA, small molecules, etc. In some embodiments, the method comprises administering to the subject one or more nucleic acid molecules contemplated herein. In some embodiments, the one or more nucleic acid molecules comprise a nucleic acid molecule system having at least two nucleic acid molecules.

[0195] In some embodiments of the method for treating NAION in a subject, the agent for upregulating OSK expression comprises at least one nucleic acid molecule encoding OSK as described herein above. The nucleic acid molecule encoding OSK does not comprise a nucleic acid sequence encoding c-myc. The nucleic acid molecule encoding OSK may be an adeno-associated virus (AAV) vector. According to some embodiments, the method further comprises administering to the subject a nucleic acid molecule encoding a reverse tetracycline-controlled transactivator (rtTA). The nucleic acid molecule encoding the reverse tetracycline-controlled transactivator (rtTA) may be an AAV vector. In some embodiments, the AAV vector comprising the nucleic acid molecule encoding the reverse tetracycline-controlled transactivator (rtTA) is not the same AAV vector as the AAV vector comprising the nucleic acid molecules encoding OCT4, SOX2, and KLF4.

[0196] The nucleic acid molecules encoding OCT4, SOX2, and KLF4 are operably linked to an inducible promoter. In some embodiments, the inducible promoter is induced by a tetracycline class antibiotic. Tetracycline class antibiotics are well known in the art and include, for example, tetracycline, chlortetracycline, oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, doxycycline, tigecycline, eravacycline, sarecycline, and omadacycline. Doxycycline is an exemplary tetracycline class antibiotic. In some embodiments, the inducible promoter is a tetracycline class antibiotic response element (TRE), including, for example, the TRE2 promoter.

[0197] The reverse tetracycline-controlled transactivator (rtTA) can be rtTA3, rtTA4, or a combination thereof. In some embodiments, the nucleic acid molecule encoding rtTA is operably linked to a constitutive promoter, including one or more of the following promoters: CP1, CMV, EF1 alpha, SV40, PGK1, Ubc, human beta-actin, CAG, Ac5, polyhedrin, TEF1, GDS, CaM3 5S, Ubi, H1, and / or U6. In some embodiments, the nucleic acid molecule encoding rtTA is operably linked to a CMV promoter.

[0198] In some embodiments, the AAV vector is serotype 2 (AAV2). In some embodiments, the AAV vector is a hybrid vector comprising capsid proteins from one or more serotypes, including AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, and AAV9 (e.g., AAV2 / 2, AAV2 / 6, AAV2 / 1, AAV2 / 5, AAV2 / 7, AAV2 / 8, and AAV2 / 9).

[0199] In some embodiments, the AAV vectors comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 comprise a self-cleaving peptide, for example, a 2A peptide.

[0200] Different AAV serotypes are used to optimize transduction of specific target cells or target specific cell types within specific target tissues (e.g., RGCs). AAV particles can contain viral proteins and viral nucleic acids of the same serotype or any natural or artificial sequence variant of AAV. For example, AAV particles can contain AAV2 capsid protein and at least one, preferably two AAV2 ITRs.

[0201] In some embodiments, AAV vectors comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 comprise inverted terminal repeats (ITRs) flanking a first nucleic acid. In some embodiments, AAV vectors comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 comprise inverted terminal repeats (ITRs) flanking a second nucleic acid. In some embodiments, AAV vectors comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 comprise inverted terminal repeats (ITRs) flanking a third nucleic acid. In some embodiments, AAV vectors comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 comprise inverted terminal repeats (ITRs) flanking one or more combinations of a first nucleic acid, a second nucleic acid, and / or a third nucleic acid. In some embodiments, the distance between two inverted terminal repeats (ITRs) is less than 5.0 kilobases (kb) (e.g., less than 4.9 kb, less than 4.8 kb, less than 4.7 kb, less than 4.6 kb, less than 4.5 kb, less than 4.4 kb, less than 4.3 kb, less than 4.2 kb, less than 4.1 kb, less than 4 kb, less than 3.5 kb, less than 3 kb, less than 2.5 kb, less than 2 kb, less than 1.5 kb, less than 1 kb, or less than 0.5 kb). In some embodiments, the distance between two ITRs is 4.7 kb or less.

[0202] The method may further comprise administering an inducer to the subject. The inducer may include, for example, tetracycline-controlled transactivator (tTA). In certain embodiments, the inducer can induce the expression of a first nucleic acid (e.g., OCT4), a second nucleic acid (e.g., SOX2), a third nucleic acid (e.g., KLF4), or any combination thereof from an inducible promoter in the absence of tetracycline (e.g., doxycycline).

[0203] In some embodiments, an AAV-OSK vector containing nucleic acid molecules encoding OCT4, SOX2, and KLF4 contains nucleic acid elements in a specific order. For example, an AAV vector containing nucleic acid molecules encoding OCT4, SOX2, and KLF4 can contain elements in the following order: a) a first inverted terminal repeat (ITR) sequence, b) a TRE2 promoter sequence, c) an OCT4 sequence, d) a P2A cleavage sequence, e) a SOX2 sequence, f) a T2A cleavage sequence, g) a KLF4 sequence, h) an SV-40-derived terminator sequence, and i) a second inverted terminal repeat (ITR) sequence, as described, for example, in U.S. Patent Application No. 17 / 280,384, published as International Publication No. WO2020 / 069373, entitled "CELLULAR REPROGRAMMING TO REVERSE AGING AND PROMOTE ORGAN AND TISSUE REGENERATION," which is incorporated herein by reference in its entirety.

[0204] In certain embodiments, the encoded OCT4 comprises an amino acid sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:2. In certain embodiments, the nucleic acid sequence encoding OCT4 is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:1. In certain embodiments, the encoded SOX2 comprises an amino acid sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:4. In certain embodiments, the nucleic acid sequence encoding SOX2 is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:3. In certain embodiments, the encoded KLF4 comprises an amino acid sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 6. In certain embodiments, the nucleic acid sequence encoding KLF4 is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) to SEQ ID NO: 5. In some embodiments, the nucleic acid sequence encoding OCT4 is SEQ ID NO: 1, the nucleic acid sequence encoding SOX2 is SEQ ID NO: 3, and the nucleic acid sequence encoding KLF4 is SEQ ID NO: 5.

[0205] In some embodiments, the P2A sequence encodes a polypeptide having the sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 9). In some embodiments, the P2A sequence is GCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCT (SEQ ID NO: 8).

[0206] In some embodiments, the T2A sequence encodes the polypeptide of SEQ ID NO: 11. In some embodiments, the T2A sequence is GAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCA (SEQ ID NO: 10).

[0207] In some embodiments, the TRE2 promoter sequence is SEQ ID NO: 7. In some embodiments, the TRE2 promoter sequence comprises at least one minimal CMV promoter sequence. In some embodiments, the at least one minimal SV40 promoter sequence is SEQ ID NO: 12.

[0208] In some embodiments, the terminator sequence from SV-40 is SEQ ID NO:12.

[0209] In some embodiments, the ITR sequence is SEQ ID NO:16.

[0210] In some embodiments, the nucleic acid molecule encoding OCT4, SOX2, and KLF4 comprises SEQ ID NO: 13 or 14. In some embodiments, the nucleic acid molecule encoding OCT4, SOX2, and KLF4 comprises SEQ ID NO: 15.

[0211] The AAV vectors containing nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the AAV vector containing a nucleic acid molecule encoding rtTA are administered sequentially or simultaneously.

[0212] The nucleic acid molecules disclosed herein can be administered to a subject by any suitable route, including, but not limited to, intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, topical, or intradermal routes. In certain embodiments, the compositions are formulated for administration by intravenous or subcutaneous injection. In some embodiments, a dual vector system comprising an AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 and an AAV vector comprising a nucleic acid molecule encoding rtTA is administered intravitreally.

[0213] In some embodiments, nucleic acid molecules encoding OCT4, SOX2, and KLF4 and nucleic acid molecules encoding rtTA (e.g., rtTA3, rtTA4, etc.) are administered at a ratio (nucleic acid molecule:nucleic acid molecule) of about 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.5, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1. In some embodiments, the nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the nucleic acid molecule encoding rtTA (e.g., rtTA3, rtTA4, etc.) are at a ratio of about 1:2, 1:1.9, 1:1.8, 1:1.7, 1:1.6, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, 1:1, 1:0.9, 1:0.8, 1:0.7, 1: In some embodiments, the nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the nucleic acid molecule encoding rtTA are administered in a ratio of about 1:1.

[0214] In some embodiments, the AAV vectors comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the AAV vectors comprising nucleic acid molecules encoding rtTA (e.g., rtTA3, rtTA4, etc.) are expressed at a ratio of about 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1 In some embodiments, the AAV vectors comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the AAV vector comprising a nucleic acid molecule encoding rtTA are administered at a ratio of about 1:1 (vg:vg). In some embodiments, an AAV vector comprising a nucleic acid molecule encoding OCT4, SOX2, and KLF4 and an AAV vector comprising a nucleic acid molecule encoding an rtTA (e.g., rtTA3, rtTA4, etc.) are administered at a ratio (vg / vg) of about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.5, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the AAV vectors comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the AAV vectors comprising nucleic acid molecules encoding rtTA (e.g., rtTA3, rtTA4, etc.) are expressed at a ratio of about 1:2, 1:1.9, 1:1.8, 1:1.7, 1:1.6, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, 1:1, 1:0.9, 1:0.8, 1:1.9 ... :0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, 1:0.1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1 or 2:1 ratio (vg / vg).In some embodiments, the AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the AAV vector comprising nucleic acid molecule encoding rtTA are administered in a ratio of about 1:1 (vg:vg). According to some embodiments of the disclosed methods, the AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 comprises SEQ ID NO: 13, 14, or 35. According to some embodiments, the AAV vector comprising nucleic acid molecule encoding rtTA comprises SEQ ID NO: 19, 26, 36, or 37. According to some embodiments of the disclosed methods, the AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 comprises SEQ ID NO: 13, 14, or 35, and the AAV vector comprising nucleic acid molecule encoding rtTA comprises SEQ ID NO: 19, 26, 36, or 37. According to some embodiments of the disclosed methods, the AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 comprises SEQ ID NO: 35, and the AAV vector comprising nucleic acid molecule encoding rtTA comprises SEQ ID NO: 36 or 37.

[0215] In some embodiments, the AAV vector comprising the nucleic acid molecules encoding OCT4, SOX2, and KLF4 is an AAV2-TRE-OSK vector, and the AAV vector comprising the nucleic acid molecule encoding rtTA is AAV2-CMV-rtTA3. The AAV composition may comprise the AAV2-TRE-OSK vector and the pAAV2-CMV-rtTA3VP16 vector. The method may comprise the AAV2-TRE-OSK vector and the pAAV2-CMV-rtTA3VP16 vector in the same or separate compositions.

[0216] In some embodiments, the AAV vector comprising the nucleic acid molecules encoding OCT4, SOX2, and KLF4 is an AAV2-TRE-OSK vector, and the AAV vector comprising the nucleic acid molecule encoding rtTA is AAV2-CMV-rtTA4. The AAV composition may comprise the AAV2-TRE-OSK vector and the AAV2-CMV-rtTA4 vector. The method may comprise the AAV2-TRE-OSK vector and the AAV2-CMV-rtTA4 vector in the same composition or in separate compositions.

[0217] The concentration of AAV2-TRE-OSK vector administered according to the disclosed methods is approximately 1 x 10 10 vg / mL ~ approx. 2×10 13 Amounts in the range of vg / mL, e.g., 1 x 10 12 vg / mL ~ approx. 2×10 12 For example, an effective amount of the AAV2-TRE-OSK vector may range from about 1.0 x 10 12 vg / mL ~ approx. 1.1×10 12 vg / mL, approximately 1.1×10 12 vg / mL ~ approx. 1.2×10 12 vg / mL, approximately 1.2×10 12 vg / mL ~ approx. 1.3×10 12 vg / mL, approximately 1.3×10 12 vg / mL ~ approx. 1.4×10 12 vg / mL, approximately 1.4×10 12 vg / mL ~ approx. 1.5×10 12 vg / mL, approximately 1.5×10 12 vg / mL ~ approx. 1.6×10 12 vg / mL, approximately 1.6×10 12 vg / mL ~ approx. 1.7×10 12 vg / mL, approximately 1.7×10 12 vg / mL ~ approx. 1.8×10 12 vg / mL, approximately 1.8×10 12 vg / mL ~ approx. 1.9×10 12 vg / mL, approximately 1.9×10 12 vg / mL ~ approx. 2.0×10 12 vg / mL, and any and all increments therebetween.

