Treatment of ocular conditions

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 Kruppel-like factor 4 (KLF4). In particular, non-arteritis anterior ischemic optic neuropathy (NAION) may be treated or prevented.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is an international patent application that claims priority to U.S. Provisional Patent Application No. 63 / 499,864, filed on May 3, 2023, and U.S. Provisional Patent Application No. 63 / 478,843, filed on January 6, 2023, the entire contents of which are incorporated herein by reference.

[0003] Incorporated by reference into the sequence listing

[0004] This application contains a sequence listing submitted via EFS-Web. The file is named 061189-501001WO_SequenceListing_ST26, was created on January 4, 2024, is 92,025 bytes in size, and its contents are hereby incorporated by reference in their entirety. Technical Field

[0005] The present disclosure relates to gene therapy for ocular disorders that delivers vitality-restoring factors to cells, particularly retinal ganglion cells. Background Art

[0006] Ischemic optic neuropathy is the most common acute optic nerve disorder in patients over 50 years of age. Ischemic optic neuropathy is generally categorized as anterior (affecting the optic disc) or posterior (back of the eyeball), and as arteritic or nonarteritic. Both arteritic and nonarteritic ischemic optic neuropathy typically affect the anterior segment.

[0007] Non-arteritic anterior ischemic optic neuropathy (NAION) is the most common form of ischemic optic neuropathy. It is an idiopathic ischemic injury to the optic nerve head and is characterized by acute, unilateral, 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 annually and is the most common cause of acute optic neuropathy in patients over 50 years of age. Approximately 6,000 new cases occur annually. Men and women are affected almost equally. [www_eyewiki_aao_org / Non-Arteritic_Anterior_Ischemic_Optic_Neuropathy_(NAION)]

[0008] There is currently no known, proven treatment for NAION. Numerous clinical trials have investigated dozens of different therapies, but none have convincingly improved vision outcomes in patients with NAION. The present disclosure addresses this need for treatment. Summary of the Invention

[0009] The present disclosure relates to and provides a method 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 molecule comprising a nucleic acid sequence encoding Kruppel-like factor 4 (KLF4). In some embodiments, the nucleic acid sequences encoding OCT4, SOX2, and KLF4 are located 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.

[0010] 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, rtTA is rtTA-L, rtTA3, or rtTA4. The AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the AAV vector comprising a nucleic acid molecule encoding a reverse tetracycline-controlled transactivator (rtTA) may be present in a single AAV composition or as separate AAV compositions.

[0011] In some embodiments of the disclosed methods, a 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 response element (TRE). In some embodiments, the tetracycline antibiotic is doxycycline. In some embodiments, the inducible promoter is a TRE2 promoter.

[0012] 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.

[0013] 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).

[0014] 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.

[0015] 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.

[0016] In some embodiments of the disclosed methods, the nucleic acid molecules encoding OCT4, SOX2, and KLF4 are flanked by inverted terminal repeats (ITRs), and wherein the distance between the ITRs is 4.7 kb or less.

[0017] In some embodiments, the method further comprises administering an induction agent to the subject.

[0018] 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 contain nucleic acid molecules encoding OCT4, SOX2, and KLF4. The nucleic acid molecule encoding the reverse tetracycline-controlled transactivator (rtTA) can be an AAV vector comprising SEQ ID NO: 36 or SEQ ID NO: 37.

[0019] 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) an SV-40-derived terminator sequence; and i) a second inverted terminal repeat (ITR) sequence.

[0020] 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.

[0021] In some embodiments, the nucleic acid sequence encoding T2A is GAGGGCAGGGG AAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCA (SEQ ID NO: 10).

[0022] In some embodiments, the TRE promoter sequence is SEQ ID NO:7.

[0023] In some embodiments, the SV-40 derived terminator sequence is SEQ ID NO:12.

[0024] In some embodiments, the first ITR sequence is SEQ ID NO:16.

[0025] In some embodiments, the nucleic acid molecule encoding OCT4, SOX2, and KLF4 comprises SEQ ID NO:13.

[0026] In some embodiments, the nucleic acid molecule encoding OCT4, SOX2, and KLF4 comprises SEQ ID NO:14.

[0027] In some embodiments, nucleic acid molecules encoding OCT4, SOX2, and KLF4 and a nucleic acid molecule encoding rtTA are administered sequentially or simultaneously.

[0028] In some embodiments, nucleic acid molecules encoding OCT4, SOX2, and KLF4 are administered intravitreally.

[0029] In some embodiments, the nucleic acid molecule encoding rtTA is administered intravitreally.

[0030] In some embodiments, nucleic acid molecules encoding OCT4, SOX2, and KLF4 and nucleic acid molecules encoding rtTA are administered in a ratio of about 1:1.

[0031] In some embodiments, the AAV vector 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).

[0032] 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.

[0033] In some embodiments, the effective amount of the AAV2-TRE-OSK vector is about 1×10 9 vg / eye is about 1×10 14 vg / eye range.

[0034] In some embodiments, the effective amount of the pAAV2-CMV-rtTA3VP16 vector is about 1×10 9vg / eye is about 1×10 14 vg / eye range.

[0035] In some embodiments, the effective amount of the AAV2-CMV-rtTA4 vector is about 1×10 9 vg / eye is about 1×10 14 vg / eye range.

[0036] In some embodiments, nucleic acid molecules encoding OCT4, SOX2, and KLF4 are administered to a subject by left eye (OS) injection, right eye (OD) injection, or both eyes (OU) injection.

[0037] In some embodiments, the nucleic acid molecule encoding rtTA is administered to the subject by left eye (OS) injection, right eye (OD) injection, or both eyes (OU) injection.

[0038] In some embodiments, the method further comprises administering to the subject an effective amount of an antibiotic. In some embodiments, the antibiotic is administered at least one day prior to administering the nucleic acid molecule encoding rtTA. In some embodiments, the antibiotic is administered at the same time as administering the nucleic acid molecule encoding rtTA. In some embodiments, the antibiotic is administered at least one day after administering the nucleic acid molecule encoding rtTA.

[0039] In certain aspects, provided herein are methods for recombinantly producing AAV, the methods comprising introducing a vector into a cell under conditions that produce AAV, wherein the vector comprises one or more nucleic acid sequences encoding a) OCT4, SOX2, and KLF4. In some embodiments, the cell comprises a population of HEK293T cells.

[0040] Further provided herein are methods for producing AAV comprising modifying cells 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. In some embodiments, the cells comprise a population of HEK293T cells.

[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 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 encode one or more transcription factors, such as Nanog.

[0042] 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 comprising a polynucleotide encoding three transcription factors, wherein the transcription factors consist of OCT4, SOX2, and KLF4.

[0043] 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 comprising polynucleotides encoding four or more transcription factors, wherein the transcription factors comprise OCT4, SOX2, and KLF4, but do not comprise c-Myc. In some embodiments, the polynucleotide does not encode one or more transcription factors, such as Nanog.

[0044] In some embodiments, the composition does not reprogram the subject's cells, tissues, or organs to a pluripotent state. In some embodiments, the composition rejuvenates at least one cell, tissue, or organ of 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, rejuvenating at least one cell, tissue, or organ includes increasing the repair and / or regeneration of the cell, tissue, or organ. In some embodiments, rejuvenating at least one cell, tissue, or organ includes restoring the epigenetic information of the subject. In some embodiments, rejuvenating at least one cell, tissue, or organ comprises restoring epigenetic information lost in the cell, tissue, or organ due to aging, injury, disease, or any combination thereof. In some embodiments, rejuvenating at least one cell, tissue, or organ comprises reconstructing the epigenetic state of the cell, tissue, or organ to an epigenetic state closer to that of fertilization or terminal differentiation. In some embodiments, rejuvenating at least one cell, tissue, or organ comprises increasing the number of healthy axons in the subject. In some embodiments, rejuvenating at least one cell, tissue, or organ comprises preventing damage to healthy axons in the subject.

[0045] 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 a TRE3G promoter, a TRE2 promoter, a P tight promoter, and a tetracycline response element (TRE).

[0046] In some embodiments, the methods described herein further comprise administering an inducer to the subject to induce expression of OCT4, SOX2, and KLF4 in the subject. In some embodiments, the inducer comprises a tetracycline 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 located in an expression vector.

[0047] In some embodiments, the composition and the inducer are administered sequentially or simultaneously. In some embodiments, the composition is administered before the inducer. In some embodiments, the composition is administered after the inducer. In some embodiments, the composition and the inducer are administered simultaneously.

[0048] In some embodiments, the composition is administered at 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 to the inducer. In some embodiments, the composition is administered at a ratio of greater than about 100: 1. In some embodiments, the composition is administered at a ratio of less than about 1: 100 to the inducer. In some embodiments, the composition is administered at a ratio of about 1: 1 to the inducer.

[0049] In some embodiments, the polynucleotide comprises a self-cleaving peptide, such as a 2A peptide.

[0050] In some embodiments, the polynucleotide comprises inverted terminal repeats (ITRs).

[0051] In some embodiments, the expression vector is a viral expression vector selected from the group consisting of a lentivirus, a retrovirus, an adenovirus, an alphavirus, a vaccinia virus, and an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is serotype 2 (AAV2).

[0052] In some embodiments, the polynucleotide comprises nucleic acid elements in the following order:

[0053] a. first inverted terminal repeat (ITR) sequence;

[0054] b. TRE3G promoter sequence;

[0055] c.OCT4 sequence;

[0056] d. P2A cleavage sequence;

[0057] e. SOX2 sequence;

[0058] f.T2A cleavage sequence;

[0059] g.KLF4 sequence;

[0060] h. SV-40 derived terminator sequence; and

[0061] i. Second inverted terminal repeat (ITR) sequence.

[0062] 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.

[0063] In some embodiments, i) OCT4 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2; ii) SOX2 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:4; and / or iii) KLF4 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:6.

[0064] 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.

[0065] In some embodiments, i) the polynucleotide comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1; ii) the polynucleotide comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 3; and / or iii) the polynucleotide comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 5.

[0066] In some embodiments, i) the polynucleotide comprises a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 1; ii) the polynucleotide comprises a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 3; and / or iii) the polynucleotide comprises a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 5.

[0067] In some embodiments, i) the polynucleotide comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 1; ii) the polynucleotide comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 3; and / or iii) the polynucleotide comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 5.

[0068] 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.

[0069] In some embodiments, the composition is administered systemically. In some embodiments, the composition is administered topically to a tissue or organ. In some embodiments, the composition is administered intravitreally. In some embodiments, the composition is administered to a subject by injection into the left eye (OS), right eye (OD), or both eyes (OU).

[0070] In some embodiments, administration of the composition improves retinal ganglion cell (RGC) function and / or restores visual function in the subject.

[0071] In some embodiments, prevention or treatment of NAION can be measured by electroretinography (pERG). In some embodiments, prevention or treatment of NAION can be measured by electroretinography (pERG).