[0218] The concentration of pAAV2-CMV-rtTA3VP16 vector administered according to the disclosed methods is approximately 1 x 10 10 vg / mL ~ approx. 2×10 13 Amounts in the range of vg / mL, e.g., 1 x 10 13 vg / mL ~ approx. 2×10 13 For example, an effective amount of the pAAV2-CMV-rtTA3VP16 vector may range from about 1.0 x 10 13 vg / mL ~ approx. 1.1×10 13 vg / mL, approximately 1.1×10 13 vg / mL ~ approx. 1.2×10 13 vg / mL, approximately 1.2×10 13 vg / mL ~ approx. 1.3×10 13 vg / mL, approximately 1.3×10 13 vg / mL ~ approx. 1.4×10 13 vg / mL, approximately 1.4×10 13 vg / mL ~ approx. 1.5×10 13 vg / mL, approximately 1.5×10 13 vg / mL ~ approx. 1.6×10 13 vg / mL, approximately 1.6×10 13 vg / mL ~ approx. 1.7×10 13 vg / mL, approximately 1.7×10 13 vg / mL ~ approx. 1.8×10 13 vg / mL, approximately 1.8×10 13 vg / mL ~ approx. 1.9×10 13 vg / mL, approximately 1.9×10 13 vg / mL ~ approx. 2.0×10 13 vg / mL, and any and all increments therebetween.

[0219] The concentration of AAV2-CMV-rtTA4 vector administered according to the disclosed methods is approximately 1 x 10 10 vg / mL ~ approx. 2×10 13 vg / mL, e.g., about 1 x 10 13 vg / mL ~ approx. 2×10 13 For example, an effective amount of the AAV2-CMV-rtTA4 vector may range from about 1.0 x 10 13vg / mL ~ approx. 1.1×10 13 vg / mL, approximately 1.1×10 13 vg / mL ~ approx. 1.2×10 13 vg / mL, approximately 1.2×10 13 vg / mL ~ approx. 1.3×10 13 vg / mL, approximately 1.3×10 13 vg / mL ~ approx. 1.4×10 13 vg / mL, approximately 1.4×10 13 vg / mL ~ approx. 1.5×10 13 vg / mL, approximately 1.5×10 13 vg / mL ~ approx. 1.6×10 13 vg / mL, approximately 1.6×10 13 vg / mL ~ approx. 1.7×10 13 vg / mL, approximately 1.7×10 13 vg / mL ~ approx. 1.8×10 13 vg / mL, approximately 1.8×10 13 vg / mL ~ approx. 1.9×10 13 vg / mL, approximately 1.9×10 13 vg / mL ~ approx. 2.0×10 13 vg / mL, and any and all increments therebetween.

[0220] According to some embodiments, an effective amount of the AAV2-TRE-OSK vector administered according to the disclosed methods comprises an amount of vector administered per eye. 9 vg / eye ~ approx. 1×10 14 In some embodiments, an effective amount of an AAV2-TRE-OSK vector may comprise an amount of vector in the range of about 1 x 10 vg / eye. 11 vg / eye ~ approx. 10×10 11 For example, a dose of the AAV2-TRE-OSK vector may be about 1 x 10 11 vg / eye ~ approx. 2×10 11 vg / eye, approx. 2×10 11 vg / eye ~ approx. 3×10 11 vg / eye, approx. 3 x 10 11 vg / eye ~ approx. 4×10 11 vg / eye, approx. 4 x 10 11 vg / eye ~ approx. 5×10 11vg / eye, approx. 5 x 10 11 vg / eye ~ approx. 6×10 11 vg / eye, approx. 6 x 10 11 vg / eye ~ approx. 7×10 11 vg / eye, approx. 7 x 10 11 vg / eye ~ approx. 8×10 11 vg / eye, approx. 8 x 10 11 vg / eye ~ approx. 9×10 11 vg / eye, approx. 9 x 10 11 vg / eye ~ approx. 10×10 11 vg / eye, including any and all increments therebetween. In some embodiments, the effective dose of the AAV2-TRE-OSK vector is about 3.06 x 10 11 vg / eye.

[0221] According to some embodiments, an effective amount of the AAV2-TRE-OSK vector administered according to the disclosed methods comprises an amount of vector administered per eye. 9 vg / eye ~ approx. 1×10 12 In some embodiments, an effective amount of an AAV2-TRE-OSK vector may comprise an amount of vector in the range of about 1 x 10 vg / eye. 9 vg / eye ~ approx. 1×10 12 For example, a dose of the AAV2-TRE-OSK vector may be about 1 x 10 9 vg / eye ~ approx. 4×10 9 vg / eye, approx. 4 x 10 9 vg / eye ~ approx. 7×10 9 vg / eye, approx. 7 x 10 9 vg / eye ~ approx. 1×10 10 vg / eye, approx. 1×10 10 vg / eye ~ approx. 4×10 10 vg / eye, approx. 4 x 10 10 vg / eye ~ approx. 7×10 10 vg / eye, approx. 7 x 10 10 vg / eye ~ approx. 1×10 11 vg / eye, approx. 1×10 11 vg / eye ~ approx. 4×10 11 vg / eye, approx. 4 x 10 11 vg / eye ~ approx. 7×10 11 vg / eye, approx. 7 x 1011 vg / eye ~ approx. 1×10 12 vg / eye, including any and all increments therebetween. In some embodiments, the effective dose of the AAV2-TRE-OSK vector is about 3.06 x 10 11 vg / eye.

[0222] An effective amount of the pAAV2-CMV-rtTA3VP16 vector administered according to the disclosed methods includes the amount of vector administered per eye. An effective amount of the pAAV2-CMV-rtTA3VP16 vector is approximately 1 x 10 9 vg / eye ~ approx. 1×10 14 In some embodiments, an effective amount of the pAAV2-CMV-rtTA3VP16 vector may comprise an amount of vector in the range of about 1 x 10 vg / eye. 11 vg / eye ~ approx. 10×10 11 For example, the dose of the pAAV2-CMV-rtTA3VP16 vector may be approximately 1 x 10 11 vg / eye ~ approx. 2×10 11 vg / eye, approx. 2×10 11 vg / eye ~ approx. 3×10 11 vg / eye, approx. 3 x 10 11 vg / eye ~ approx. 4×10 11 vg / eye, approx. 4 x 10 11 vg / eye ~ approx. 5×10 11 vg / eye, approx. 5 x 10 11 vg / eye ~ approx. 6×10 11 vg / eye, approx. 6 x 10 11 vg / eye ~ approx. 7×10 11 vg / eye, approx. 7 x 10 11 vg / eye ~ approx. 8×10 11 vg / eye, approx. 8 x 10 11 vg / eye ~ approx. 9×10 11 vg / eye, approx. 9 x 10 11 vg / eye ~ approx. 10×10 11 vg / eye, including any and all increments therebetween. In some embodiments, the effective dose of the pAAV2-CMV-rtTA3VP16 vector is about 2.66 x 10 11 vg / eye.

[0223] An effective amount of the pAAV2-CMV-rtTA3VP16 vector administered according to the disclosed methods includes the amount of vector administered per eye. An effective amount of the pAAV2-CMV-rtTA3VP16 vector is approximately 1 x 10 9 vg / eye ~ approx. 1×10 12 In some embodiments, an effective amount of the pAAV2-CMV-rtTA3VP16 vector may comprise an amount of vector in the range of about 1 x 10 vg / eye. 9 vg / eye ~ approx. 1×10 12 For example, the dose of the pAAV2-CMV-rtTA3VP16 vector may be approximately 1 x 10 9 vg / eye ~ approx. 4×10 9 vg / eye, approx. 4 x 10 9 vg / eye ~ approx. 7×10 9 vg / eye, approx. 7 x 10 9 vg / eye ~ approx. 1×10 10 vg / eye, approx. 1×10 10 vg / eye ~ approx. 4×10 10 vg / eye, approx. 4 x 10 10 vg / eye ~ approx. 7×10 10 vg / eye, approx. 7 x 10 10 vg / eye ~ approx. 1×10 11 vg / eye, approx. 1×10 11 vg / eye ~ approx. 4×10 11 vg / eye, approx. 4 x 10 11 vg / eye ~ approx. 7×10 11 vg / eye, approx. 7 x 10 11 vg / eye ~ approx. 1×10 12 vg / eye, including any and all increments therebetween. In some embodiments, the effective dose of the pAAV2-CMV-rtTA3VP16 vector is about 2.66 x 10 11 vg / eye.

[0224] An effective amount of the AAV2-CMV-rtTA4 vector administered according to the disclosed methods comprises the amount of vector injected per eye. An effective amount is about 1 x 10 9 vg / eye ~ approx. 1×10 14In some embodiments, an effective amount of the AAV2-CMV-rtTA4 vector may comprise an amount of vector in the range of about 1 x 10 vg / eye. 11 vg / eye ~ approx. 10×10 11 For example, a dose of AAV2-CMV-rtTA4 vector may be in the range of about 1 x 10 vg / eye. 11 vg / eye ~ approx. 2×10 11 vg / eye, approx. 2×10 11 vg / eye ~ approx. 3×10 11 vg / eye, approx. 3 x 10 11 vg / eye ~ approx. 4×10 11 vg / eye, approx. 4 x 10 11 vg / eye ~ approx. 5×10 11 vg / eye, approx. 5 x 10 11 vg / eye ~ approx. 6×10 11 vg / eye, approx. 6 x 10 11 vg / eye ~ approx. 7×10 11 vg / eye, approx. 7 x 10 11 vg / eye ~ approx. 8×10 11 vg / eye, approx. 8 x 10 11 vg / eye ~ approx. 9×10 11 vg / eye, approx. 9 x 10 11 vg / eye ~ approx. 10×10 11 vg / eye, including any and all increments therebetween. In some embodiments, the effective amount of AAV2-CMV-rtTA4 vector is about 2.66 x 10 11 vg / eye.

[0225] An effective amount of the AAV2-CMV-rtTA4 vector administered according to the disclosed methods comprises the amount of vector injected per eye. An effective amount is about 1 x 10 9 vg / eye ~ approx. 1×10 12 In some embodiments, an effective amount of the AAV2-CMV-rtTA4 vector may comprise an amount of vector in the range of about 1 x 10 vg / eye. 9 vg / eye ~ approx. 1×10 12 For example, a dose of AAV2-CMV-rtTA4 vector may be in the range of about 1 x 10 vg / eye. 9 vg / eye ~ approx. 4×10 9 vg / eye, approx. 4 x 109 vg / eye ~ approx. 7×10 9 vg / eye, approx. 7 x 10 9 vg / eye ~ approx. 1×10 10 vg / eye, approx. 1×10 10 vg / eye ~ approx. 4×10 10 vg / eye, approx. 4 x 10 10 vg / eye ~ approx. 7×10 10 vg / eye, approx. 7 x 10 10 vg / eye ~ approx. 1×10 11 vg / eye, approx. 1×10 11 vg / eye ~ approx. 4×10 11 vg / eye, approx. 4 x 10 11 vg / eye ~ approx. 7×10 11 vg / eye, approx. 7 x 10 11 vg / eye ~ approx. 1×10 12 vg / eye, including any and all increments therebetween. In some embodiments, the effective amount of AAV2-CMV-rtTA4 vector is about 2.66 x 10 11 vg / eye.

[0226] Method embodiments include administering to a subject an AAV composition comprising an AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 and an AAV vector comprising a nucleic acid molecule encoding rtTA via one or more appropriate routes, including left eye (OS) injection, right eye (OD) injection, or both eyes (OU) injection. Administration may include one or more injections; for example, administration may include one injection comprising an AAV composition comprising an AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 and an AAV vector comprising a nucleic acid molecule encoding rtTA. In other aspects, administration may include two (or more) injections, for example, one injection comprising an AAV composition comprising an AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4, and one injection comprising an AAV vector comprising a nucleic acid molecule encoding rtTA. The injections comprising the AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the injections comprising the AAV vector comprising a nucleic acid molecule encoding rtTA may be administered sequentially or simultaneously. For example, an injection containing an AAV composition comprising an AAV vector comprising a nucleic acid molecule encoding OCT4, SOX2, and KLF4 can be administered before, simultaneously with, or after an injection containing an AAV vector comprising a nucleic acid molecule encoding rtTA.

[0227] Method embodiments further include administering to the subject an effective amount of an antibiotic. In some embodiments, the antibiotic comprises tetracycline or doxycycline. In some embodiments, the antibiotic is administered at least one day before administering the AAV vector comprising the nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the AAV vector comprising the nucleic acid molecule encoding rtTA.

[0228] When administering an AAV vector comprising a nucleic acid molecule encoding OCT4, SOX2, and KLF4 and an AAV vector comprising a nucleic acid molecule encoding rtTA, an antibiotic may be administered. The antibiotic may be administered at least one day after administration of an AAV composition comprising an AAV vector comprising a nucleic acid molecule encoding OCT4, SOX2, and KLF4 and an AAV vector comprising a nucleic acid molecule encoding rtTA. The antibiotic may be administered two, three, four, five, six, or more days after administration of an AAV composition comprising an AAV vector comprising a nucleic acid molecule encoding OCT4, SOX2, and KLF4 and an AAV vector comprising a nucleic acid molecule encoding rtTA.

[0229] The disclosed methods for treating NAION include administering to a subject in need thereof an effective amount of an AAV genome comprising one or more nucleic acid molecule sequences expressing OCT4, SOX2, and KLF4, and an AAV genome comprising a nucleic acid sequence encoding transactivator 3 or transactivator 4.

[0230] AAV-OSK vector preparation method Provided herein are methods for the recombinant preparation of AAV. In some embodiments, the methods include introducing one or more vectors contemplated herein into cells under conditions such that AAV is produced. The cells may include a population of cells. The population of cells may include any suitable cells understood in the art, including, for example, HEK293 cells, HEK293T cells, COS cells, CHO cells, BHK cells, HeLa cells, etc. The one or more vectors may be introduced into the cells using one or more suitable techniques, including, for example, transfection, transduction, and / or infection. Exemplary methods for recombinant preparation of AAV include transient transfection (e.g., with one or more transfer plasmids comprising a first vector, a second vector, and optionally a third vector described herein), viral infection (e.g., with one or more recombinant helper viruses, e.g., adenovirus, poxvirus (such as vaccinia virus), herpesvirus (including HSV, cytomegalovirus, or baculovirus comprising a first vector, a second vector, and optionally a third vector described herein)), and transfection or infection of a stable producer cell line (e.g., a stable producer cell, such as a mammalian or insect cell, comprising a Rep nucleotide sequence encoding one or more AAV Rep proteins and / or a Cap nucleotide sequence encoding one or more AAV capsid proteins described herein, and an AAV genome delivered in the form of a plasmid or recombinant helper virus described herein). The first vector may comprise one or more nucleic acid sequences expressing OCT4, SOX2, and / or KLF4 encoded by one or more of SEQ ID NOs: 13, 14, 15, and 35. The second vector may comprise one or more nucleic acid sequences expressing transactivator 3, e.g., SEQ ID NO: 21 or 36. Alternatively, the second vector may comprise one or more nucleic acid sequences expressing transactivator 4, e.g., SEQ ID NO: 28 or 37.

[0231] Also provided herein is a method for producing AAV, comprising modifying a cell to express one or more plasmids. The one or more plasmids may include one or more AAV2 Rep-Cap plasmids, one or more helper plasmids, and one or more transfer plasmids. The one or more transfer plasmids may include a first transfer plasmid containing one or more nucleic acids encoding one or more of OCT4, SOX2, and KLF4, a second transfer plasmid containing one or more nucleic acids encoding transactivator 3, or a transfer plasmid containing one or more nucleic acids encoding one or more of OCT4, SOX2, KLF4, and transactivator 3. The one or more transfer plasmids may also include a first transfer plasmid containing one or more nucleic acids encoding one or more of OCT4, SOX2, and KLF4, a second transfer plasmid containing one or more nucleic acids encoding transactivator 4, or a transfer plasmid containing one or more nucleic acids encoding one or more of OCT4, SOX2, KLF4, and transactivator 4.

[0232] definition The singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0233] As used herein, the term "about" refers to a range of values ​​that includes the specified value and that would be reasonably considered by a person skilled in the art to be comparable to the specified value. For example, about refers to within a standard deviation using measurements generally accepted in the art. For example, about refers to a range of up to ±10%, ±5%, ±3%, or ±1% of the specified value.

[0234] The term "at least" followed by a number is used herein to indicate the start of a range beginning with that number (which may be an open-ended or open-ended range, depending on the variable being defined). For example, "at least 1" means 1 or more than 1.

[0235] The term "up to" followed by a number is used herein to indicate the end of a range ending with that number (which may be a range with a lower limit of 1 or 0, or a range with no lower limit, depending on the variable being defined). For example, "up to 4" means 4 or less than 4, and "up to 40%" means 40% or less than 40%. When a range is given herein as "(first number) to (second number)" or "(first number) to (second number)," this means a range whose lower limit is the first number and whose upper limit is the second number. For example, 25 to 100 mm means a range whose lower limit is 25 mm and whose upper limit is 100 mm.

[0236] The term "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a certain percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence, without considering conservative amino acid substitutions as part of sequence identity. Methods for aligning sequences for comparison are well known in the art. After alignment, the number of matches is determined by counting the number of positions where identical nucleotides or amino acid residues exist in both sequences. The percent sequence identity is determined by dividing the number of matches in the alignment by the length of the reference sequence and then multiplying the result by 100. For example, a peptide sequence that has 1166 matches when aligned with a reference sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166 ÷ 1554 × 100 = 75.0). As used herein, gaps in the alignment do not reduce the percent sequence identity. Unless otherwise specified, optimal alignment of sequences for comparison is performed by the global alignment algorithm of Needleman and Wunsch, Mol. Biol. 48:443 (1970) implemented by EMBOSS Needle (World Wide Web ebi.ac.uk / Tools / psa / emboss_needle / ) (Madeira et al. Nucleic Acids Res. 50(W1):W276-W279(2022)).Other alignment methods may be used, including, but not limited to, Devereux, et al., Nucleic Acids Res. 12:387-95 (1984), Atschul et al. J. Mo. Biol. 215:403-10 (1990) (BLAST), Carrillo and Lipman Siam J. Appl. Math. 48(5) (1988), Computational Molecular Biology (Lesk, AM, ed., 1989), Biocomputing Informatics and Genome Projects, (Smith, DW, ed., 1993), Computer Analysis of Sequence Data, Part I, (Griffin and Griffin, eds., 1994), Sequence Analysis in Molecular Biology (von Heinje, 2012), Sequence Analysis Primer (Gribskov and Devereux, J., eds. 1993). Sequence identity is calculated using the Needleman-Wunsch implementation provided by the National Library of Medicine (World Wide Web blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=GlobalAln).

[0237] For example, sequence identity can be determined by standard methods commonly used to compare the similarity of two polypeptides or two polynucleotide sequences. Using a computer program such as EMBOSS Needle or BLAST, two polypeptides or two polynucleotide sequences are aligned for optimal matching of their respective residues (along the entire length of one or both sequences, or along a predetermined portion of one or both sequences). The program provides a default opening penalty and a default gap penalty, as well as a scoring matrix such as PAM250 (a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, sup.3 (1978)) that can be used in conjunction with the computer program.

[0238] Throughout this specification and the claims that follow, unless the context requires otherwise, the terms "comprise," and variations such as "comprises" and "comprising," as well as "has" or "having" or "includes" or "including," are to be understood to imply the inclusion of a stated element or step or group of elements or steps, but not the exclusion of any other element or step or group of elements or steps. "Consisting essentially of" or "consists essentially of" indicates the exclusion of elements or steps that materially affect the basic and novel characteristics of the claimed invention.

[0239] "Subjects" to which administration is contemplated include, but are not limited to, humans (i.e., male or female of any age, e.g., a pediatric subject (e.g., infant, child, adolescent) or an adult subject (e.g., young adult, middle-aged adult, or elderly adult)) and / or other non-human animals, such as mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), commercially relevant mammals (including, e.g., cows, pigs, horses, sheep, goats, cats, and / or dogs) and birds (including, e.g., commercially relevant birds, e.g., chickens, ducks, geese, and / or turkeys). In certain embodiments, the animal is a mammal. The animal may be male or female and at any stage of development. The non-human animal may be a transgenic animal. array [Table 3] JPEG2026501545000005.jpg205159JPEG2026501545000006.jpg196159JPEG2026501545000007.jpg198159JPEG2026501545000008.j pg197159JPEG2026501545000009.jpg201159JPEG2026501545000010.jpg196159JPEG2026501545000011.jpg201159JPEG2026501545 000012.jpg194159JPEG2026501545000013.jpg199159JPEG2026501545000014.jpg198159JPEG2026501545000015.jpg198159JPEG20 26501545000016.jpg196159JPEG2026501545000017.jpg198159JPEG2026501545000018.jpg198159JPEG2026501545000019.jpg42159 [Table 4] JPEG2026501545000021.jpg200159JPEG2026501545000022.jpg201159JPEG2026501545000023.jpg202159 JPEG2026501545000024.jpg201159JPEG2026501545000025.jpg203159JPEG2026501545000026.jpg121159 [Table 5] JPEG2026501545000028.jpg205159JPEG2026501545000029.jpg202159JPEG2026501545000030.jpg19915 9JPEG2026501545000031.jpg208159JPEG2026501545000032.jpg199159JPEG2026501545000033.jpg96159 [Example]

[0240] Example 1 - Methods for producing vectors This study aimed to develop a method for producing batches of vector and to evaluate the stability of the prepared batches.

[0241] AAV production protocol material cell: HEK293T cells were used to generate vectors as described herein. Cells were grown in DMEM medium (Invitrogen, Catalog No. 11995073) containing 10% fetal bovine serum (FBS) (Invitrogen HI FBS, Catalog No. 16140), penicillin / streptomycin (Invitrogen, Catalog No. 15140-122), and glutamine (Invitrogen, Catalog No. 25030).

[0242] Plasmids: The recombinant AAV2-TRE-OSK plasmid (shown in Figure 1) was prepared using the following components: (1) a nucleic acid sequence encoding an AAV2 capsid protein or fragment thereof, (2) a nucleic acid encoding a functional rep gene, (3) a recombinant AAV transfer vector containing AAV2 inverted terminal repeats (SEQ ID NO: 16, SEQ ID NO: 32) flanking a transgene (SEQ ID NO: 13) encoding OCT4, KLF4, and SOX2 operably linked to an inducible TRE promoter (TRE3G, SEQ ID NO: 7), and (4) a helper vector carrying the rAAV2 Rep-Cap protein. Plasmids were obtained from Stratagene / Agilent (Stratagene catalog number: 240071). AAV2 Rep-Cap plasmids included the pAAV-RC plasmid (Stratagene catalog number 240071). In some cases, AAV2 hybrid vectors were used, such as AAV2 / 1, AAV2 / 2, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, and AAV2 / 9, which contain capsid proteins from AAV1, 2, 3, 5, 6, 7, 8, and 9 serotypes. The helper plasmids included pHelper plasmids (Stratagene, catalog no. 240071), which carried adenovirus-derived genes for introducing helper functions. As shown in Figure 1, the entire AAV2-TRE3G-OSK-SV40pA vector is 7250 base pairs in length, with two inverted terminal repeats (ITRs) flanking the OSK sequence.