[0072] Also 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, wherein the gene therapy vector comprises 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 Kruppel-like factor 4 (KLF4), operably linked to at least one promoter (or operably linked to a recombination site, blunt-end ligation, or homologous site for genome editing to place the one or more nucleic acid molecules under the control of an endogenous promoter).

[0073] In some embodiments, the ischemic optic neuropathy is non-arteritic anterior ischemic optic neuropathy (NAION).

[0074] In some embodiments, the ischemic optic neuropathy is arteritic anterior ischemic optic neuropathy (A-AION).

[0075] In some embodiments, the ischemic optic neuropathy is posterior ischemic optic neuropathy (PION).

[0076] In some embodiments, the vector is administered intravitreally.

[0077] In some embodiments, when administered to the eye in vivo, the administration method delivers the one or more nucleic acid molecules to retinal ganglion cells.

[0078] In some embodiments, 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 octamer-binding transcription factor 4 (OCT4), sex-determining region (Y)-box 2 (SOX2), and Kruppel-like factor 4 (KLF4).

[0079] In some embodiments, the vector is a viral vector.

[0080] In some embodiments, the carrier is a liposome or lipid nanoparticle (LNP).

[0081] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector, optionally an oculotrophic AAV vector.

[0082] In some embodiments, the AAV vector is an AAV serotype 2 (AAV2) vector or a variant thereof.

[0083] 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 specific for the eye listed in Table 2.

[0084] In some embodiments, the one or more nucleic acid molecules is an AAV genome comprising flanking inverted terminal repeats (ITRs).

[0085] In some embodiments, the carrier is an LNP comprising an ionizable lipid, a helper lipid, a sterol, and a poly(ethylene glycol)-lipid (PEG-lipid).

[0086] In some embodiments, the promoter is an inducible promoter.

[0087] In some embodiments, the inducible promoter is a tetracycline response element (TRE) promoter, optionally a Tet-On promoter, optionally a TRE3G promoter.

[0088] In some embodiments, the method further comprises sequentially or concurrently 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 control the TRE promoter.

[0089] In some embodiments, the rtTA is selected from the rtTA listed in Table 1, optionally with MS RLDKSKIINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG (SEQ ID NO:20) an rtTA having at least 80%, at least 90%, at least 85% or 100% identity, and optionally comprising one or more amino acid substitutions selected from V9I, G12S, F67S, G72V, G72P and R171K, optionally comprising the amino acid acidic substitutions G12S, F67S and R171K or the amino acid substitutions V9I, G12S, F67S and R171K.

[0090] In some embodiments, the method further comprises administering a tetracycline inducer, optionally doxycycline or tetracycline.

[0091] In some embodiments, the vector is a polycistronic vector and the one or more nucleic acid molecules is a nucleic acid molecule comprising open reading frames encoding OCT4, SOX2, and KLF4.

[0092] In some embodiments, the open reading frame does not encode other proteins.

[0093] In some embodiments, OCT4, SOX2, and KLF4 are linked by a self-cleaving peptide (optionally a 2A peptide).

[0094] In some embodiments, the open reading frame encodes OCT4, SOX2, and KLF4 in 5' to 3' order.

[0095] In some embodiments, the method comprises administering an inducing agent to the subject for a period of time sufficient to rejuvenate the retinal ganglion cells but not to induce pluripotency in the cells.

[0096] In some embodiments, OCT4 is at least 80%, at least 90%, at least 85%, or 100% identical to SEQ ID NO:2; SOX2 is at least 80%, at least 90%, at least 85%, or 100% identical to SEQ ID NO:4, and / or KLF4 is at least 80%, at least 90%, at least 85%, or 100% identical to SEQ ID NO:6.

[0097] In some embodiments, the method improves retinal ganglion cell (RGC) function and / or restores visual function in the subject.

[0098] In some embodiments, wherein preventing or treating NAION is measured by electroretinography (pERG).

[0099] In another aspect, the present disclosure provides a gene therapy vector for use in any of the foregoing methods.

[0100] Further aspects and embodiments are provided in the detailed description below. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 is an illustrative vector map of TRE3G-OSK-SV40pA, an AAV2 vector encoding OSK (SEQ ID NO: 15).

[0103] Figure 2 An illustrative vector diagram of pAAV2-CMV-rtTA3VP16 (SEQ ID NO: 21) is depicted. This vector is a non-limiting example of a vector encoding rtTA (other examples of rtTA include, but are not limited to, rtTA-L and rtTA4).

[0104] Figure 3 An illustrative vector diagram of pAAV2-CMV-rtTA4 (SEQ ID NO: 28) is depicted. This vector is a non-limiting example of a vector encoding rtTA.

[0105] Figure 4 A schematic diagram depicting a non-limiting example of a Tet-ON system expressing OCT4, SOX2, and KLF4 (OSK) in the presence of tetracycline.

[0106] Figure 5 Schematic diagram of the study design for non-arteritic anterior ischemic optic neuropathy (NAION) in nonhuman primates (NHPs).

[0107] Figure 6Depicted are pattern electroretinogram (pERG) measurements showing that induction of nonarteritic anterior ischemic optic neuropathy (NAION) results in a decrease in the pERG signal. The pERG uses a contrast-reversing pattern stimulus (checkerboard) to assess macular retinal ganglion cell (RGC) activity. Changes in the pERG waveform indicate RGC dysfunction.

[0108] Figure 7 Graph comparing the absolute amplitude from p50 to n95 when treated with vehicle (left bar at each time point) or OSK (controlled by Dox; as a Tet-on system; right bar at each time point) before laser treatment (i.e., prevention study). pERG signals were measured on different days and compared under different treatments.

[0109] Figure 8 Graph comparing the absolute amplitude from p50 to n95 when treated with vehicle (left bar at each time point) or OSK (controlled by Dox; as a Tet-on system; right bar at each time point) after laser treatment (i.e., rescue study). pERG signals were measured on different days and compared under different treatments.

[0110] Figure 9 Depicted is the correlation between the initial damage to the optic nerve head due to laser (x-axis, degree of optic disc edema measured 8 days after laser treatment) and the degree of pattern electroretinogram (pERG, y-axis) defects at 5 weeks.

[0111] Figure 10A showed a correlation between the degree of early disc edema and pERG deficits in vehicle-treated RGCs at week 5 ( Figure 9 same). Figure 10B showed a reduced correlation between the degree of early optic disc edema and pERG deficits in OSK-treated NHPs at week 5, indicating reduced RGC cell damage.

[0112] Figure 11 Depicted is the correlation between the initial damage to the optic nerve head due to laser (x-axis, degree of optic disc edema measured 8 days after laser treatment) and the degree of axonal density damage at autopsy (9 weeks).

[0113] Figure 12A showed a correlation between the extent of early optic disc edema and axonal density impairment in vehicle-treated NHPs at the end of the study ( Figure 11 same). Figure 12B showed that the correlation between the degree of early optic disc edema and axonal density damage was reduced in OSK-treated NHPs at the end of the study, suggesting prevention or reversal of axonal damage. DETAILED DESCRIPTION

[0114] The disclosed methods may be more readily understood by reference to the following detailed description taken in conjunction with the accompanying drawings, which form a part of this disclosure. It should be understood that the disclosed methods are not limited to the specific methods described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed methods.

[0115] Unless expressly stated otherwise, any description of possible mechanisms or modes of action or reasons for improvement is illustrative only, and the disclosed methods are not limited by the correctness or incorrectness of any such suggested mechanisms or modes of action or reasons for improvement.

[0116] Herein, descriptions relate to compositions and methods of using the compositions. When the disclosure describes or claims a feature or embodiment relating to a composition, such feature or embodiment also applies to the method of using the composition. Similarly, when the disclosure describes or claims a feature or embodiment relating to a method of using a composition, such feature or embodiment also applies to the composition.

[0117] It should be understood that certain features of the methods herein described herein in the context of separate embodiments for clarity may also be provided in combination with a single embodiment. Conversely, various features of the disclosed methods described herein in the context of a single embodiment for clarity may also be provided separately or in any sub-combination.

[0118] Unless otherwise defined, all technical and scientific terms used herein are the same as those generally understood by those of ordinary skill in the art. In the event of conflict, this document (including definitions) shall prevail. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used when practicing or testing 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 merely illustrative and not intended to be limiting. The terms used herein are only for the purpose of describing specific embodiments and are not intended to be restrictive.

[0119] As used herein, the terms "comprising," "including," "having," "has," "may," "containing," and variations thereof are intended to serve as open transitional phrases, terms, or words that do not exclude the possibility of other actions or configurations. The term "comprising" is intended to include instances encompassed by the terms "consisting essentially of" and "consisting of; similarly, the term "consisting essentially of" is intended to include instances 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 set forth herein, whether or not explicitly stated.

[0120] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0121] For the recitation of numerical ranges herein, each number therebetween is expressly contemplated with equal precision. For example, for a range of 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for a range of 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.

[0122] Some quantitative expressions given herein are not qualified with the term "about." It should be understood that, regardless of whether the term "about" is explicitly used, each given quantity is intended to refer to the actual given value and also to the approximate value of the given value that would reasonably be inferred based on ordinary skill in the art, including approximate values ​​resulting from the experimental and / or measurement conditions of the value.

[0123] As used herein, the term "cell" refers not only to a single cell but also to the specific tissue or organ from which it originates.

[0124] "Gene expression" refers to the extent to which some or all genes in a cell or tissue are transcribed into RNA. In some cases, the RNA is translated into protein by the cell. The epigenome determines gene expression patterns.

[0125] The terms "disorder," "disease," and "condition" are used interchangeably. As used herein, an "ocular disease" or "eye disease" is a disease or disorder of the eye. An example of an ocular disease is non-arteritic anterior ischemic optic neuropathy.

[0126] Any suitable method can be used to measure ocular function. Non-limiting examples include visual acuity testing, pattern electroretinogram (pERG), and pathology.

[0127] As used herein, "cause of cellular senescence" includes loss or alteration of epigenetic information. As used herein, the terms "effective amount" and "therapeutically effective amount" refer to an amount that, when a compound or composition is administered to a subject, is effective in at least partially treating the condition from which the subject is suffering.

[0128] As used herein, a "functional" or "active" protein is one that retains its biological activity (e.g., is able to act as a transcription factor or inducer). In contrast, a non-functional or inactive protein is one that is unable to perform one or more of its wild-type functions.

[0129] The term "gene" refers to a nucleic acid fragment that expresses a protein, including regulatory sequences preceding the coding sequence (5' non-coding sequences) and following the coding sequence (3' non-coding sequences). A "native gene" refers to a gene found in nature that has its own regulatory sequences. A "chimeric gene" or "chimeric construct" refers to any gene or construct that is not a native gene and contains regulatory sequences and coding sequences that are not found together in nature. Thus, a chimeric gene or chimeric construct may contain regulatory sequences and coding sequences from different sources, or regulatory sequences and coding sequences from the same source but arranged in a manner 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 present in the host organism but is introduced into the host organism through gene transfer. Foreign genes can include native genes or chimeric genes inserted into non-native organisms. A "transgene" is a gene introduced into the genome through a transformation procedure.