[0243] The first expression vector encoding OCT4, SOX2, and KLF4 comprises the nucleic acid sequence set forth in SEQ ID NO: 15. The recombinant AAV vector may comprise a nucleic acid encoding an inducing agent.

[0244] The recombinant pAAV2-CMV-rtTA for Tet-On plasmid, pAAV2-CMV-rtTA3VP16 (shown in Figure 2) was prepared using a similar approach as above, but using a transfer plasmid with a CMV constitutive promoter (SEQ ID NO: 17) operably linked to the rtTA3 (SEQ ID NO: 19) inducer with three vp16 domains at its 3' end. An alternative recombinant pAAV2-CMV-rtTA, pAAV2-CMV-rtTA4(V16) (shown in Figure 3) may be prepared similarly.

[0245] PEI solution (1 μg / μl, Polysciences, catalog number 23966-2) was prepared by dissolving PEI powder in water, heated to 80° C., cooled to room temperature, neutralized to pH 7.0, filter-sterilized, aliquoted, and stored at −20° C. Transfection efficiency was tested as each new batch was prepared.

[0246] method Vector Generation: On day 1, HEK293T cells were plated onto ten 15 cm dishes and transfected for 24 hours. Cells were seeded in 25 mL of medium per 15 cm dish. To obtain the 5E12 viral genome (vg) yield, cells were split ten times onto 15 cm dishes to a density of 70-90% (standard transfection density). To prevent cell death, the medium was replaced with 5% FBS to slow growth and reduce the purification time to one dish per split.

[0247] On day 2, 1 hour prior to transfection, the medium was replaced with 20 mL of freshly warmed medium. DNA solutions were prepared in 50 mL FALCON® tubes. The amounts of DNA and reagents per dish are shown in Table 3 below. The amount of DNA was calculated based on the inverted terminal repeat (ITR) size of the plasmid. Tubes of master mix were prepared for five 15 cm dishes. All plasmids were diluted to 1 μg / μl in sterile water as shown in Table 3 below.

[0248] A 10 mL sample of DMEM (phenol red-free, Invitrogen catalog number 31053-036) was prepared. A 785 µL aliquot of the PEI solution was added and the medium was mixed. This was then incubated at room temperature for 20-30 minutes. [Table 6]

[0249] Next, 2 mL of the DNA-PEI mixture was added to each of ten 15 cm dishes. The transfected cells were then incubated.

[0250] On day 3, the medium was removed and 25 mL of freshly warmed medium was added. Serum-containing medium was added for AAV2 / 2 and AAV2 / 6. Serum-free medium was used for AAV2 / 1, AAV2 / 5, AAV2 / 7, AAV2 / 8, and AAV2 / 9.

[0251] On day 5, the cells were scraped off with a cell scraper and transferred to a 50 mL tube. The cell suspension was then centrifuged at 1000 relative centrifugal force (rcf) for 5 minutes. The supernatant was then discarded.

[0252] Transfection Procedure: All cells were combined into one 50 mL tube, washed with PBS, and centrifuged at 1000 rpm for 5 minutes, and then the supernatant was discarded.

[0253] The cell pellet was resuspended in a volume of hypotonic buffer five times the packed cell volume (approximately 25 mL). It was then incubated on ice for 10 minutes. Next, 0.11% by volume of 10x renaturation buffer was added and mixed by pipetting (typically a volume of 3.3 mL).

[0254] The nuclei were then spun down at 2000 rcf for 10 minutes, yielding approximately 1 mL of nuclear pellet per ten 15 cm dishes, which were stored at -80°C for further purification.

[0255] Preparation of solutions: 5M NaCl salt solution A mass of 292.2 g of NaCl was added to 200 ml of deionized (DI) water in a 2 L bottle. The mixture was shaken to mix and poured into a large graduated cylinder. DI water was then added to 1 L and poured back into the 2 L bottle. A stir bar was added, and the volume was heated and stirred until dissolved. Alternatively, the volume was autoclaved. The solution was then cooled and sterile filtered through a 0.2 μm filter.

[0256] 40% PEG-8000, 2.5M NaCl (5x stock solution) In a 1 L graduated cylinder, 400 g of PEG-8000 and 500 mL of 5 M NaCl were mixed. Deionized water was added to 1 L. This typically required approximately 100 mL of water. The solution was transferred to a 2 L flask and shaken to mix. A large stir bar was added, and the solution was heated in a 55°C water bath overnight. The next day, the solution was sterile filtered through a 0.2 μm filter. This filtration step typically takes approximately 30 minutes.

[0257] Recovery of AAV from media and cells: The media and cells were harvested without the use of trypsin. The media was collected with a Pipetman and sterile filtered through a 0.2 μm filter. The cells were collected and spun down. All remaining supernatant was added to the media, which had been sterile filtered through a 0.2 μm filter. Cells were harvested in one of several ways. Cells were harvested with a cell scraper. Alternatively, cells were "blasted" with calcium- and magnesium-free PBS at a volume of 10 mL per two dishes. A 40% solution of PEG 8000, adjusted to pH 7.4, was added to a final concentration of 12%. Approximately 25 mL was used per 100 mL of media / cells. The solution was stirred in a cold room for 1 hour and left without rotation for 3 hours or left overnight in the cold room. The next day, the PEG mixture was centrifuged at 3000 x g for 20 minutes. The supernatant was discarded. The pellet was resuspended in approximately 7 mL or less of 1x PBS. The pellet was initially suspended in approximately 5 mL, and then a maximum volume of 2 mL was added once it was mostly suspended. A 1:10,000 benzonase solution was then added and the mixture was incubated for 45 minutes at 37° C. If desired, the mixture was spun down at 2415×g for 10 minutes at 4° C. The supernatant was transferred to a new tube.

[0258] Concentration of AAV by ultracentrifugation: Preparation of solutions A 1000x stock solution of 1 M MgCl2 was prepared and used to make MK buffer. A 1000x stock solution of 2.5 M KCl was prepared and also used to make MK buffer.

[0259] A 1M solution of NaCl / phosphate buffered saline (PBS) MK buffer was prepared by dissolving 58.4 g of NaCl in 1 mL of 1 M MgCl and 1 mL of 2.5 M KCl. Then, 1x PBS (Ca - Mg - Dulbecco's phosphate buffered saline (DPBS, Gibco) was added to a final volume of 1 L. The solution was sterilized by passing it through a 0.22 μm filter and stored at 4° C. The solution was brought to a final concentration of 1 M NaCl, 1 mM MgCl, and 2.5 mM KCl.

[0260] 1x PBS-MK buffer was prepared by dissolving 1 mL of 1 M MgCl2 and 1 mL of 2.5 M KCl in two 500 mL bottles of Ca-Mg-DPBS (Gibco). The solution was sterilized by passing through a 0.22 μm filter and stored at 4°C. The solution was brought to a final concentration of 1 mM MgCl2 and 2.5 mM KCl.

[0261] A 0.001% Pluronic-F68 (formulation buffer) solution was prepared by adding 500 μL of sterile 1000X Pluronic F-68 (1% solution) to 500 mL of 1X DPBS (Gibco, TC stock). The solution was stored at 4°C for up to 1 month or aliquoted and stored at -80°C for up to 1 year.

[0262] A 0.001% PLURONIC®-F68 + 5% sorbitol (freezing buffer) solution was prepared by adding 25 g of sorbitol to 500 μL of sterile 1000× PLURONIC® F-68 (1% solution) and adding it to 500 mL of 1× DPBS (Gibco, TC stock). The solution was stored at 4°C for up to 1 month or aliquoted and stored at -80°C for up to 1 year.

[0263] As shown in Table 4, a 15% iodixanol solution was prepared by mixing 30 mL of 60% iodixanol with 90 mL of 1 M NaCl / PBS-MK buffer. A 25% iodixanol solution was prepared by mixing 112.5 mL of 60% iodixanol, 157.5 mL of 1x PBS-MK buffer, and 900 μL of phenol red. A 40% iodixanol solution was prepared by mixing 202.5 mL of 60% iodixanol with 67.5 mL of 1x PBS-MK buffer. A 60% iodixanol solution was prepared by mixing 150 mL of 60% iodixanol with 675 μL of phenol red. [Table 7] Note that all solutions were sterile filtered through a 0.2 um filter.

[0264] To create the supergradient, the benzonase-treated supernatant was added to a Beckman Optiseal tube. If the volumes were not equal, 1 M NaCl, PBS-MK mix was added to equalize them to a final volume of approximately 7 mL.

[0265] The tubes were filled from the bottom using a 10 mL syringe and a long hypodermic needle. The same syringe was reused for each solution, but the needle was changed for each sample to prevent cross-contamination between AAV preparations. A minimum volume of 5 mL was used for any layer.

[0266] The tubes were equilibrated to 5-10 μg. The tubes were balanced in pairs. First, they were sorted so that the most similar tubes were paired together. Then, PBS was added to the lighter tube of each pair. The PBS was added by attaching the tip to the side of the tube to prevent droplets from disturbing the layer. The cap was replaced, and the tube was loaded into the ultracentrifuge.

[0267] The tubes were then spun in an ultracentrifuge at maximum speed (50,000 rpm = 242,000 x g) for 1 hour using a VTi50 rotor. Fractions were collected from the ultracentrifuge tubes by puncturing the bottom with an 18-gauge needle. The black stopper at the top was removed before puncturing with the needle; otherwise, air bubbles would form that would disrupt the layers. The majority of the 60% fraction was removed. The remainder of the 60% layer was then collected in a 50 mL tube along with the 40% fraction. Sample collection was complete when there was a significant change in color or the solution became cloudy.

[0268] Separation of fractions on protein gels Samples were denatured with 4x LDS containing 2.5% β-mercaptoethanol at 70°C for 10 minutes. To prepare 20 μL aliquots, 10 μL of sample was mixed with 5 μL of water and 5 μL of LDS containing 2.5% β-mercaptoethanol. Additional running buffer (200 mL of 10x Tris-glycine SDS buffer, 1800 mL of millipure water) was prepared as needed.

[0269] The samples were then loaded onto a Tris-glycine gel. Either a 4-12% or a 4-20% gradient gel was used; either was suitable for AAV capsids.

[0270] Assemble the gel box and load 20 µL of sample into the wells of the gel. Run the gel at 225 V for 30-45 minutes until the blue dye reaches the bottom of the gel.

[0271] The gel was then stained with SYPRO® Red and imaged. First, a staining solution was prepared by adding 10 μL of SYPRO® Red to 50 mL of staining solution (7.5% acetic acid, 5000x SYPRO® Red). This was sufficient for one gel. The gel was stained for 1 hour, covered, at room temperature on a slow shaker. The staining solution was then removed and dye-free acetic acid solution was added. The gel was destained by incubating for 1-5 minutes. The gel was imaged on a gel dock using the EtBr setting (UV).

[0272] The good fractions were then combined and washed with 1x PBS containing 0.001% F68. In some cases, approximately 20 mL of PBS+F68 was added to bring the final volume to just under 30 mL to dilute the iodixanol, which facilitates passage through the filter.

[0273] The samples were centrifuged at 4700 g for 5 minutes. After everything was through, the samples were further washed three times with 15 mL per wash to prepare a clear final solution. The samples were then aliquoted into labeled tubes (virus name, payload, date) and stored at 4°C for up to one week. For longer storage, a final eluate was prepared in 1x PBS containing 0.001% F68 and 5% sorbitol. The samples were frozen at -80°C.

[0274] Viral titer determination by qPCR Viral titers can be determined by various molecular biology techniques, including quantitative PCR (qPCR). Viral titers can be reported as genome copies (GC) per ml or vector genomes (VG) per ml. GC per ml is equivalent to VG per ml.

[0275] AAV2-TRE-OSK virus was analyzed and titer quantified by qPCR using the following primers: TRE3G F:AACGTATCTACAGTTTACTCCCTATC and TRE3G R:GGTAGGAAGTGGTACGGAAAG. The titer of the batch used was 1.53 x 10 12 Intraocular injection of 200 μL of AAV2-TRE-OSK was found to yield approximately 2.66 × 10 vg / mL per eye. 11 Equivalent to vg.