[0130] By "homolog" or "homolog" is meant a sequence (e.g., a nucleic acid or amino acid sequence) that has a certain 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%, 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 sequences or orthologous sequences. Paralogous sequences are produced by the duplication of genes within the genome of a species, while orthologous sequences diverge after the speciation event. Functional homologs retain one or more biological activities of the wild-type protein. In certain embodiments, the functional homologs of OCT4, KLF4 or SOX2 retain 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 the wild-type counterpart.

[0131] The term "tissue" refers to any biological tissue (including a group of cells, a body part or an organ) or a part thereof, including blood vessels and / or lymphatic vessels, of a subject, to which the compounds, particles and / or compositions disclosed herein are delivered. The tissue may be abnormal, damaged or unhealthy tissue and may require treatment. The tissue may also be normal or healthy tissue, but the risk of becoming abnormal or unhealthy is higher than normal and may require prevention. In certain embodiments, the tissue is considered healthy, but its performance or survival under current or future conditions is not ideal. In certain embodiments, the tissue is the central nervous system. In certain embodiments, the cells or tissue are from the eye. In certain embodiments, the tissue is damaged (e.g., due to congenital defects, injuries, accidents or iatrogenic injuries), diseased and / or aging. In certain embodiments, the tissue is deep tissue accessible to a fiber optic probe.

[0132] In the context of damaged tissue, the term "tissue repair" refers to restoration of tissue structure, function, or a combination thereof following tissue injury. Tissue repair includes tissue regeneration, cell growth, tissue replacement, and / or rewiring (reprogramming) of existing tissue.

[0133] The term "tissue regeneration" refers to the generation of new tissue or cells within a tissue that are of the same type as the tissue of interest (eg, the same type as the damaged tissue or cells). In some embodiments, the methods provided herein promote organ regeneration.

[0134] The term "tissue replacement" refers to the production of a tissue that is a different type than the tissue of interest (eg, replacing damaged tissue with connective tissue).

[0135] As used herein, the term "treat" refers to reversing, alleviating a disease or disorder or one or more symptoms thereof, delaying its onset or inhibiting its progression, as described herein. In certain embodiments, treatment may be performed after the onset of one or more symptoms. In other embodiments, treatment may be performed in the absence of symptoms. In other embodiments, treatment may be performed to improve one or more of retinal perfusion, intraocular pressure, retinal layer thickness, RGC survival rate, retinal electrical response, macular nerve electrical response, optic nerve activity, retinal neural light response, retinal layer thickness (OCT), retinal RGC cell survival rate, visual acuity, etc. For example, susceptible individuals may be treated before the onset of symptoms. Treatment may also be continued after the symptoms disappear, for example, to prevent or delay recurrence.

[0136] The term "variant" refers to a sequence that contains modifications relative to the wild-type sequence. Non-limiting modifications of amino acid sequences include insertions, deletions, and point mutations. Non-limiting modifications of nucleic acid sequences include frameshift mutations, nucleotide insertions, and nucleotide deletions.

[0137] The term "WPRE" refers to the Woodchuck Hepatitis Virus (WHP) post-transcriptional regulatory element (WPRE). The WPRE creates a tertiary structure in a nucleic acid (e.g., an expression vector) and is capable of enhancing transgene expression (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.

[0138] Gene therapy

[0139] Gene therapy for eye diseases

[0140] Ocular diseases encompass a wide range of conditions that can severely impact vision and overall quality of life. Conventional treatment options aim to relieve symptoms or slow disease progression, and in some cases, surgical intervention is required. These approaches are not always curative, and despite the best available treatments, many patients continue to experience significant vision loss. Gene therapy has the potential to revolutionize ophthalmic care, providing new treatment options for patients who currently have limited options. The unique characteristics of the eye, including its immune-privileged status, small size, and compartmentalized structure, facilitate the efficient delivery and maintenance of gene therapy components without provoking an excessive immune response. This allows for local administration of therapeutic agents and minimizes the risk of systemic exposure. The eye can be assessed using non-invasive imaging techniques such as optical coherence tomography, funduscopy, angiography, and next-generation two-photon microscopy, facilitating real-time monitoring of treatment outcomes and safety. Examples of gene therapy for 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), the entire contents of which are incorporated herein by reference.

[0141] In an embodiment, the present disclosure provides a method for treating an eye disease or condition, including but not limited to a disease or condition associated with retinal ganglion cell damage. Specifically, the examples provided below demonstrate that the methods described herein are effective in treating a variety of eye diseases or conditions associated with eye damage, which are experimentally modeled by laser-induced damage. Without being bound by theory, the experimental laser heats blood vessels (e.g., arteries) in the eye, causing vascular damage, which in turn deprives retinal ganglion cells and other cells of oxygen, leading to ischemic cell damage; therefore, by demonstrating examples of preventing or treating such damage via a vitality restoration mechanism, it is shown that the method can be used to treat various ischemic optic neuropathies, including but not limited to non-arteritic anterior ischemic optic neuropathy.

[0142] Eye diseases

[0143] NAION

[0144] Nonarteritic anterior ischemic optic neuropathy (NAION) accounts for 95% of all anterior ischemic optic neuropathy (AION) and is the most common cause of acute optic neuropathy in people over 50 years of age, affecting approximately 2 to 10 people per 100,000 (approximately 1,500 to 6,000 new cases per year in the United States). Currently, there is no universally accepted treatment or secondary prevention for NAION, but some patients have traditionally been treated with steroids.

[0145] NAION refers to the interruption of blood flow to the small blood vessels that supply the front of the optic nerve. Vision loss from NAION is painless, rapid, and usually permanent. Risk factors for NAION include atherosclerosis (because this can impair blood flow through the blood vessels that supply the optic nerve) and a "tight" optic nerve. An optic nerve with a small or missing optic cup is also called a "risky optic disc" because it forms a "tight" channel through the sclera as it enters the eye. This tight channel through the sclera is thought to put further pressure on the small blood vessels that supply the optic nerve. This process ultimately leads to the optic nerve losing adequate blood flow and causing ischemic optic neuropathy. Attempts to treat NAION include radial nerve axonotomy to relieve mechanical pressure on the optic nerve and its supporting blood vessels. This surgery has all the risks of intraocular surgery and is difficult to perform. It is not uncommon for these structures to suffer collateral damage. In some embodiments, the ischemic optic neuropathy treated or prevented is non-arteritic anterior ischemic optic neuropathy (NAION).

[0146] A-AION

[0147] Arteritic AION (A-AION) is an eye disease caused by inflammation of the arteries that supply blood to the optic nerve. A-AION accounts for 5-10% of AION. The inflammation is caused by a disease called giant cell arteritis (GCA) or temporal arteritis, which can cause inflammation of medium and large arteries. If not diagnosed and treated quickly, GCA may be fatal and may damage the entire optic nerve head, leading to permanent, severe vision loss. The incidence of A-AION in women is 3 times that of men and most commonly affects people over 55 years old. In the embodiments, the ischemic optic neuropathy treated or prevented is arteritic anterior ischemic optic neuropathy (A-AION).

[0148] PION

[0149] Posterior ischemic optic neuropathy (PION) is a potentially devastating disease characterized by acute, painless vision loss in one or both eyes. PION can be divided into three categories: arteritic PION due to giant cell arteritis, non-arteritic PION, and perioperative PION. PION is caused by reduced blood flow and oxygen content to the intraorbital optic nerve. This may be due to a variety of risk factors: low blood pressure due to massive blood loss causing reduced arterial perfusion pressure, increased peripheral vascular resistance leading to reduced downstream blood flow, orbital edema leading to increased peripheral venous pressure, increased intraocular pressure, or decreased blood oxygen carrying capacity. In some embodiments, the ischemic optic neuropathy treated or prevented is posterior ischemic optic neuropathy (PION).

[0150] Physiological measurements

[0151] Various physiological measurements can be used to assess the effectiveness of the treatment and prevention methods described herein, including:

[0152] retinal ganglion cells

[0153] Retinal ganglion cells (RGCs) are neurons located near the inner surface of the retina (ganglion cell layer) of the eye. They receive visual information from photoreceptors via two interneuron types: bipolar cells and amacrine cells. Together, RGCs transmit both image-forming and non-image-forming visual information from the retina to several regions of the thalamus, hypothalamus, and midbrain or diencephalon.

[0154] RGCs vary widely in size, connectivity, and response to visual stimuli, but all share the characteristic of long axons extending into the brain. These axons form the optic nerve, optic chiasm, and optic tracts. A small subset of RGCs contribute little or nothing to vision but are themselves light-sensitive; their axons form the retinohypothalamic tract and influence circadian rhythms and the pupillary light reflex, which adjusts the size of the pupil. RGC types include dwarf RGCs, fimbriae RGCs, small stratified RGCs, large bistratified RGCs, smooth single-stratified RGCs, recursive single- and bistratified RGCs, spinous RGCs, large sparse RGCs, and intrinsically photosensitive RGCs containing melanopsin. RGC degeneration is the underlying cause of several conditions that lead to serious vision problems, including glaucoma, hereditary optic neuropathies, ischemic optic neuropathies, and demyelinating diseases.

[0155] In embodiments, treating or preventing an ocular disease or disorder may comprise treating or preventing RGC damage, 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 (preventing disease) or increase axon density or number (treating disease).

[0156] Pattern electroretinogram

[0157] The pattern electroretinogram (pERG) provides an objective measure of central retinal function. The pERG consists of two major components: a positive response at approximately 50 ms (P50) and a larger negative response at approximately 95 ms (N95). The P50 component is affected by macular dysfunction, with a concomitant decrease in the N95 component. The pERG complements the Ganzfeld ERG in the evaluation of patients with retinal diseases. 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, in which selective loss of the N95 is observed. Both macular dysfunction and optic nerve disease result in abnormalities in visually evoked cortical potentials (VEPs), and the pERG facilitates more meaningful VEP interpretation. The pERG allows for the assessment of retinal electrical activity related to RGC function.

[0158] In embodiments, treating or preventing an eye disease or condition may comprise treating or preventing RGC damage, assessing pERG. In embodiments, the methods described herein prevent a decrease in p50 amplitude (preventing disease) or increase p50 amplitude (treating disease). In embodiments, the methods described herein prevent a decrease in p50-n95 amplitude (preventing disease) or increase p50-n95 amplitude (treating disease).

[0159] Visual acuity test

[0160] Visual acuity testing is an eye exam that checks a subject's ability to see the details of letters or symbols at a specific distance. Visual acuity refers to a subject's ability to distinguish the shapes and details of what they see. Visual acuity is only one factor in a subject's overall vision. Other factors include color vision, peripheral vision, and depth perception. Examples of visual acuity tests include the Snellen eye chart, dynamic visual acuity test, pinhole acuity test, Cardiff visual acuity test, best corrected visual acuity (BCVA) using electronic visual acuity (EVA) with the E-ETDRS algorithm, and random E charts. Visual acuity tests can be used to help diagnose common conditions that affect vision, including nearsightedness, farsightedness, astigmatism, presbyopia, and color blindness.

[0161] In some embodiments, the methods described herein prevent a decrease in visual acuity amplitude (preventing a disease) or increase visual acuity amplitude (treating a disease).

[0162] Slit lamp examination

[0163] A slit lamp exam is a test that allows for the visual inspection and evaluation of every part of a person's eye. A slit lamp exam is performed using a slit lamp microscope with a bright light source and can be used to examine and evaluate different parts of a person's eye. The slit lamp light can be adjusted to allow for viewing through the various layers of the person's eye. A slit lamp exam can be used to examine and evaluate the overall health of a person's eye and diagnose any problems or symptoms. During a slit lamp exam, the cornea, sclera, conjunctiva, pupil, iris, lens, retina, and optic nerve can be examined. A slit lamp exam can be used to screen for eye conditions, including cataracts, glaucoma, dry eye, corneal scratches, corneal disease, macular degeneration, and retinitis pigmentosa.

[0164] Nucleic Acids

[0165] The present disclosure provides nucleic acid molecules comprising a nucleic acid sequence encoding OCT4, a nucleic acid sequence encoding SOX2, a nucleic acid sequence encoding KLF4, or a combination thereof, and in the absence of an exogenous nucleic acid sequence encoding c-Myc. The nucleic acid molecule can 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, 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. 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% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical 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, such as a protein from one or more mammals, including one or more primates (e.g., cynomolgus monkeys, rhesus monkeys). If two or more of OCT4, SOX2, and KLF4 are on a single nucleic acid molecule, they may be in any order. Terms such as "first," "second," and "third" do not necessarily imply the order of genes on a nucleic acid molecule.

[0166] 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-stranded or single-stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotides. This includes single-stranded and double-stranded molecules, i.e., DNA-DNA, DNA-RNA, and RNA-RNA hybrids.

[0167] The nucleic acids described herein can be synthesized by standard methods known in the art, for example, by using an automated DNA synthesizer (e.g., those commercially available from Biosearch, Applied Biosystems, etc.). Such DNA sequences can be incorporated into a variety of vectors having a suitable RNA polymerase promoter (e.g., T7 or SP6 polymerase promoter). The vector can be introduced into the body so that it is taken up by the cell and guides the transcription of the nucleic acid molecule. Such vectors can remain free or integrated into the chromosome as long as they can be transcribed. Such vectors can be constructed by recombinant DNA technology methods standard in the art. The vector can be a plasmid, a virus, or other vector known in the art for replication and expression in mammalian cells. The expression of the sequence can be achieved by any promoter known in the art that works 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 a recombinant DNA construct that can be administered to a subject.

[0168] Nucleic acid molecules may include 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, repressors, signal sequences, polyadenylation sequences, introns, 5" and 3" non-coding regions, etc. "Recombinant nucleic acid molecules" or "engineered nucleic acid molecules" are nucleic acid molecules that have been manipulated by molecular biology, i.e., non-naturally occurring nucleic acid molecules or genetically engineered nucleic acid molecules. In addition, the term "recombinant DNA molecule" or "engineered nucleic acid" refers to a non-naturally occurring nucleic acid sequence, or a nucleic acid sequence that can be made by artificially combining two originally separate nucleic acid sequence fragments, i.e., by joining together DNA fragments that are not normally adjacent. "Recombinantly produced" means that the artificial combination is usually achieved by chemical synthesis methods or by artificial manipulation of isolated nucleic acid segments, such as by genetic engineering techniques using restriction enzymes, ligases, and similar recombinant techniques, such as those described by Sambrook et al., Molecular Cloning, 2nd ed., 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, Vols. I and II (D. N. Glover, ed.) IREL Press, Oxford, (1985); each of which is incorporated herein by reference.

[0169] Such manipulations can be performed to replace codons with redundant codons encoding identical or conservative amino acids, and sequence recognition sites are typically introduced or removed. Alternatively, nucleic acid fragments with desired functions can be linked together to generate a single genetic entity comprising a combination of desired functions not found in nature. Restriction enzyme recognition sites are typically 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, can also be incorporated by design.

[0170] As used herein, a "terminator" or "terminator sequence" is a nucleic acid (s, engineered nucleic acid) sequence that stops transcription. A terminator can be unidirectional or bidirectional. It consists of a DNA sequence that participates in the specific termination of an RNA transcript by RNA polymerase. A terminator sequence prevents transcriptional activation of a downstream nucleic acid sequence by an upstream promoter. Therefore, in certain embodiments, terminators that terminate the production of an RNA transcript are contemplated.

[0171] The most commonly used terminator type is the forward terminator. When the forward transcription terminator is placed downstream of the nucleic acid sequence that is usually transcribed, it will cause transcription to stop. In some embodiments, a bidirectional transcription terminator can be used, which usually causes transcription to stop on both the forward strand and the reverse strand. In some embodiments, a reverse transcription terminator can be used, which usually only stops transcription on the reverse strand.

[0172] Non-limiting examples of mammalian terminator sequences include the bovine growth hormone terminator and viral terminator 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 that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 12.

[0173] In certain embodiments, the nucleic acid molecules of the present disclosure comprise an SV40-derived terminator sequence. 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.

[0174] In certain embodiments, the nucleic acid molecules of the present disclosure comprise a separator sequence that can be used to generate two separate amino acid sequences from one 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).

[0175] OSK

[0176] "OCT4" may also be referred to as octamer-binding transcription factor 4, OCT3, OCT3 / 4, POU 5F1, or POU 5-like homeobox 1, and is a transcription factor involved in embryonic development and cell fate determination. Similar to other OCT transcription factors, OCT4 is characterized by having a bipartite DNA binding domain called the POU domain. As used herein, the OCT4 transcription factor, homolog, or variant thereof may be derived from any species, including humans. In certain embodiments, the nucleic acid encoding human OCT4 (e.g., an engineered nucleic acid) is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to the nucleic acid (e.g., an engineered nucleic acid) described in NCBI RefSeq with 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 a nucleic acid (e.g., an 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 OCT4 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) 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) described in NCBI RefSeq with 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 encoding OCT4 transcription factors (e.g., engineered nucleic acids) can be found in public databases, including GenBank. For a detailed description of the crystal structure and structure-function analysis of OCT 4, see Remenyi et al., Genes Dev. 17(16): 2048–2059 (2003), Yesudhas et al., PLos One 11(1): e0147240 (2016), and Michael et al., Science 368(6498): 1460-1465 (2020); the entire contents of which are incorporated herein by reference. Yesudhas et al. identified key residues and hydrogen bonds in OCT4 that may facilitate protein-protein and protein-DNA interactions.

[0177] "SRY-box 2" or "SOX2" is a member of the SRY-related HMG-box (SOX) family of transcription factors. SOX2 is associated with promoting embryonic development. Members of the SOX (SRY-related HMG-box) transcription factor family are characterized by a high-mobility group 5 (HMG)-box DNA sequence. The HMG box is a DNA binding domain that is highly conserved among eukaryotic species. As used herein, the SOX2 transcription factor, homolog, or variant thereof may be derived from any species, including humans. In certain embodiments, the nucleic acid encoding SOX2 (e.g., an engineered nucleic acid) 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 described in NCBI RefSeq with accession number NM_011443.4 (e.g., an engineered nucleic acid). 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) described in NCBI RefSeq with 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 encoding human SOX2 (e.g., an engineered nucleic acid) 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 with accession number NP_003097.1. In some cases, 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 cases, 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 a detailed description 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); the entire contents of which are incorporated herein by reference. Yesudhas et al. identified key residues and hydrogen bonds in SOX2 that may promote protein-protein and protein-DNA interactions.

[0178] "KLF4" may also be referred to as Kruppel-like factor 4, EZF or GKLF, and is a zinc finger transcription factor. KLF4 is associated with the regulation of differentiation and proliferation and is capable of interacting with coactivators, including members of the p300-CBP coactivator family. As used herein, the KLF4 transcription factor, homolog (e.g., functional homolog) or variant thereof may be derived from any species, including humans. In certain embodiments, the nucleic acid encoding human KLF4 (e.g., engineered nucleic acid) 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 described in the NCBI RefSeq database with accession number NM_004235.5 or NM_001314052.1 (e.g., engineered nucleic acid). Non-limiting examples of KLF4 variants include Krueppel-like factor 4 transcript variant 1 and Krueppel-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%) homologous 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%) homologous 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%) homologous 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 a detailed description of the KLF4 crystal structure and structure-function analysis, see Borisova et al., iScience 25(1):103525 (2021), Schuetz et al., Cell Mol Life Sci. 68(18):3121-3131 (2011), and Liu et al., Nucleic Acids Res. 42(8):4859-4867 (2014); the entire contents of which are incorporated herein by reference. Borisova et al. identified amino acid substitutions in the KLF4 zinc finger domain that enhance protein function.

[0179] The term "c-Myc" or "Myc" refers to a nuclear phosphoprotein associated with the cell cycle process. c-Myc can form a heterodimer with the transcription factor MAX, and the heterodimer can bind to the E-box sequence on the nucleic acid (e.g., engineered nucleic acid) to regulate the transcription of the target gene. In certain embodiments, the nucleotide 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 the sequence described in the NCBI RefSeq database with 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 NP 002458.2 or NP 001341799.1. In certain embodiments, the method comprises inducing the expression of OCT4; KLF4; SOX2; or any combination thereof in the absence of induced c-Myc expression or the absence of activated c-Myc. The absence of induced c-Myc expression may refer to the absence of significant induction of c-Myc expression compared to the endogenous c-Myc expression level in the cell, tissue, subject, or any combination thereof. The absence of significant induction of c-Myc expression compared to the endogenous c-Myc expression level in the cell, tissue, subject, or any combination thereof may refer to an increase in c-Myc expression of 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 the endogenous c-Myc expression level in the cell, tissue, subject, or any combination thereof. The absence of activated c-Myc expression may refer to the absence of significant activation of c-Myc (e.g., activity) relative to endogenous c-Myc activity in the cell, tissue, subject, or any combination thereof. Absence of significant induction of c-Myc activity compared to endogenous c-Myc activity in a cell, tissue, subject, or any combination thereof can refer to an increase in c-Myc activity of less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or any value in between, compared to endogenous c-Myc activity in a cell, tissue, subject, or any combination thereof.

[0180] constitutive promoter

[0181] The term "promoter" refers to the control region of a nucleic acid sequence, within which the transcription initiation and rate of the remainder of the nucleic acid sequence are controlled. The promoter may also contain subregions to which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. The promoter may be constitutive, inducible, activatable, repressive, tissue-specific, or any combination thereof. The promoter drives the expression or transcription of the nucleic acid sequence it regulates. In this article, the promoter is considered to be "operably connected" when it is in the correct functional position and direction relative to the nucleic acid sequence it regulates, to control ("drive") the transcription initiation of the sequence, the expression of the sequence, or a combination thereof.