[0276] The pAAV2-CMV-rtTA3VP16 virus was analyzed and the titer quantified by qPCR using the following primers: WPRE F: CACTGACAATTCCGTGGTGT and WPRE R: GAGATCCGACTCGTCTGAGG. The titer of the batch used was 1.33 x 10 13 Intraocular injection of 20 μL of pAAV2-CMV-rtTA3VP16 resulted in approximately 2.11 × 10 vg / mL per eye. 11 Equivalent to vg.

[0277] Samples were prepared by aliquoting 12.5 μl of master mix into tubes. Either ThermoFisher's fast advanced TAQMAN® Master Mix or IDT Primetime Master Mix was used. To each tube, 0.0625 μl of primer 1, 0.0625 μl of primer 2, 0.125 μl of probe, 1 μl of virus, and 11.3 μl of water were added for a total volume of 25 μl.

[0278] Example 2 - Methods for treating NAION in non-human primates This study aimed to determine whether epigenetic reprogramming improves RGC function and restores visual function (pERG) in a non-human primate (NHP) model of NAION (non-arteritic anterior ischemic optic neuropathy). The neuroprotective efficacy of the doxycycline-responsive dual vector system AAV2-TRE-OSK (SEQ ID NO: 35) / pAAV2-CMV-rtTA3VP16 (SEQ ID NO: 36) was evaluated in an experimental photothrombotic model of NAION. The vector system was administered intravitreally (IVT) to African green monkeys, induced by laser excitation of a systemically administered fluorophore at the optic nerve head (ONH). A schematic diagram of the NHP NAION study design is shown in Figure 5.

[0279] method Target Recruitment: Monkeys with normal slit-lamp and fundus examinations, color fundus photography (CFP), optical coherence tomography (OCT), confocal scanning laser ophthalmoscopy (cSLO), pattern electroretinogram (pERG), and pattern visual evoked potentials (pVEP) were recruited for this study. For baseline screening and all subsequent procedures, anesthesia was achieved effectively using intramuscular ketamine (8 mg / kg) and xylazine (1.6 mg / kg), and pupil dilation was achieved with topical 10% phenylephrine and / or 1% cyclopentolate.

[0280] Test substance: Subjects were administered a composition containing a dual vector system including the AAV2-TRE-OSK vector and the pAAV2-CMV-rtTA3VP16 vector. The AAV2-TRE-OSK vector was administered at approximately 1.53 x 10 12 The pAAV2-CMV-rtTA3VP16 vector was prepared at a concentration of approximately 1.33 × 10 13 The two vectors were administered at a volume ratio of 10:1 (v / v) or a viral genome ratio (vg / vg) of approximately 1:1.

[0281] The pAAV2-CMV-rtTA3VP16 vector contains a CMV promoter controlling expression of the reverse tetracycline-controlled transactivator (rtTA) fused to three copies of virion protein 16 (VP16). The encoded rtTA3VP16 protein contains the three VP16 domains plus the amino acid substitutions V9I, G12S, F67S, and R171K. References to pAAV2-CMV-rtTA3(VP16), AAV2-CMV-rtTA3VP16, AAV2-CMV-rtTA3(VP16), and pAAV2-rtTA3-Kan all refer to the same vector, pAAV2-CMV-rtTA3VP16.

[0282] Intravitreal administration: Eyes received a single intravitreal (IVT) injection of the test article or vehicle according to treatment assignment (Table 5). For IVT administration, 0.5% proparacaine hydrochloride was instilled into the eye, followed by administration of 5% betadine solution using a speculum and rinsing with 0.9% sterile saline. A 220 μL injection was made into the central vitreous using a 31-gauge, 0.375-inch needle inserted in an inferotemporal direction, approximately 2.5 mm posterior to the limbus, at the level of the ora serrata. Following injection, 1% topical atropine and topical triple antibiotic neomycin, polymyxin, and bacitracin ophthalmic ointment (or equivalent) were administered. [Table 8]

[0283] Doxycycline administration The monkeys continued to receive oral doxycycline (5 mg / kg) in their food (sliced ​​bananas) starting one day before dosing and continuing until the end of the study (Table 6). [Table 9] JPEG2026501545000038.jpg70159

[0284] immunosuppression Monkeys were administered methylprednisolone (40 mg IM) weekly starting on day 0 (Table 6). If inflammation exceeding 2+ anterior segment and / or vitreous cells was observed on subsequent examination, subconjunctival dexamethasone (100 mL of 40 mg / mL) was administered, with repeat dosing determined by clinical observation. If inflammation did not respond to dexamethasone, methylprednisolone was increased to 80 mg IM weekly. Administered steroids were applied consistently to all treated animals.

[0285] NAION induction Monkeys were divided into two cohorts based on examination and weight criteria. After achieving mydriasis and placing a saphenous vein catheter, rose bengal (2.5 mg / kg, 0.1 ml / kg of 25 mg / ml) was administered intravenously (IV), and 25 seconds later, laser spot application to the ONH was initiated. Laser spots were applied with an Iridex Oculight TX 532 nm laser using a 0.9x contact laser lens. Four spots were placed in four quadrants of the ONH, each with a pulse duration of 6 seconds, a spot size of 500 μm, and a power of 100 mW. The first spot was applied to the superior temporal side of the ONH, the second to the inferior temporal side, the third to the inferior nasal side, and the fourth to the superior nasal side. Branching retinal veins and arteries were avoided during each spot placement. Immediate post-laser CFP imaging was performed to document the response to the laser photothrombosis procedure.

[0286] Intraocular pressure testing Intraocular pressure (IOP) was measured in the oculus uterque (OU), i.e., both eyes, using a TonoVet tonometer set to the canine (d) calibration setting. At each ophthalmic examination time point (Table 6), three measurements were taken from each eye, and the mean IOP was defined.

[0287] Vitreous humor collection At the designated time points (Table 6), a 27-gauge needle was introduced into the central vitreous via temporal scleral puncture at the level of the ora serrata using a 1.0 mL syringe, and vitreous fluid (approximately 170 μL) was collected in the OU. An aliquot of vitreous fluid was transferred to pre-labeled cryotubes, flash-frozen, and stored and shipped for analysis.

[0288] Full-field electroretinogram (ffERG) Slit-lamp biomicroscopy and retinoscopy were performed on both eyes (OU) at the indicated time points (Table 6). The LKC UTAS full-field ERG device and emWin software (version 9.8.0) were used. Eyes were dark-adapted for 30 minutes immediately after sedation and topical instillation of 10% phenylephrine and 1% cyclopentolate. An additional mydriatic was administered before stimulus exposure. ffERG was performed at OD after OS (or OS after OD in a random order). After sequential unilateral trials, dark-adapted rod-specific responses were elicited by a dim white flash using an LED Ganzfeld stimulator bowl. Mixed rod and cone responses were obtained using a standard bright white flash under dark-adapted conditions. To assess the photopic function of cone photoreceptors, monkeys were allowed to light-adapt to ambient room light for 10 minutes, after which a strobe white flash stimulus was presented to the dilated eye using maximum flash intensity. The following ISCEV stimulus standards for toxicity studies were applied: Dark adaptation 0.158 cd-s m 2 (-12 dB) stimulation (rod-driven response, b-wave, measured in bipolar cells) Dark adaptation 2.51 cd-s m 2 (0 dB) stimulation (rod- ​​and cone-driven responses of both photoreceptors, a-wave, and bipolar cells, b-wave) Light adaptation 2.51 cd-s m 2 (0 dB) stimulation (cone-driven responses of both photoreceptors, a-wave, and bipolar cells, b-wave) 2.51 cd / s m 2 Light-adapted 30Hz flicker stimulus at (0dB) (cone-driven response)

[0289] Stimulus induction was represented by markers. The time-integrated luminance of the stimulus and background was recorded as absolute values. Monkeys underwent dark adaptation testing before light adaptation testing and received stimulus exposures in the order of increasing stimulus intensity for a given adaptation. To avoid bleaching and loss of adaptation, a single stimulus exposure preceded the flicker stimulus exposure. Waveforms were analyzed for a- and b-wave amplitude and latency. B-wave amplitude was measured from the a-wave trough to the b-wave peak, or, if the a-wave was absent, from the pre-stimulus baseline to the b-wave peak. A non-human primate ophthalmology scoring system was used to evaluate ocular pathological changes in response to NAION induction and therapeutic intervention, with summary scores derived from the test components. The incidence of papilledema and flame hemorrhage was also assessed.

[0290] Imaging Color anterior segment and fundus photographs were taken using a Topcon TRC-50EX retinal camera equipped with Canon 6D digital imaging hardware and New Vision Fundus Image Analysis System software.

[0291] Optical Coherence Tomography (OCT) and Confocal Scanning Laser Ophthalmoscopy (cSLO) At the indicated time points (Table 6), cSLO and OCT were performed using a Heidelberg Spectralis HRA OCT with HEYEX image capture and analysis software. cSLO infrared (IR) and autofluorescence (AF) images were acquired with a 30° field of view centered on the fovea, and follow-up imaging functions were referenced to the baseline image. Global OCT volume scans of the optic nerve and entire macula were performed through the posterior pole. Images were qualitatively evaluated by quantitatively analyzing calculated retinal thickness. For the macular-centered posterior pole scans, ganglion cell layer (GCL) and retinal nerve fiber layer (RNFL) thickness maps were generated, and thickness data were exported to an electronic spreadsheet. Peripapillary retinal nerve fiber layer (pRNFL) thickness was determined for each quadrant (superior, inferior, auricular, and nasal). HEYEX raw data files were generated for further analysis.

[0292] Pattern ERG and VEP At the indicated time points (Table 6), pattern ERG (pERG) and pattern VEP (pVEP) recordings were performed in the OU. The monkeys were positioned using DTL electrodes, an additional active electrode was placed at the inion at the midline of the back of the skull, and a ground electrode was placed in the armpit. Using a VERIS retinal projection stimulator, an alternating black-and-white checkerboard pattern was projected at 2 Hz with a luminance of 100 cd / m² and 80% contrast onto a 45% visual field generated by a VERIS multifocal ERG device. The grid size was varied in logarithmic steps, and recordings at each grid size were repeated twice to determine latency and amplitude. pVEP amplitude and pERG N95 amplitude were measured. Figure 6 shows representative pERG measurements demonstrating the induction of NAOIN after laser treatment of the optic nerve head in NHPs.

[0293] Clinical observations General health was assessed twice daily by cage observation beginning 1 week before dosing and continuing until the end of the study.

[0294] A thorough clinical examination Detailed clinical observations and physical examinations were performed at the designated time points (Table 6). Respiratory rate, heart rate, blood pressure, auscultation, and skin integrity were also assessed. Body temperature was measured using a digital rectal thermometer.

[0295] body weight Body weights were collected at ophthalmological examination intervals (Table 6).

[0296] CBC with differential Blood (0.5 mL) was transferred directly into K2EDTA lavender top vacutainer tubes (Greiner MiniCollect EDTA tube reference number 450475) and kept on ice until CBC analysis with differential on a Hemavet analyzer.

[0297] DNA methylation blood Blood was transferred directly to a 0.5 mL microcentrifuge tube anticoagulated with K2EDTA. Samples were then lysed and DNA isolated on QIAMP MiniElute columns according to the manufacturer's instructions (Qiagen). DNA samples were analyzed for DNA methylation, a measure of biological age.

[0298] serum At the designated time points (Table 6), blood (3 ml) was transferred directly into serum separator tubes (red caps) and allowed to stand at room temperature for 30–60 minutes, followed by centrifugation at 3000 rpm for 10 minutes at 4°C. Two serum aliquots (approximately 0.5 ml × 2) were obtained and carefully transferred into labeled 1.8 ml cryotubes, stored below −70°C, and shipped to the designated laboratory for nAb analysis.