[0182] In some embodiments, the promoter is a eukaryotic promoter. The promoter can promote universal expression or tissue-specific expression of an operably connected nucleic acid sequence from any species (including people). In some embodiments, the promoter is a eukaryotic promoter. Non-limiting examples of eukaryotic promoters include TDH3, PGK1, PKC1, TDH 2, PYK1, TPI1, AT1, CMV, EF1 α, SV40, PGK1 (people or mice), Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMK IIa, GAL1, GAL10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1 and U6, as known to those of ordinary skill in the art (see, for example, Addgene website: blog.addgene.org / plasmids-101-the-promoter-region).

[0183] 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-globin intron, human synapsin, EF1α, SV40 promoter, PGK1, Ubc, CAG, human β-actin gene promoter, RSV promoter, EFS promoter, and promoters comprising an upstream activation 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 the document are incorporated herein by reference.

[0184] Non-limiting examples of constitutive promoters include CP1, CMV, EF1α, 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), rhodopsin kinase promoter (rk promoter). In some cases, the constitutive promoter is the Rosa26 promoter. In some cases, 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.

[0185] In certain embodiments, nucleic acid molecules of the present disclosure comprise a constitutive promoter, such as one or more of CP1, CMV, EF1α, SV40, PGK1, Ubc, human beta actin, CAG, Ac5, polyhedrin, TEF1, GDS, CaM3 5S, Ubi, H1, and / or U6 promoters. The constitutive promoter can 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 more than one constitutive promoter.

[0186] Inducible promoter

[0187] An "inducible promoter" is a promoter characterized by initiating or enhancing transcriptional activity when in the presence of, under the influence of, or in contact with an inducing agent. The inducing agent can be an endogenous or generally exogenous condition, compound, agent, or protein that contacts the engineered nucleic acid in a manner that actively induces 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).

[0188] Inducible promoters for use in accordance with the present disclosure include any inducible promoter described herein or known to those of ordinary 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 protein (TetR or TetRKRAB), tetracycline operator sequence (tetO) and tetracycline transactivator fusion protein (tTA), and tetracycline operator sequence (tetO) and reverse tetracycline transactivator fusion protein (rtTA)), steroid-regulated promoters, and the like. The promoters of the invention include promoters based on the rat glucocorticoid receptor, the human estrogen receptor, the moth ecdysone receptor, and promoters from the steroid / retinoic acid / thyroid 25 receptor superfamily, metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and chelate metal ions) genes from yeast, mouse, and human), pathogenesis-regulated promoters (e.g., inducible 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.

[0189] In the field of genetic engineering, precise control of gene expression is a valuable tool for studying, manipulating and controlling development and other physiological processes. Gene expression is a complex biological process involving many specific protein-protein interactions. Strictly regulated inducible gene expression systems or "gene switches" can be used for various applications, such as gene therapy, large-scale production of proteins in cells, cell-based high-throughput screening assays, functional genomics, and the regulation of traits in transgenic plants and animals. Inducible promoters are useful because they can be used to turn on or off the expression of genes operably connected thereto at certain stages of organism development, in specific tissues, or at certain stages of treatment. Examples of inducible promoters and on / off gene expression systems are 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 instability domains, riboswitches, and hormone-activated promoters. Many other systems have been described and can be easily selected by those skilled in the art. Specific examples of inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionein (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 that are regulated by specific physiological states (e.g., temperature, acute phase, a specific differentiation state of the cell), or those that are regulated only in replicating cells. Any type of inducible promoter that is tightly regulated and specific for a particular target ocular cell type can be used. Exemplary inducible promoters for ocular therapy 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), the entire contents of which are incorporated herein by reference. Exemplary inducible promoters for mammalian cells are described in US20200283778, Kalunke 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), the entire contents of which are incorporated herein by reference.

[0190] Additional non-limiting examples of inducible promoters include mifepristone-responsive promoters (e.g., GAL4-Elb promoter) and coumermycin-responsive promoters. See, e.g., Zhao et al., Hum Gene Ther. 2003 Nov 20; 14(17): 1619-29.

[0191] In some embodiments, the nucleic acid molecules of the present disclosure include inducible promoters. In some embodiments, the nucleic acid molecules have an inducible promoter. In this case, the expression of OCT4, SOX2 and KLF4 is controlled by the same inducible promoter. In some embodiments, the nucleic acid molecules have more than one inducible promoter. Inducible promoters may include tetracycline response elements (TRE) (e.g., TRE3G promoter, TRE2 promoter or P tight promoter), mifepristone response promoters (e.g., GAL4-Elb promoter) or coumermycin responsiveness). As an example, TRE (e.g., TRE3G) promoters may include 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, for example, U.S. Published Application No. 2021-0403923A, and International Publication No. WO2020 / 069339.

[0192] In certain embodiments, the inducing agent is capable of inducing expression of a first (e.g., OCT4), a second (e.g., SOX2), a 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; U.S. 7541446B2; US5650298A; US8383364B2; Zhou et al., Gene Ther. 13:1382–90 (2006) and in an application titled “MUTANT REVERSE TETRACYCLINE TRANSACTIVATORS FOR EXPRESSION OF GENE”. GENES); each of which is incorporated herein by reference in its entirety. In certain embodiments, rtTA is rtTA3 comprising 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, rtTA3 is encoded by a nucleic acid sequence 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, rtTA is rtTA4 and 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% (eg, at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:26.

[0193] In certain embodiments, the inducing agent is capable of inducing expression of the first nucleic acid (e.g., OCT4), the second nucleic acid (e.g., SOX2), the third nucleic acid (e.g., KLF4), or any combination thereof from an inducible promoter in the absence of tetracycline (e.g., doxycycline).

[0194] In certain embodiments, the inducing agent is tetracycline-controlled transactivator (tTA).

[0195] Tissue-specific promoter

[0196] Non-limiting examples of tissue-specific promoters include eye-specific promoters. Non-limiting examples of eye-specific promoters include human GRK1 (rhodopsin kinase) promoter, human CRX (cone-rod 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 variant of glial fibrillary acidic protein (GFAP), monocyte chemoattractant protein-1 (Mcp1), short promoter variant of mouse cone arrestin (mCAR), short promoter variant of human neurofilament heavy polypeptide, and human NRL promoter (neural retinal 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 Khani et al., Invest Ophthalmol Vis Sci. 48(9):3954-3961 (2007); the reference is incorporated herein by reference in its entirety. Non-limiting examples of RGC-specific promoters include 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), microtubule-depolymerizing protein (stathmin) 2 (STMN2), perilocalizing protein (PRPH1), cartilage acidic protein 1 (CRTAC1), RNA binding protein-mRNA processing factor (RBPMS), Ras-associated protein Rab-13 (RAB13), ATPase Na+ / K+ transport subunit beta 1 (ATP1B1), fatty acid binding protein 3 (FABP3), mouse γ-synuclein, human γ-synuclein genes, all of which are described in Hanlon et 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); the references are incorporated herein by reference in their entirety.

[0197] In some embodiments, the promoters disclosed herein are suitable for use in AAV vectors. See, for example, U.S. Patent Application Publication No. 2018 / 0155789, which is incorporated herein by reference in its entirety for this purpose.

[0198] rtTA system

[0199] "Tetracycline" refers to tetracycline antibiotic compounds, including but not limited to tetracycline, chlortetracycline, oxytetracycline, demeclocyline, lymecycline, meclocycline, methacycline, minocycline, rolitetr acycline, doxycycline, tigecycline, eravacycline, sarecycline, and omadacycline. For 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.

[0200] In certain embodiments, the inducible promoter comprises a tetracycline (Tet) response element. For example, the inducible promoter can 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 can 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.

[0201] As used herein, "reverse tetracycline transcription activator" ("rtTA") is an inducing agent that binds to a TRE promoter (e.g., TRE3G, TRE2 promoter, or P tight promoter) in the presence of tetracycline (e.g., doxycycline) and is capable of driving expression of a transgene operably linked to the TRE promoter. rtTA typically comprises a mutant tetracycline repressor DNA binding protein (TetR) and a transcriptional activation domain (see, e.g., Gossen et al., Science. 1995 Jun 23; 268(5218): 1766-9 and any transcriptional activation domains listed herein). The mutant TetR domain is capable of binding to the TRE promoter when bound to tetracycline. See, for example, 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 “Mutated reverse tetracycline transactivator for expressing genes,” each of which is incorporated herein by reference in its entirety.

[0202] As used herein, the "Tet-Off" system is an inducible system that is capable of inhibiting the expression of a specific transgene in the presence of tetracycline (e.g., doxycycline (DOX)). Conversely, the Tet-Off system is capable of inducing the expression of a specific transgene in the absence of tetracycline (e.g., doxycycline, DOX). In certain embodiments, the 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 transgene with the tetracycline-responsive promoter (e.g., TRE3G, Ptight, or TRE2) and the tetracycline-controlled transcriptional activator can 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 “Mutated Reverse Tetracycline Transactivator for Expressing Genes,” each of which is incorporated herein by reference in its entirety.

[0203] As used herein, the "Tet-On" system is an inducible system that is capable of inducing the expression of a specific transgene in the presence of tetracycline (e.g., doxycycline (DOX)). In certain embodiments, the Tet-On system comprises a tetracycline-responsive promoter operably linked to a transgene (e.g., encoding OCT4; KLF4; SOX2; or any combination thereof) and a reverse tetracycline-controlled transactivator (rtTA). For example, rtTA can be any of the rtTa systems listed in Table 1 or a variant thereof. In certain embodiments, the nucleic acid encoding rtTA3 comprises a sequence that is 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 that 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: 20. In certain embodiments, the nucleic acid encoding rtTA4 comprises a sequence that 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: 26. In certain embodiments, rtTA4 comprises an amino acid sequence that 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: 27. Expression cassettes encoding a tetracycline-responsive promoter (e.g., a promoter comprising a TRE, including TRE3G, Ptight, and TRE2) and a reverse tetracycline-controlled transcriptional activator can 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.

[0204] As used herein, a "TRE promoter" is 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, the TRE promoter further comprises a minimal promoter located downstream of the tet-O sequence. A minimal promoter is a promoter that comprises the minimal elements of a promoter (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 Tet-O). For example, a minimal promoter can be a minimal CMV 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: 17 or 24. For example, the TRE promoter can be a TRE3G promoter (e.g., a TRE3G 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:7).

[0205] Table 1 - Representative rtTA Systems

[0206]

[0207] Polycistronic vectors

[0208] A "polycistronic vector" is a vector that encodes more than one amino acid sequence (e.g., a vector encoding OCT4 and KLF4, OCT4 and SOX2, KLF4 and SOX2, or OCT4, SOX2, and KLF4 (OSK)). Polycistronic 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) can be linked or separated to produce unlinked proteins. For example, an internal ribosome entry site (IRES) or a polypeptide cleavage signal can be placed 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 that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 10. The P2A peptide can comprise a sequence that is at least 70% (eg, at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:8.