[0299] Eye removal and preparation At the end of the study (Table 6), after confirming the quality of in vivo imaging and electrophysiology, the monkeys were sedated with intramuscular ketamine (8 mg / kg) and xylazine (1.6 mg / kg), and then euthanized with pentobarbital sodium (100 mg / kg IV). The eyeballs (OU) were removed after placing a suture marker at the 12 o'clock position. Excess tissue from the eyeballs with attached optic nerves was trimmed, and approximately 300 μl of Davidson's fixative was injected intraocularly. The eyeballs were placed in Davidson's fixative at room temperature for 24 hours. The eyeballs were transferred to phosphate-buffered saline (PBS) containing 0.05% sodium azide and stored in a container maintained at 4°C for histological and immunohistochemical processing and analysis by a certified veterinary pathologist. The eyeballs were then shipped to a designated histology laboratory. Additional quantitative scoring of the histological images was performed based on qualitative findings.

[0300] Axon quantification Axon quantification was performed using a workflow consisting of three computer programs. The first program identifies the entire optic nerve against the background of the glass slide, improving the speed and accuracy of the following programs. The second program is used to distinguish nerve bundles from connective tissue, eliminating the false count of connective tissue elements in axonal forms, thereby improving the accuracy of the final application. The final program locates axons and classifies them into three categories: healthy, subhealthy, and degenerated axons. The final program also generates numerical data, such as the number and area of ​​each axon category, which allows for the calculation of density and / or proportional endpoints.

[0301] pERG results NAION-like damage was induced in all NHPs (African green monkeys, N = 20) on day 0 by intravenous injection of rose bengal followed by laser treatment of the ONH of the OS eye. On day -28, pretreated NHPs (n = 6) received an intravitreal (IVT) injection of OSK (doxycycline-inducible AAV2-OSK, AAV2-TRE-OSK + pAAV2-CMV-rtTA3VP16 at a 1:1 ratio) or vehicle (n = 4) into the OS eye. Post-treatment, NHPs received an IVT injection of OSK (n = 6) or vehicle (n = 4) into the OS eye on day +1. All NHPs received oral doxycycline daily throughout the experiment. Both eyes were examined for multiple imaging and functional measurements of retinal ganglion cells (RGCs) at baseline and then at weekly intervals until day +35 (corresponding to day 42) after laser treatment.

[0302] Pretreatment with OSK increased pERG amplitude and partially reversed NAION-like pERG deficits compared with vehicle-treated control eyes at endpoint (p50-n95 amplitude: 5.36 ± 0.91 vs. 3.95 ± 1.01, ns, 5 weeks after laser treatment) (Figure 7). Treatment with OSK demonstrated significant recovery of pERG function by 5 weeks after laser treatment (p50-n95 amplitude: 4.60 ± 0.24 vs. 2.89 ± 0.79, P = 0.039) (Figure 8).

[0303] To further analyze the impact of OSK treatment on NAION, a correlation analysis was performed to compare the degree of early optic disc edema with the degree of pERG deficit 5 weeks after laser treatment. Optic disc edema was calculated by measuring optic disc size on day 8 and subtracting the baseline optic disc size. Optic disc edema is an indicator of early laser damage. pERG is measured as the absolute amplitude between p50 and n95. pERG deficits were calculated by measuring the absolute amplitude at week 5 and subtracting the baseline absolute amplitude. pERG deficits are an indicator of long-term damage to retinal ganglion cells (RGCs). In vehicle-treated NHPs, the degree of early optic disc edema correlated with the degree of pERG deficit 5 weeks after laser treatment, indicating the long-term effects of early laser damage (Figures 9 and 10A). Strikingly, OSK-treated NHPs showed a reduced correlation between the degree of initial optic disc edema and the degree of pERG deficit at 5 weeks after laser treatment, indicating reduced RGC damage ( Fig. 10B ).

[0304] A similar correlation analysis was performed at the end of the experiment, comparing the degree of initial optic disc edema with the degree of axon density damage at the end of the study. In vehicle-treated NHPs, the degree of initial optic disc edema correlated with the degree of axon density damage at the end of the study, indicating the long-term effects of the initial laser injury (Figures 11 and 12A). Notably, OSK-treated NHPs showed a decreased correlation between the degree of initial optic disc edema and the degree of axon density damage at the end of the study, indicating protection against or reversal of axon density damage (Figure 12B). [Table 10]

[0305] In vehicle-treated NHPs, the degree of initial optic disc edema correlated with the degree of axon density at the end of the study, indicating a long-term effect of the initial laser injury. The degree of initial optic disc edema was less predictive (less correlated) of axon density at the end of the study in OSK-treated NHPs, due to protection and / or reversal of axonal injury. In vehicle-treated NHPs, the degree of initial optic disc edema correlated with the magnitude of the pERG effect at the end of the study, indicating a long-term effect of the initial laser injury. The degree of initial optic disc edema was less predictive (less correlated) of the magnitude of the pERG effect at the end of the study in OSK-treated NHPs, due to protection and / or reversal of axonal injury.

[0306] Example 3 - Clinical Trials This clinical trial will evaluate the efficacy of a single dose of ER-100 administered to patients with glaucoma or NAION. ER-100 is a dual vector AAV2 containing a 1:1 ratio of AAV2-CMV-rtTA-L to AAV2-TRE-OSK administered intravitreally (IVT). ER-100 is administered with oral doxycycline (a widely used antibiotic) to activate OSK expression.

[0307] Men and women at least 50 years of age with clinical symptoms consistent with NAION or at least 35 years of age with clinical symptoms consistent with glaucoma will be enrolled. Subjects will receive intravitreal administration of a doxycycline-responsive dual vector system. Subjects will receive oral doxycycline starting on the day of virus administration and continuing through Day 56 of the clinical trial.

[0308] This study is a first-in-human (FIH) multicenter trial that includes a dose-escalation phase (Phase 1a) followed by an expansion phase (Phase 1b). ER-100 will be administered open-label to one eye. Systemic doxycycline will be administered for 8 weeks, followed by placebo doxycycline for an additional 6 weeks, with subjects blinded to when doxycycline was discontinued. Only subjects will be blinded to the placebo doxycycline.

[0309] The primary objective of this study is to determine the safety, tolerability, and dose-limiting toxicities of ER-100 when administered as a single intravitreal (IVT) injection to patients with optic neuropathy (glaucoma, NAION). The following endpoints will be monitored: incidence of treatment-emergent adverse events (TEAEs), incidence of protocol-defined dose-limiting toxicities for glaucoma and NAION at different dose levels, change from baseline in safety laboratory tests, and negative change from baseline in ocular assessments. These endpoints will be measured during the doxycycline timeframe (through Day 56) and the placebo timeframe (Days 57-84) or no treatment (after Day 84).

[0310] One secondary objective of this clinical trial is to investigate the efficacy of a single intravenous injection of ER-100 in patients with optic neuropathy (glaucoma, NAION). The following endpoints will be measured and compared with the change from baseline in the treated eye: intraocular pressure (IOP), best-corrected visual acuity (BCVA) using electronic visual acuity (EVA) with the E-ETDRS algorithm, Humphrey automated visual field (Humphrey automated visual field), quantitative contrast sensitivity function (qCSF), pattern electroretinogram (pERG), full-field electroretinogram (ffERG), optical coherence tomography (OCT): retinal nerve fiber layer, and optical coherence tomography (OCT): ganglion cell layer. Anatomical changes in the optic nerve head and retina observed by color fundus examination / photography will also be documented. These endpoints will be measured during the doxycycline timeframe (up to day 56) and the placebo timeframe (days 57-84) or after treatment-free treatment (after day 84).

[0311] Additional secondary objectives of this clinical trial are to characterize the immune response, viral shedding, and kinetics of ER-100. The following endpoints will be monitored during the doxycycline timeframe (through day 56) and the placebo timeframe (days 57-84) or no treatment (after day 84): levels of neutralizing antibodies to AAV2, as measured by ELISPOT assay (including IFγ, rtTA-L, OSK, and capsid proteins); levels of anti-drug antibodies to rtTA-L and OSK; ER-100 viral shedding measured by qPCR in tears, nasal swabs, saliva, and blood; and biodistribution of ER-100 vector DNA. These endpoints will be measured during the doxycycline timeframe (through day 56) and the placebo timeframe (days 57-84) or no treatment (after day 84).

[0312] The results are expected to show that expression of OSK in RGCs in subjects with NAOIN results in increased pERG amplitude, improved visual acuity, reduced visual field defects, and overall improved ocular function.

[0313] Enumerated Embodiment Set I Clause 1. A method for preventing or treating non-arteritic anterior ischemic optic neuropathy in a subject, the method comprising administering to the subject a nucleic acid molecule comprising a nucleic acid sequence encoding OCT4, a nucleic acid sequence encoding SOX2, and a nucleic acid sequence encoding KLF4.

[0314] Clause 2. The method of clause 1, further comprising administering to said subject a nucleic acid molecule comprising a nucleic acid sequence encoding a reverse tetracycline-controlled transactivator (rtTA).

[0315] Clause 3. The method of clause 1 or clause 2, wherein the nucleic acid molecule comprising a nucleic acid sequence encoding OCT4, a nucleic acid sequence encoding SOX2, and a nucleic acid sequence encoding KLF4 is operably linked to an inducible promoter.

[0316] Clause 4. The method of clause 3, wherein said inducible promoter comprises a tetracycline class antibiotic response element (TRE).

[0317] Clause 5. The method of clause 4, wherein the tetracycline class antibiotic is doxycycline.

[0318] Clause 6. The method of clause 4, wherein said inducible promoter is a TRE2 promoter.

[0319] Clause 7. The method of any one of clauses 2 to 6, wherein said rtTA is rtTA3 or rtTA4.

[0320] Clause 8. The method of any one of clauses 2 to 7, wherein said nucleic acid molecule comprising a nucleic acid sequence encoding rtTA is operably linked to a CMV promoter.

[0321] Clause 9. The method of any one of the preceding clauses, wherein the nucleic acid molecule comprising a nucleic acid sequence encoding OCT4, a nucleic acid sequence encoding SOX2, and a nucleic acid sequence encoding KLF4 is an adeno-associated virus (AAV) vector.

[0322] Clause 10. The method of clause 9, wherein the AAV vector is serotype 2 (AAV2).

[0323] Clause 11. The method of any one of the preceding clauses, wherein the nucleic acid molecule comprising a nucleic acid sequence encoding OCT4, a nucleic acid sequence encoding SOX2, and a nucleic acid sequence encoding KLF4 does not comprise a nucleic acid sequence encoding c-Myc.

[0324] Clause 12. The method of any one of the preceding clauses, wherein the nucleic acid molecule comprising a nucleic acid sequence encoding OCT4, a nucleic acid sequence encoding SOX2, and a nucleic acid sequence encoding KLF4 comprises a nucleic acid sequence encoding a self-cleaving peptide.

[0325] Clause 13. The method of clause 12, wherein said self-cleaving peptide is a 2A peptide.

[0326] Clause 14. The method of any one of the preceding clauses, wherein the nucleic acid molecules encoding OCT4, SOX2, and KLF4 are flanked by inverted terminal repeats (ITRs), and the distance between the ITRs is 4.7 kb or less.

[0327] Clause 15. The method of any one of the preceding clauses, further comprising administering to said subject an inducer.

[0328] Clause 16. The method of any one of clauses 2 to 15, wherein said nucleic acid molecule encoding reverse tetracycline-controlled transactivator (rtTA) is an AAV vector that does not include said nucleic acid molecules encoding OCT4, SOX2, and KLF4.

[0329] Clause 17. The nucleic acid molecule encoding OCT4, SOX2, and KLF4 is present in the following order:

[0330] a) the first inverted terminal repeat (ITR) sequence (SEQ ID NO: 16);

[0331] b) TRE promoter sequence (SEQ ID NO: 7);

[0332] c) a nucleic acid sequence encoding OCT4 (SEQ ID NO: 1);

[0333] d) a nucleic acid sequence encoding P2A (SEQ ID NO: 8);

[0334] e) a nucleic acid sequence encoding SOX2 (SEQ ID NO: 3);

[0335] f) a nucleic acid sequence encoding T2A (SEQ ID NO: 10);

[0336] g) a nucleic acid sequence encoding KLF4 (SEQ ID NO: 5);

[0337] h) a terminator sequence derived from SV-40 (SEQ ID NO: 12), and

[0338] i) comprising a nucleic acid element with a second inverted terminal repeat (ITR) sequence (SEQ ID NO: 32).

[0339] Clause 18. The method of any one of the preceding clauses, wherein said nucleic acid sequence encoding OCT4 comprises SEQ ID NO:1.

[0340] Clause 19. The method of any one of the preceding clauses, wherein said nucleic acid sequence encoding SOX2 comprises SEQ ID NO:3.

[0341] Clause 20. The method of any one of the preceding clauses, wherein said KLF4 is a human KLF4 protein.

[0342] Clause 21. The method of any one of the preceding clauses, wherein said nucleic acid sequence encoding KLF4 comprises SEQ ID NO:5.

[0343] Clause 22. The method of Clause 17, wherein said nucleic acid sequence encoding P2A comprises SEQ ID NO:8.

[0344] Clause 23. The method of clause 17, wherein said P2A comprises the amino acid sequence of SEQ ID NO:9.

[0345] Clause 24. The method of clause 17, wherein said T2A comprises the amino acid sequence of SEQ ID NO:11.

[0346] Clause 25. The method of Clause 17, wherein the nucleic acid sequence encoding T2A is GAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCA (SEQ ID NO: 10).

[0347] Clause 26. The method of clause 17, wherein the TRE promoter sequence is SEQ ID NO:7.

[0348] Clause 27. The method of Clause 17, wherein the SV-40 derived terminator sequence is SEQ ID NO:12.

[0349] Clause 28. The method of clause 17, wherein said first ITR sequence is SEQ ID NO: 16.

[0350] Clause 29. The method of clause 17, wherein said nucleic acid sequence encoding OCT4, SOX2, and KLF4 comprises SEQ ID NO: 13.

[0351] Clause 30. The method of Clause 17, wherein said nucleic acid sequence encoding OCT4, SOX2, and KLF4 comprises SEQ ID NO:14.

[0352] Clause 31. The method of any one of clauses 2 to 30, wherein said nucleic acid molecules encoding OCT4, SOX2, and KLF4 and said nucleic acid molecule encoding rtTA are administered sequentially or simultaneously.

[0353] Clause 32. The method of any one of the preceding clauses, wherein said nucleic acid molecules encoding OCT4, SOX2, and KLF4 are administered intravitreally.

[0354] Clause 33. The method of any one of clauses 2 to 32, wherein said nucleic acid molecule encoding rtTA is administered intravitreally.

[0355] Clause 34. The method of any one of clauses 2 to 31, wherein said nucleic acid molecules encoding OCT4, SOX2, and KLF4 and said nucleic acid molecule encoding rtTA are administered in a ratio of about 1:1.

[0356] Clause 35. The method of any one of clauses 16 to 34, wherein the AAV vector comprising the nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the AAV vector comprising the nucleic acid molecule encoding rtTA are administered in a ratio of about 1:1 (vg:vg).

[0357] Clause 36. The method of any one of the preceding clauses, wherein the nucleic acid molecule encoding OCT4, SOX2, and KLF4 is an AAV2-TRE-OSK vector comprising a nucleic acid molecule comprising SEQ ID NO: 35, and the nucleic acid molecule encoding rtTA is a pAAV2-CMV-rtTA3VP16 vector comprising a nucleic acid molecule comprising SEQ ID NO: 36, or an AAV2-CMV-rtTA4 vector comprising a nucleic acid molecule comprising SEQ ID NO: 37.

[0358] Clause 37. The nucleic acid molecule encoding a reverse tetracycline-controlled transactivator (rtTA) comprises the following order:

[0359] a) the first inverted terminal repeat (ITR) sequence (SEQ ID NO: 22);

[0360] b) CMV promoter sequence (SEQ ID NO: 17);

[0361] c) a CMV enhancer sequence (SEQ ID NO: 18);

[0362] d) a nucleic acid sequence encoding rtTA3 (SEQ ID NO: 19);

[0363] e) the WPRE sequence (SEQ ID NO: 23), and

[0364] f) The method of clause 16, wherein the AAV vector comprises a nucleic acid element at the second inverted terminal repeat (ITR) sequence (SEQ ID NO: 33).

[0365] Clause 38. The nucleic acid molecule encoding a reverse tetracycline-controlled transactivator (rtTA) comprises the following order:

[0366] a) the first inverted terminal repeat (ITR) sequence (SEQ ID NO: 29);

[0367] b) CMV promoter sequence (SEQ ID NO: 24);

[0368] c) a CMV enhancer sequence (SEQ ID NO: 25);

[0369] d) a nucleic acid sequence encoding rtTA4 (SEQ ID NO: 26);

[0370] e) the WPRE sequence (SEQ ID NO: 31), and

[0371] f) SV40 sequence (SEQ ID NO: 30), and

[0372] e) The method of clause 16, wherein the AAV vector comprises a nucleic acid element at the second inverted terminal repeat (ITR) sequence (SEQ ID NO: 34).

[0373] Clause 39. The effective amount of the AAV2-TRE-OSK vector is about 1 x 10 9 vg / eye ~ approx. 1×10 14 37. The method of claim 36, wherein the concentration is within the range of vg / eye.

[0374] Clause 40. The effective amount of the AAV2-CMV-rtTA3 vector is about 1 x 10 9 vg / eye ~ approx. 1×10 14 37. The method of claim 36, wherein the concentration is within the range of vg / eye.

[0375] Clause 41. The effective amount of the AAV2-CMV-rtTA4 vector is about 1 x 10 9 vg / eye ~ approx. 1×10 14 37. The method of claim 36, wherein the concentration is within the range of vg / eye.

[0376] Clause 42. The method of any one of the preceding clauses, wherein the nucleic acid molecules encoding OCT4, SOX2, and KLF4 are administered to the subject by left eye (OS) injection, by right eye (OD) injection, or by both eyes (OU) injection.

[0377] Clause 43. The method of any one of clauses 2 to 42, wherein the nucleic acid molecule encoding rtTA is administered to the subject by left eye (OS) injection, by right eye (OD) injection, or by both eyes (OU) injection.

[0378] Clause 44. The method of any one of the preceding clauses, further comprising administering to said subject an effective amount of an antibiotic.

[0379] Clause 45. The method of clause 44, wherein said antibiotic is administered at least one day before administering said nucleic acid molecule encoding rtTA.

[0380] Clause 46. The method of Clause 44, wherein said antibiotic is administered at the time said nucleic acid molecule encoding rtTA is administered.

[0381] Clause 47. The method of Clause 44, wherein said antibiotic is administered at least one day after administration of said nucleic acid molecule encoding rtTA.

[0382] Clause 48. A method for the recombinant preparation of AAV, comprising introducing a vector into a cell under conditions in which said AAV is produced, said vector comprising a) one or more nucleic acid sequences encoding OCT4, SOX2, and KLF4.

[0383] Clause 49. The method of Clause 48, wherein said cells comprise a population of HEK293T cells.

[0384] Clause 50. A method for producing AAV, comprising modifying a cell to express one or more plasmids, comprising one or more AAV2 Rep-Cap plasmids, one or more helper plasmids, and one or more transfer plasmids, wherein the one or more transfer plasmids comprise one or more nucleic acids encoding OCT4, SOX2, and KLF4.

[0385] Clause 51. The method of Clause 50, wherein said cells comprise a population of HEK293T cells.

[0386] Clause 52. A method for the prevention or treatment of non-arteritic anterior ischemic optic neuropathy (NAION) in a subject in need thereof, comprising administering to said subject a pharmaceutically effective amount of a composition comprising an expression vector comprising a polynucleotide encoding OCT4, SOX2, and KLF4, but not c-Myc.

[0387] Clause 53. The method of Clause 52, wherein said composition does not reprogram cells, tissues, or organs to a pluripotent state in said subject.

[0388] Clause 54. The method of Clause 52, wherein said composition does not induce c-Myc expression in said subject.

[0389] Clause 55. The method of Clause 52, wherein said polynucleotide comprises DNA, RNA, or a combination thereof.

[0390] Clause 56. The method of Clause 55, wherein the DNA comprises plasmid DNA.

[0391] Clause 57. The method of Clause 55, wherein said RNA comprises mRNA.

[0392] Clause 58. The method of Clause 52, wherein said polynucleotide comprises an inducible promoter.

[0393] Clause 59. The method of Clause 58, wherein said inducible promoter comprises a tetracycline response element (TRE).

[0394] Clause 60. The method of clause 52, further comprising administering to said subject an inducer to induce expression of OCT4, SOX2, and KLF4 in said subject.

[0395] Clause 61. The method of Clause 60, wherein the inducer comprises a tetracycline class antibiotic.

[0396] Clause 62. The method of clause 61, wherein the tetracycline class antibiotic is doxycycline.

[0397] Clause 63. The method of Clause 60, wherein said inducing agent comprises a reverse tetracycline-controlled transactivator (rtTA) or a polynucleotide encoding said rtTA.

[0398] Clause 64. The method of Clause 63, wherein the polynucleotide encoding the rtTA is in an expression vector.

[0399] Clause 65. The method of claim 60, wherein the composition and the inducer are administered sequentially or simultaneously.

[0400] Clause 66. The method of Clause 60, wherein said composition and said inducer are administered in a ratio of about 1:1.

[0401] Clause 67. The method of Clause 52, wherein said expression vector further comprises a polynucleotide encoding a self-cleaving peptide.

[0402] Clause 68. The method of clause 67, wherein the self-cleaving peptide is a 2A peptide.

[0403] Clause 69. The method of Clause 52, wherein said polynucleotide comprises an inverted terminal repeat (ITR).

[0404] Clause 70. The method of Clause 52, wherein said expression vector is a viral expression vector, and said viral vector is a lentiviral, retroviral, adenoviral, alphavirus, vaccinia virus, and adeno-associated viral (AAV) vector.

[0405] Clause 71. The method of Clause 70, wherein the AAV vector is serotype 2 (AAV2).

[0406] Clause 72. The polynucleotide comprises the following sequence:

[0407] a. a first inverted terminal repeat (ITR) sequence;

[0408] b. TRE3G promoter sequence,

[0409] c.OCT4 sequence,

[0410] d. P2A cleavage sequence,

[0411] e.SOX2 sequence,

[0412] f. T2A cleavage sequence,

[0413] g.KLF4 sequence,

[0414] h. a terminator sequence derived from SV-40, and

[0415] i. The method of clause 52, comprising a nucleic acid element at the second inverted terminal repeat (ITR) sequence.

[0416] Clause 73. A method according to clause 52, comprising:

[0417] i) OCT4 comprises an amino acid sequence having at least 90% identity to SEQ ID NO:2;

[0418] ii) SOX2 comprises an amino acid sequence having at least 90% identity to SEQ ID NO:4;

[0419] iii) The method, wherein KLF4 comprises an amino acid sequence having at least 90% identity to SEQ ID NO:6.

[0420] 74. A method according to clause 52, comprising:

[0421] i) the polynucleotide comprises a nucleic acid sequence having at least 75% identity to SEQ ID NO: 1;

[0422] ii) the polynucleotide comprises a nucleic acid sequence having at least 75% identity to SEQ ID NO: 3, and / or

[0423] iii) the polynucleotide comprises a nucleic acid sequence having at least 75% identity to SEQ ID NO:5.

[0424] Clause 75. The method of clause 52, wherein the composition is administered intravitreally.

[0425] Clause 76. The method of clause 52, wherein the composition is administered to the subject by left eye (OS) injection, by right eye (OD) injection, or by both eyes (OU) injection.

[0426] Clause 77. The method of Clause 52, wherein administering the composition improves retinal ganglion cell (RGC) function and / or restores visual function in the subject.