[0209] In some embodiments, the expression vectors of the present disclosure are multicistronic expression vectors.

[0210] In certain embodiments, the nucleic acid molecule is a viral vector (e.g., a lentiviral, retroviral, or adeno-associated viral (AAV) vector). The AAV vectors of the present disclosure typically comprise inverted terminal repeats (ITRs) flanking a 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).

[0211] In certain embodiments, the nucleic acid molecules of the present disclosure (e.g., expression vectors encoding OCT4, KLF4, SOX2, inducers, or a combination thereof) may further comprise a nucleic acid sequence encoding a selection agent (e.g., antibiotics 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, FLAG tag, HA tag, His tag, Myc tag, V5 tag, etc.).

[0212] In some embodiments, the expression vector encoding OCT4, KLF4, and SOX2 comprises the sequence provided 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 Figure 1 The expression vector may be a viral vector. The viral vector may be an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector, a herpes virus vector, or the like.

[0213] Delivery vehicle

[0214] viral vectors

[0215] A "recombinant virus" is a virus (e.g., a lentivirus, adenovirus, retrovirus, herpes virus, alphavirus, vaccinia virus, or adeno-associated virus (AAV)) that is isolated from its natural environment (e.g., from a host cell, tissue, or subject) or artificially produced.

[0216] A variety of adeno-associated virus (AAV) serotypes have been identified, including 12 human serotypes and more than 100 non-human primate serotypes (Howarth et al., 2010, Cell Biol Toxicol 26: 1-10). Of these serotypes, human serotype 2 was the first AAV 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. In addition, AAV serotypes for treating ocular diseases are described in the following: Ghoraba et al., Clin. Opthalmol. 16: 1753-1771 (2022), Bordet et al., Drug Discovery Today 24, 8: 1685-1693 (2019) and Dalkara et al., Sci. Transl. Med. 5, 189 (2013); the entire contents of which are incorporated herein by reference. Exemplary AAV serotypes for ocular therapy are described in U.S. Patent No. 9,567,376, U.S. Patent No. 10,383,922 and U.S. Patent No. 10,426,844, the entire contents of which are incorporated herein by reference. In addition, non-natural engineered variants and chimeric AAVs may also be useful. Specifically, the capsid protein may be a variant comprising one or more amino acid substitutions that enhance transduction efficiency.

[0217] "AAV" or "adeno-associated virus" is a non-enveloped virus that can carry and deliver nucleic acids (e.g., encoding OCT4; KLF4; SOX2; or engineered nucleic acids of any combination thereof) and belongs to the genus Dependoparvovirus. In some cases, AAV is capable of delivering nucleic acids encoding inducers. In general, AAV does not integrate into the genome. The tissue-specific targeting ability of AAV is generally determined by the AAV capsid serotype (see, for example, Table 2 below for examples of AAV serotypes and their practicality 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).

[0218] Table 2. Non-limiting examples of AAV serotypes and their use in specific tissues

[0219]

[0220] As used herein, the term "AAV vector" is 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 (alone or in combination), or an expression cassette encoding rtTA or tTA). The AAV vector may further comprise a promoter sequence.

[0221] "Inverted terminal repeats" or "ITRs" are nucleic acid sequences that are reverse complementary to each other. Generally, in AAV vectors, ITRs flank a cassette (e.g., an expression cassette comprising a nucleic acid encoding OCT4, KLF4, SOX2, or any combination thereof). For example, the ITRs flanking the OSK cassette may comprise SEQ ID NOs: 16 and 32. Similarly, in some cases, the pAAV2-CMV-rtTA3VP16 vector disclosed herein may comprise ITRs comprising SEQ ID NOs: 22 and 33, and the AAV2-CMV-rtTA4 vector disclosed herein may comprise ITRs comprising SEQ ID NOs: 29 and 34. In some cases, the cassette encodes an inducer. AAV ITRs include ITRs from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV variants thereof.

[0222] In one aspect, the present disclosure provides recombinant viruses. The recombinant virus may include one or more lentiviruses, adenoviruses, retroviruses, herpes viruses, alphaviruses, vaccinia viruses, or adeno-associated viruses (AAVs), comprising any expression vector described herein. The use of recombinant lentiviruses for treating ocular diseases is described in Arsenijevic Y. et al., Pharmaceutics 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 the documents 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, e.g., OCT4, SOX2, KLF4, and another transcription factor.

[0223] Lipid nanoparticles (LNPs)

[0224] The compositions of the present disclosure can be introduced into suitable host cells using delivery vehicles (e.g., liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc.). Specifically, any nucleic acid (e.g., engineered nucleic acid) (e.g., expression vector) capable of inducing expression of OCT4, KLF4, and / or SOX2; any engineered protein; any chemical agent that activates OCT4, KLF4, and / or SOX2 (e.g., induces its expression); any antibody that activates OCT4, KLF4, and / or SOX2 (e.g., induces its expression); engineered cells; and / or any recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) can be encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, etc. In some embodiments, any nucleic acid (e.g., engineered nucleic acid) (e.g., expression vector) capable of inducing expression of OCT4, KLF4, SOX2, or any combination thereof; any engineered protein; any chemical agent that activates OCT4, KLF4, SOX2, or any combination thereof (e.g., induces expression thereof); any antibody that activates OCT4, KLF4, SOX2, or any combination thereof (e.g., induces expression thereof); 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, or nanoparticle, etc. The inducer (e.g., a nucleic acid encoding an inducer or a protein encoding an inducer and / or a recombinant virus encoding an inducer) and / or a chemical agent capable of modulating the activity of an inducer can be encapsulated in a lipid particle, liposome, vesicle, nanosphere, or nanoparticle, etc.

[0225] In some embodiments, any nucleic acid, engineered protein, chemical agent, antibody, and / or recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) is formulated in a lipid nanoparticle. See, e.g., Cullis and Hope Mol Ther. 2017 Jul 5;25(7):1467-1475. In some embodiments, the lipid nanoparticle comprises one or more membrane fusion proteins that deliver the plasmid directly to the cytoplasm, or factors OCT4; KLF4; SOX2; or any combination thereof can be fused directly to the targeting protein with or without nanoparticle encapsulation. In some embodiments, the lipid nanoparticle is a Fusogenix lipid nanoparticle. In some embodiments, the lipid nanoparticle is a "wrapped liposome" (WL). See, e.g., Yamauchi et al., Biochim Biophys Acta. 2006 Jan;1758(1):90-7. In some embodiments, the lipid nanoparticle is a pegylated liposome (e.g., DOXIL TM) (e.g., Allen and Hansen, Biochim Biophys Acta. 1991 Jul 1;1066(1):29-36), 1,2-dioleoyl-sn-glycero-3 phosphatidylethanolamine (DOPE), a 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 and Huang, Biochim Biophys Acta. 1994 Jan 19;1189(2):195-203). In some embodiments, the lipid nanoparticle or fusion protein comprises a molecule or protein that mimics the methods used by viruses for intracellular delivery of macromolecules (e.g., Kobayashi et al., Bioconjug Chem. 2009 May 20;20(5):953-9), for example, using a variety of pH-sensitive peptides, such as vesicular stomatitis virus protein (VSV G), bacteriophage coat protein and / or shGALA, and / or fusion-associated small transmembrane (FAST) proteins, such as avian reovirus (ARV), Nelson Bay reovirus (NBV) and baboon reovirus (BBV), aquatic reovirus (AQV) and reptilian reovirus (R RV), and / or caerulein targeting peptides. See, e.g., Peisajovich et al., Eur J Biochem. 2002 Sep;269(17):4342-50; Sakurai et al., 2011. See also, Targeted Intracellular Therapeutic Delivery Using Liposomes Formulated with Multifunctional FAST proteins, Western University Thesis, 2012.

[0226] In some embodiments, nucleic acids encoding OCT4, KLF4, SOX2 or a combination thereof (e.g., RNA or DNA, including plasmids) 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, lipid nanoparticles comprise viral membrane proteins. Without being bound by a particular theory, lipid nanoparticles may be non-toxic because they comprise membrane fusion proteins of non-viral membrane fusion proteins. Non-limiting examples of membrane fusion proteins include membrane fusion proteins disclosed in U.S. Patent Nos. 7,851,595, 8,252,901, International Application Publication No. WO 2012 / 040825, and International Application Publication No. WO 2002 / 044206.

[0227] In some embodiments, the compositions of the present disclosure (e.g., comprising nucleic acids encoding OCT4, KLF4, SOX2, or a combination thereof) are delivered non-virally. Methods for non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycationic or lipid:nucleic acid conjugates, naked nucleic acids (e.g., RNA or DNA), artificial viral particles, and agent-enhanced nucleic acid (e.g., RNA or DNA) uptake.

[0228] In some embodiments, the delivery vehicle targets the cargo. For example, any nucleic acid, engineered protein, chemical agent, antibody, and / or recombinant virus described herein (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) can be delivered via nanoparticles that deliver the cargo to a specific tissue or cell type. For example, nanoparticles coated with galactose polymers are known to release their cargo in aging cells due to their endogenous β-galactosidase activity. See, for example, Lozano-Torres et al., J Am Chem Soc. Jul 5, 2017; 139(26): 8808-8811.

[0229] In some embodiments, the delivery of nucleic acids not on viral vectors includes administration of naked nucleic acids, electroporation, use of nanoparticles, and / or use of liposomes. As non-limiting examples, the engineered nucleic acids of the present disclosure (e.g., RNA, including mRNA, or DNA) can be formulated in nanoparticles for delivery. See, for example, Dong et al., Nano Lett. February 10, 2016; 16(2):842-8. In some embodiments, the nanoparticles comprise acetylated galactose. See, for example, Lozano-Torres et al., J Am Chem Soc. July 5, 2017; 139(26):8808-8811. In some embodiments, engineered nucleic acids (e.g., RNA, including mRNA, or DNA) are electroporated or transfected into cells. In certain embodiments, engineered nucleic acids are delivered as naked nucleic acids (e.g., naked DNA or naked RNA).

[0230] In some embodiments, the engineered nucleic acid formulated for delivery in nanoparticles is not an AAV vector. Vector backbones suitable for formulation in nanoparticles include, but are not limited to, NANOPLASMID TM 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 a particular theory, the NTC'8' series mammalian expression vectors may be useful because they are typically cleared by cells within a few weeks. The NTC'8' series mammalian expression vectors contain a CMV promoter that can be operably linked to a sequence encoding OCT4, KLF4, SOX2, or a combination thereof. Without being bound by a particular theory, NANOPLASMID TM The vector may be less immunogenic than other vectors, have higher expression levels, and can express for a longer period of time, which may facilitate long-term expression of operably linked nucleic acids. TM The vector can be used for long-term expression of OCT4, KLF4, SOX2, or a combination thereof.

[0231] In some embodiments, proteins encoding OCT4, SOX2, and / or KLF4 and / or inducers are formulated in 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 in nanoparticles (e.g., for nuclear delivery). In certain embodiments, the nanoparticles further comprise a protein encoding an inducer. For example, chitosan [poly(N-acetylglucosamine)] is a biodegradable polysaccharide and can be used to formulate nanoparticles by several methods. In some embodiments, chitosan polymeric nanoparticles are loaded with proteins encoding OCT4, SOX2, and / or KLF4 and / or inducers and delivered to the nucleus. See, e.g., Tammam et al., Oncotarget. 2016 Jun 21;7(25):37728-37739.

[0232] Delivery channels

[0233] As used herein, the terms "administer," "administering," or "administration" refer to introducing into a subject any composition described herein; any nucleic acid capable of inducing expression of OCT4, KLF4, and / or SOX2; any nucleic acid capable of inducing expression of one or more transcription factors selected from the group consisting of OCT4, KLF4, SOX2, and any combination thereof; any engineered protein described herein; any chemical agent that activates OCT4, KLF4, and / or SOX2 (e.g., induces expression thereof); any chemical agent that activates one or more transcription factors selected from OCT4, KLF4, SOX2, and any combination thereof (e.g., induces expression thereof); any antibody that activates OCT4, KLF4, and / or SOX2, and any combination thereof (e.g., induces expression thereof); and / or any recombinant virus described herein (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV), alone or in combination, into 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 composition described herein, comprising any nucleic acid capable of inducing expression of one or more transcription factors selected from OCT4, KLF4, SOX2, and any combination thereof; any chemical agent that activates OCT4, KLF4, and / or SOX2 (e.g., induces expression of, for example, tetracycline); any engineered protein encoding OCT4, SOX2, KLF4, or any combination thereof; any chemical agent that activates OCT4, KLF4, SOX2, or any combination thereof (e.g., induces expression of, for example, tetracycline); any antibody that activates OCT4, KLF4, and / or SOX2 (e.g., induces expression thereof); and / or any recombinant virus described herein (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) can be administered intravitreally, intraocularly, subconjunctivally, or subretinaly, alone or in combination. In other aspects, administration can be intravenous, intradermal, intraarterial, intralesional, intratumoral, intracranial, intraarticular, intraprostatic, intrapleural, intranasal, intravitreal, intravaginal, intrarectal, topical, intratumoral, intramuscular, intraperitoneal, subcutaneous, subconjunctival, intracapsular, transmucosal, intrapericardial, intraumbilical, intraocular, oral, external, topical, systemic, injection, infusion, continuous infusion, local perfusion directly infiltrating target cells, via a catheter, in a cream, in a lipid composition (e.g., liposomes), or by other methods known to those skilled in the art, or any combination of the foregoing methods (see, e.g., Remington's Pharmaceutical Sciences (1990), incorporated herein by reference).In some embodiments, a composition 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 is also administered to cells, tissues, organs, and / or subjects using any suitable method (e.g., intravitreally, intraocularly, subconjunctivally, or subretinally).

[0234] Treatment options for NAION

[0235] Further provided herein is a method for treating non-arteritic anterior ischemic optic neuropathy (NAION) in a subject in need thereof, wherein the method comprises administering to the subject one or more agents for raising OCT4, SOX2, KLF4 and / or one or more combinations thereof. One or more agents will not raise c-Myc. One or more agents that raise OCT4, SOX2, KLF4 (OSK) may include one or more means for inducing OSK expression, including DNA, RNA, small molecules, etc. In some embodiments, the method comprises administering to the subject one or more nucleic acid molecules as described herein. In some embodiments, the one or more nucleic acid molecules include a nucleic acid molecule system having at least two nucleic acid molecules.

[0236] In some embodiments of the method for treating NAION of a subject, the agent for raising OSK expression includes at least one nucleic acid molecule encoding OSK as described above. The nucleic acid molecule encoding OSK does not include a nucleic acid sequence encoding c-myc. The nucleic acid molecule encoding OSK can be an adeno-associated virus (AAV) vector. According to some embodiments, the method further includes 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) can be an AAV vector. In some embodiments, the AAV vector comprising a nucleic acid molecule encoding a reverse tetracycline-controlled transactivator (rtTA) is not the same AAV vector as the AAV vector comprising a nucleic acid molecule encoding OCT4, SOX2, and KLF4.

[0237] 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 tetracycline antibiotics. Tetracycline antibiotics are known in the art and include, for example, tetracycline, chlortetracycline, oxytetracycline, demeclocycline, lymeclocycline, meclocycline, methecycline, minocycline, rolicycline, doxycycline, tigecycline, eracycline, sarecycline and omadacycline. Doxycycline is a typical tetracycline antibiotic. In some embodiments, the inducible promoter is a tetracycline antibiotic response element (TRE), including, for example, a TRE2 promoter.

[0238] The reverse tetracycline-controlled transcription activator (rtTA) may be rtTA3, rtTA4, or a combination thereof.

[0239] In some embodiments, the nucleic acid molecule encoding rtTA is operably linked to a constitutive promoter, including one or more of CP1, CMV, EF1α, SV40, PGK1, Ubc, human β-actin, CAG, Ac5, polyhedrin, TEF1, GDS, CaM3 5S, Ubi, H1, and / or U6 promoters. In some embodiments, the nucleic acid molecule encoding rtTA is operably linked to a CMV promoter.

[0240] 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).

[0241] In some embodiments, an AAV vector comprising a nucleic acid molecule encoding OCT4, SOX2, and KLF4 comprises a self-cleaving peptide, such as a 2A peptide.

[0242] Different AAV serotypes are used to optimize transduction of specific target cells or to target specific cell types (e.g., RGCs) within specific target tissues. AAV particles can comprise viral proteins and viral nucleic acid of the same serotype or any natural or artificial sequence variant of AAV. For example, an AAV particle can comprise an AAV2 capsid protein and at least one, preferably two, AAV2 ITRs.

[0243] In some embodiments, an AAV vector comprising a nucleic acid molecule encoding OCT4, SOX2, and KLF4 comprises inverted terminal repeats (ITRs) flanking a first nucleic acid. In some embodiments, an AAV vector comprising a nucleic acid molecule encoding OCT4, SOX2, and KLF4 comprises inverted terminal repeats (ITRs) flanking a second nucleic acid. In some embodiments, an AAV vector comprising a nucleic acid molecule encoding OCT4, SOX2, and KLF4 comprises inverted terminal repeats (ITRs) flanking a third nucleic acid. In some embodiments, an AAV vector comprising a nucleic acid molecule encoding OCT4, SOX2, and KLF4 comprises inverted terminal repeats (ITRs) flanking one or more combinations of the first, second, and / or third nucleic acids. 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.

[0244] The method may further include administering an inducing agent to the subject. The inducing agent may include, for example, a tetracycline-controlled transactivator (tTA). In certain aspects, 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).

[0245] In some embodiments, an AAV-OSK vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 comprises nucleic acid elements in a specific order. For example, an AAV vector comprising a nucleic acid molecule encoding OCT4, SOX2, and KLF4 may include 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, such as described in U.S. patent application Ser. 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.

[0246] In certain embodiments, the encoded 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. 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 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 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 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, the nucleic acid sequence encoding KLF4 is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical 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.

[0247] In some embodiments, the P2A sequence encodes a polypeptide having the sequence ATNFSLLKQAGDV EENPGP (SEQ ID NO: 9). In some embodiments, the P2A sequence is GCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCT (SEQ ID NO: 8).

[0248] In some embodiments, the T2A sequence encodes the polypeptide of SEQ ID NO: 11. In some embodiments, the T2A sequence is GAGGGCAGGGGAAGTCTTCTAACA TGCGGGGACGTGGAGGAAAATCCCGGCCCA (SEQ ID NO: 10).

[0249] 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.

[0250] In some embodiments, the SV-40 derived terminator sequence is SEQ ID NO:12.

[0251] In some embodiments, the ITR sequence is SEQ ID NO:16.

[0252] In some embodiments, the nucleic acid molecules encoding OCT4, SOX2, and KLF4 comprise SEQ ID NO: 13 or 14. In some embodiments, the nucleic acid molecules encoding OCT4, SOX2, and KLF4 comprise SEQ ID NO: 15.

[0253] An AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 and an AAV vector comprising a nucleic acid molecule encoding rtTA are administered sequentially or simultaneously.

[0254] The nucleic acid molecules disclosed herein can be administered to a subject by any appropriate route, including but not limited to intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, topical, or intradermal routes. In certain embodiments, the composition is formulated for administration by intravenous or subcutaneous injection. In some embodiments, a two-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.

[0255] In some embodiments, nucleic acid molecules encoding OCT4, SOX2 and KLF4 and nucleic acid molecules encoding rtTA (e.g., rtTA3, rtTA4, etc.) are administered in 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; 1:8; 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 (nucleic acid molecule:nucleic acid molecule). In some embodiments, nucleic acid molecules encoding OCT4, SOX2, and KLF4 and nucleic acid molecules encoding rtTA (e.g., rtTA3, rtTA4, etc.) are in 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; 1:0.6; 1:1.5 In some embodiments, the nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the nucleic acid molecule encoding rtTA are administered at a ratio of about 1:1.

[0256] In some embodiments, the AAV vector comprising a nucleic acid molecule encoding OCT4, SOX2, and KLF4 and the AAV vector comprising a nucleic acid molecule encoding rtTA (e.g., rtTA3, rtTA4, etc.) are in 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; 1:8; 1:7 In some embodiments, the AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the AAV vector comprising nucleic acid molecules encoding rtTA are administered in 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 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, an AAV vector comprising a nucleic acid molecule encoding OCT4, SOX2, and KLF4 and an AAV vector comprising a nucleic acid molecule encoding rtTA (e.g., rtTA3, rtTA4, etc.) are present in 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; 1:0 In some embodiments, the AAV vector comprising a nucleic acid molecule encoding OCT4, SOX2, and KLF4 and the AAV vector comprising a nucleic acid molecule encoding rtTA are administered at a ratio (vg / vg) of about 1:1. According to some embodiments of the disclosed method, the AAV vector comprising the nucleic acid molecules encoding OCT4, SOX2, and KLF4 comprises SEQ ID NO: 13, 14, or 35.According to some embodiments, the AAV vector comprising a 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 a nucleic acid molecule encoding OCT4, SOX2, and KLF4 comprises SEQ ID NO: 13, 14, or 35, and the AAV vector comprising a 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 a nucleic acid molecule encoding OCT4, SOX2, and KLF4 comprises SEQ ID NO: 35, and the AAV vector comprising a nucleic acid molecule encoding rtTA comprises SEQ ID NO: 36 or 37.

[0257] In some embodiments, the AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 is an AAV2-TRE-OSK vector, and the AAV vector comprising a nucleic acid molecule encoding rtTA is an AAV2-CMV-rtTA3. The AAV composition can include an AAV2-TRE-OSK vector and a pAAV2-CMV-rtTA3VP16 vector. The method can include an AAV2-TRE-OSK vector and a pAAV2-CMV-rtTA3VP16 vector in the same or separate compositions.