[0427] Clause 78. The method of clause 52, wherein said prevention or treatment of NAION is measured by electroretinogram (pERG).

[0428] Article 79. A method for preventing or treating non-arteritic anterior ischemic optic neuropathy (NAION) in a subject in need thereof, comprising administering to said subject a pharmaceutically effective amount of a composition comprising an expression vector comprising a polynucleotide encoding three transcription factors, said three transcription factors consisting of OCT4, SOX2, and KLF4.

[0429] Enumerated Embodiment Set II Clause 1. A method of treating or preventing ischemic optic neuropathy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a gene therapy vector, said gene therapy vector comprising one or more nucleic acid molecules comprising: a nucleic acid sequence encoding octamer-binding transcription factor 4 (OCT4), a nucleic acid sequence encoding sex-determining region (Y)-box 2 (SOX2), and a nucleic acid sequence encoding Krüppel-like factor 4 (KLF4), operably linked to at least one promoter (or operably linked to recombination sites, blunt ligation, or homology sites for genome editing, placing said one or more nucleic acid molecules under the control of an endogenous promoter).

[0430] Clause 2. The method of clause 1, wherein said ischemic optic neuropathy is non-arteritic anterior ischemic optic neuropathy (NAION).

[0431] Clause 3. The method of clause 1, wherein said ischemic optic neuropathy is arteritic anterior ischemic optic neuropathy (A-AION).

[0432] Clause 4. The method of clause 1, wherein said ischemic optic neuropathy is posterior ischemic optic neuropathy (PION).

[0433] Clause 5. The method of any one of clauses 1 to 4, wherein said vector is administered intravitreally.

[0434] Clause 6. The method of any one of clauses 1 to 5, wherein said method of administration delivers said one or more nucleic acid molecules to retinal ganglion cells when administered to the eye in vivo.

[0435] Clause 7. The method of any one of clauses 1 to 6, wherein the vector lacks a nucleic acid molecule encoding the Myc proto-oncogene (c-Myc) or Nanog, lacks any nucleic acid molecule encoding a transcription factor, and / or lacks any nucleic acid molecule encoding a reprogramming factor other than OCT4, SOX2, and KLF4.

[0436] Clause 8. The method of any one of clauses 1 to 7, wherein said vector is a viral vector.

[0437] Clause 9. The method of any one of clauses 1 to 8, wherein the vector is a liposome or a lipid nanoparticle (LNP).

[0438] Clause 10. The method of clause 8, wherein said viral vector is an adeno-associated viral (AAV) vector, optionally an oculotropic AAV vector.

[0439] Clause 11. The method of clause 10, wherein the AAV vector is an AAV serotype 2 (AAV2) vector or a variant thereof.

[0440] Clause 12. The method of clause 10, wherein the AAV vector is AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAVrh74, AAVdj and AAV.PHP, or a variant thereof, and optionally an AAV serotype listed in Table 2 as having specificity for the eye.

[0441] Clause 13. The method of any one of clauses 10 to 12, wherein said one or more nucleic acid molecules is an AAV genome including flanking inverted terminal repeats (ITRs).

[0442] Clause 14. The method of clause 9, wherein the vector is a LNP, and the LNP comprises an ionizable lipid, a helper lipid, a sterol, and a poly(ethylene glycol)-lipid (PEG-lipid).

[0443] Clause 15. The method of any one of clauses 1 to 14, wherein said promoter is an inducible promoter.

[0444] Clause 16. The method of clause 15, wherein said inducible promoter is a tetracycline response element (TRE) promoter, optionally a Tet-On promoter, optionally a TRE3G promoter.

[0445] Clause 17. The method of clause 16, further comprising sequentially or simultaneously administering to the subject a second gene therapy vector, optionally an adeno-associated virus (AAV), comprising a polynucleotide encoding a reverse tetracycline-controlled transactivator (rtTA), in an amount effective to positively regulate the TRE promoter.

[0446] Clause 18. The rtTA is selected from the rtTAs listed in Table 1, and optionally MSRLDKSKIINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLK 18. The method of clause 17, wherein the rtTA shares at least 80%, at least 90%, at least 85%, or 100% identity to CESGGPTDALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG (SEQ ID NO: 20), and optionally comprises one or more amino acid substitutions selected from V9I, G12S, F67S, G72V, G72P, and R171K, and optionally comprises said amino acid substitutions G12S, F67S, and R171K or said amino acid substitutions V9I, G12S, F67S, and R171K.

[0447] Clause 19. The method of any one of clauses 16 to 18, further comprising administering an inducer of the tetracycline class, optionally doxycycline or tetracycline.

[0448] Clause 20. The method of any one of claims 1 to 19, wherein the vector is a multicistronic vector and the one or more nucleic acid molecules is a single nucleic acid molecule comprising open reading frames encoding the OCT4, the SOX2, and the KLF4.

[0449] Clause 21. The method of clause 20, wherein said open reading frame does not encode any other proteins.

[0450] Clause 22. The method of clause 20 or clause 21, wherein said OCT4, said SOX2, and said KLF4 are linked by a self-cleaving peptide, optionally a 2A-peptide.

[0451] Clause 23. The method of any one of clauses 20 to 22, wherein said open reading frame encodes, in 5' to 3' order, said OCT4, said SOX2, and said KLF4.

[0452] Clause 24. The method of any one of clauses 15 to 19, comprising administering to said subject said inducer for a period of time sufficient to restore retinal ganglion cells but not to induce pluripotency of said cells.

[0453] Clause 25. The method of any one of clauses 1 to 25, wherein said OCT4 shares at least 80%, at least 90%, at least 85%, or 100% identity to SEQ ID NO:2, said SOX2 shares at least 80%, at least 90%, at least 85%, or 100% identity to SEQ ID NO:4, and / or said KLF4 shares at least 80%, at least 90%, at least 85%, or 100% identity to SEQ ID NO:6.

[0454] Clause 26. The method of any one of clauses 1 to 25, wherein the method improves retinal ganglion cell (RGC) function and / or restores visual function in said subject.

[0455] Clause 27. The method of any one of clauses 1 to 26, wherein said prevention or treatment of NAION is measured by electroretinogram (pERG).

[0456] Clause 28. A gene therapy vector for use in the method of any one of clauses 1 to 27.

[0457] * * * Incorporation by Reference The entire disclosure of each of the patent and scientific publications referenced herein is incorporated by reference for all purposes.

[0458] Reference herein to any prior publication (or information deriving therefrom) or to any known matter is not, and should not be taken as, an acknowledgment or admission, or any form of suggestion, that the prior publication (or information deriving therefrom) or known matter forms part of the common general knowledge in the art to which this specification pertains.

[0459] equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the foregoing embodiments are to be considered in all respects as illustrative and not limiting of the invention described herein. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. 1. A method for treating or preventing ischemic optic neuropathy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a gene therapy vector; The gene therapy vector comprises one or more nucleic acid molecules, the one or more nucleic acid molecules comprising: a nucleic acid sequence encoding octamer-binding transcription factor 4 (OCT4); a nucleic acid sequence encoding the sex-determining region Y-box 2 (SOX2); and comprising a nucleic acid sequence encoding Krüppel-like factor 4 (KLF4), operably linked to at least one promoter (or operably linked to recombination sites, blunt ligation, or homology sites for genome editing, placing the one or more nucleic acid molecules under the control of an endogenous promoter).

2. 2. The method of claim 1, wherein the ischemic optic neuropathy is non-arteritic anterior ischemic optic neuropathy (NAION).

3. 2. The method of claim 1, wherein the ischemic optic neuropathy is arteritic anterior ischemic optic neuropathy (A-AION).

4. 2. The method of claim 1, wherein the ischemic optic neuropathy is posterior ischemic optic neuropathy (PION).

5. The method of any one of claims 1 to 4, wherein the vector is administered intravitreally.

6. The method of any one of claims 1 to 5, wherein the administration method delivers the one or more nucleic acid molecules to retinal ganglion cells when administered to the eye in vivo.

7. 7. The method of any one of claims 1 to 6, wherein the vector lacks a nucleic acid molecule encoding Myc proto-oncogene (c-Myc) or Nanog, lacks any nucleic acid molecule encoding a transcription factor, and / or lacks any nucleic acid molecule encoding a reprogramming factor other than OCT4, SOX2, and KLF4.

8. The method according to any one of claims 1 to 7, wherein the vector is a viral vector.

9. The method according to any one of claims 1 to 8, wherein the vector is a liposome or a lipid nanoparticle (LNP).

10. 9. The method of claim 8, wherein the viral vector is an adeno-associated viral (AAV) vector, optionally an oculotropic AAV vector.

11. 11. The method of claim 10, wherein the AAV vector is an AAV serotype 2 (AAV2) vector or a variant thereof.

12. 11. The method of claim 10, wherein the AAV vector is AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAVrh74, AAVdj, and AAV.PHP, or a variant thereof, optionally an AAV serotype listed in Table 2 as having ocular specificity.

13. 13. The method of any one of claims 10 to 12, wherein the one or more nucleic acid molecules is an AAV genome including flanking inverted terminal repeats (ITRs).

14. 10. The method of claim 9, wherein the vector is an LNP, and the LNP comprises an ionizable lipid, a helper lipid, a sterol, and a poly(ethylene glycol)-lipid (PEG-lipid).

15. The method of any one of claims 1 to 14, wherein the promoter is an inducible promoter.

16. 16. The method of claim 15, wherein the inducible promoter is a tetracycline response element (TRE) promoter, optionally a Tet-On promoter, and optionally a TRE3G promoter.

17. 17. The method of claim 16, further comprising sequentially or simultaneously administering to the subject a second gene therapy vector, optionally an adeno-associated virus (AAV), comprising a polynucleotide encoding a reverse tetracycline-controlled transactivator (rtTA), in an amount effective to positively regulate the TRE promoter.

18. The rtTA is selected from the rtTAs set forth in Table 1, and optionally an rtTA sharing at least 80%, at least 90%, at least 85%, or 100% identity to HFTLGCVLEEQEHQVAKEERETPTTDSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDDLDMLPADALDDFDDLDMLPADALDDFDLDMLPG (SEQ ID NO: 20); optionally comprising one or more amino acid substitutions selected from V9I, G12S, F67S, G72V, G72P, and R171K; 18. The method of claim 17, optionally comprising the amino acid substitutions G12S, F67S and R171K or the amino acid substitutions V9I, G12S, F67S and R171K.

19. The method of any one of claims 16 to 18, further comprising administering an inducer of the tetracycline class, optionally doxycycline or tetracycline.

20. The method according to any one of claims 1 to 19, wherein the vector is a multicistronic vector, and the one or more nucleic acid molecules are a single nucleic acid molecule comprising open reading frames encoding the OCT4, the SOX2, and the KLF4.

21. 21. The method of claim 20, wherein the open reading frame does not encode any other proteins.

22. The method of claim 20 or claim 21, wherein the OCT4, the SOX2, and the KLF4 are linked by a self-cleaving peptide, optionally a 2A-peptide.

23. The method according to any one of claims 20 to 22, wherein the open reading frame encodes, in 5' to 3' order, the OCT4, the SOX2, and the KLF4.

24. 20. The method of any one of claims 15 to 19, comprising administering to the subject the inducer for a period of time sufficient to restore retinal ganglion cells but not to induce pluripotency of the cells.

25. the OCT4 shares at least 80%, at least 90%, at least 85%, or 100% identity to SEQ ID NO:2; said SOX2 shares at least 80%, at least 90%, at least 85%, or 100% identity to SEQ ID NO:4; and / or 25. The method of any one of claims 1 to 24, wherein the KLF4 shares at least 80%, at least 90%, at least 85%, or 100% identity to SEQ ID NO:

6.

26. 26. The method of any one of claims 1 to 25, wherein the method improves retinal ganglion cell (RGC) function and / or restores visual function in the subject.

27. 27. The method of any one of claims 1 to 26, wherein said prevention or treatment of NAION is measured by electroretinogram (pERG).

28. A gene therapy vector for use in the method of any one of claims 1 to 27.

Images