[0258] In some embodiments, the AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 is an AAV2-TRE-OSK vector, and the AAV vector comprising a nucleic acid molecule encoding rtTA is an AAV2-CMV-rtTA4 vector. The AAV composition may include an AAV2-TRE-OSK vector and an AAV2-CMV-rtTA4 vector. The method may include the AAV2-TRE-OSK vector and the AAV2-CMV-rtTA4 vector in the same composition or in separate compositions.

[0259] The concentration of AAV2-TRE-OSK vector administered according to the disclosed methods includes about 1×10 10 vg / mL to about 2×10 13 Amounts in the vg / mL range, such as 1×10 12 vg / mL to about 2×10 12 For example, an effective amount of an AAV2-TRE-OSK vector may include about 1.0×10 12 vg / mL to about 1.1×10 12 vg / mL, about 1.1×10 12 vg / mL to about 1.2×10 12 vg / mL, about 1.2×10 12vg / mL to about 1.3×10 12 vg / mL, about 1.3×10 12 vg / mL to about 1.4×10 12 vg / mL, about 1.4×10 12 vg / mL to about 1.5×10 12 vg / mL, about 1.5×10 12 vg / mL to about 1.6×10 12 vg / mL, about 1.6×10 12 to about 1.7×10 12 vg / mL, about 1.7×10 12 vg / mL to about 1.8×10 12 vg / mL, about 1.8×10 12 vg / mL to about 1.9×10 12 vg / mL, about 1.9×10 12 vg / mL to about 2.0×10 12 vg / mL and any and all increments therebetween.

[0260] The concentration of pAAV2-CMV-rtTA3VP16 vector administered according to the disclosed method includes about 1×10 10 vg / mL to about 2×10 13 Amounts in the range of vg / mL, for example, about 1×10 13 vg / mL to about 2×10 13 For example, an effective amount of a pAAV2-CMV-rtTA3VP16 vector may include about 1.0×10 13 vg / mL to about 1.1×10 13 vg / mL, about 1.1×10 13 vg / mL to about 1.2×10 13 vg / mL, about 1.2×10 13 vg / mL to about 1.3×10 13 vg / mL, about 1.3×10 13 vg / mL to about 1.4×10 13 vg / mL, about 1.4×10 13 vg / mL to about 1.5×10 13 vg / mL, about 1.5×10 13 vg / mL to about 1.6×10 13 vg / mL, about 1.6×10 13 to about 1.7×10 13 vg / mL, about 1.7×10 13 vg / mL to about 1.8×1013 vg / mL, about 1.8×10 13 vg / mL to about 1.9×10 13 vg / mL, about 1.9×10 13 vg / mL to about 2.0×10 13 vg / mL and any and all increments therebetween.

[0261] The concentration of AAV2-CMV-rtTA4 vector administered according to the disclosed methods includes about 1×10 10 vg / mL to about 2×10 13 Amounts in the range of vg / mL, for example, about 1×10 13 vg / mL to about 2×10 13 For example, an effective amount of an AAV2-CMV-rtTA4 vector may include about 1.0×10 13 vg / mL to about 1.1×10 13 vg / mL, about 1.1×10 13 vg / mL to about 1.2×10 13 vg / mL, about 1.2×10 13 vg / mL to about 1.3×10 13 vg / mL, about 1.3×10 13 vg / mL to about 1.4×10 13 vg / mL, about 1.4×10 13 vg / mL to about 1.5×10 13 vg / mL, about 1.5×10 13 vg / mL to about 1.6×10 13 vg / mL, about 1.6×10 13 to about 1.7×10 13 vg / mL, about 1.7×10 13 vg / mL to about 1.8×10 13 vg / mL, about 1.8×10 13 vg / mL to about 1.9×10 13 vg / mL, about 1.9×10 13 vg / mL to about 2.0×10 13 vg / mL and any and all increments therebetween.

[0262] According to some embodiments, an effective dose of an AAV2-TRE-OSK vector administered according to the disclosed methods comprises an amount of vector administered per eye. An effective dose may comprise approximately 1×10 9 vg / eye is about 1×10 14In some embodiments, an effective dose of the AA V2-TRE-OSK vector may include about 1×10 11 vg / eye to about 10×10 11 For example, a dose of AAV2-TRE-OSK vector may include about 1×10 11 vg / eye is about 2×10 11 vg / eye, about 2×10 11 vg / eye is about 3×10 11 vg / eye, about 3×10 11 vg / eye is about 4×10 11 vg / eye, about 4×10 11 vg / eye is about 5×10 11 vg / eye, about 5×10 11 vg / eye is about 6×10 11 vg / eye, about 6×10 11 vg / eye is about 7×10 11 vg / eye, about 7×10 11 vg / eye is about 8×10 11 vg / eye, about 8×10 11 vg / eye is about 9×10 11 vg / eye, about 9×10 11 vg / eye to about 10×10 11 vg / eye, including any and all increments therebetween. In some embodiments, an effective dose of an AAV2-TRE-OSK vector is approximately 3.06×10 11 vg / eye.

[0263] According to some embodiments, an effective dose of an AAV2-TRE-OSK vector administered according to the disclosed methods comprises an amount of vector administered per eye. An effective dose may comprise approximately 1×10 9 vg / eye is about 1×10 12 In some embodiments, an effective dose of an AAV2-TRE-OSK vector may include about 1×10 9 vg / eye is about 1×10 12 For example, a dose of AAV2-TRE-OSK vector may include about 1×10 9 vg / eye is about 4×10 9 vg / eye, about 4×10 9 vg / eye is about 7×10 9 vg / eye, about 7×10 9 vg / eye is about 1×10 10 vg / eye, about 1×1010 vg / eye is about 4×10 10 vg / eye, about 4×10 10 vg / eye is about 7×10 10 vg / eye, about 7×10 10 vg / eye is about 1×10 11 vg / eye, about 1×10 11 vg / eye is about 4×10 11 vg / eye, about 4×10 11 vg / eye is about 7×10 11 vg / eye, about 7×10 11 vg / eye is about 1×10 12 vg / eye, including any and all increments therebetween. In some embodiments, an effective dose of an AAV2-TRE-OSK vector is approximately 3.06×10 11 vg / eye.

[0264] An effective dose of the pAAV2-CMV-rtTA3VP16 vector administered according to the disclosed methods includes the amount of vector administered per eye. An effective dose of the pAAV2-CMV-rtTA3VP16 vector may include approximately 1×10 9 vg / eye is about 1×10 14 In some embodiments, an effective dose of pAAV2-CMV-rtTA3VP16 vector may include about 1×10 11 vg / eye to about 10×10 11 For example, a dose of pAAV2-CMV-rtTA3VP16 vector may include about 1×10 11 vg / eye is about 2×10 11 vg / eye, about 2×10 11 vg / eye is about 3×10 11 vg / eye, about 3×10 11 vg / eye is about 4×10 11 vg / eye, about 4×10 11 vg / eye is about 5×10 11 vg / eye, about 5×10 11 vg / eye is about 6×10 11 vg / eye, about 6×10 11 vg / eye is about 7×10 11 vg / eye, about 7×10 11 vg / eye is about 8×10 11 vg / eye, about 8×10 11 vg / eye is about 9×10 11 vg / eye, about 9×1011 vg / eye to about 10×10 11 vg / eye, including any and all increments therebetween. In some embodiments, an effective dose of pAAV2-CMV-rtTA3VP16 vector is approximately 2.66×10 11 vg / eye.

[0265] An effective dose of the pAAV2-CMV-rtTA3VP16 vector administered according to the disclosed methods includes the amount of vector administered per eye. An effective dose of the pAAV2-CMV-rtTA3VP16 vector may include approximately 1×10 9 vg / eye is about 1×10 12 In some embodiments, an effective dose of pAAV2-CMV-rtTA3VP16 vector may include about 1×10 9 vg / eye is about 1×10 12 For example, a dose of pAAV2-CMV-rtTA3VP16 vector may include about 1×10 9 vg / eye is about 4×10 9 vg / eye, about 4×10 9 vg / eye is about 7×10 9 vg / eye, about 7×10 9 vg / eye is about 1×10 10 vg / eye, about 1×10 10 vg / eye is about 4×10 10 vg / eye, about 4×10 10 vg / eye is about 7×10 10 vg / eye, about 7×10 10 vg / eye is about 1×10 11 vg / eye, about 1×10 11 vg / eye is about 4×10 11 vg / eye, about 4×10 11 vg / eye is about 7×10 11 vg / eye, about 7×10 11 vg / eye is about 1×10 12 vg / eye, including any and all increments therebetween. In some embodiments, an effective dose of pAAV2-CMV-rtTA3VP16 vector is approximately 2.66×10 11 vg / eye.

[0266] An effective dose of the AAV2-CMV-rtTA4 vector administered according to the disclosed methods includes the amount of vector injected per eye. An effective dose may include approximately 1×10 9 vg / eye is about 1×10 14In some embodiments, an effective dose of AAV2-CMV-rtTA4 vector may include about 1×10 11 vg / eye to about 10×10 11 For example, a dose of AAV2-CMV-rtTA4 vector may include about 1×10 11 vg / eye is about 2×10 11 vg / eye, about 2×10 11 vg / eye is about 3×10 11 vg / eye, about 3×10 11 vg / eye is about 4×10 11 vg / eye, about 4×10 11 vg / eye is about 5×10 11 vg / eye, about 5×10 11 vg / eye is about 6×10 11 vg / eye, about 6×10 11 vg / eye is about 7×10 11 vg / eye, about 7×10 11 vg / eye is about 8×10 11 vg / eye, about 8×10 11 vg / eye is about 9×10 11 vg / eye, about 9×10 11 vg / eye to about 10×10 11 vg / eye, including any and all increments therebetween. In some embodiments, an effective dose of an AAV2-CMV-rtTA4 vector is approximately 2.66×10 11 vg / eye.

[0267] An effective dose of the AAV2-CMV-rtTA4 vector administered according to the disclosed methods includes the amount of vector injected per eye. An effective dose may include approximately 1×10 9 vg / eye is about 1×10 12 In some embodiments, an effective dose of AAV2-CMV-rtTA4 vector may include about 1×10 9 vg / eye is about 1×10 12 For example, a dose of AAV2-CMV-rtTA4 vector may include about 1×10 9 vg / eye is about 4×10 9 vg / eye, about 4×10 9 vg / eye is about 7×10 9 vg / eye, about 7×10 9 vg / eye is about 1×10 10 vg / eye, about 1×1010 vg / eye is about 4×10 10 vg / eye, about 4×10 10 vg / eye is about 7×10 10 vg / eye, about 7×10 10 vg / eye is about 1×10 11 vg / eye, about 1×10 11 vg / eye is about 4×10 11 vg / eye, about 4×10 11 vg / eye is about 7×10 11 vg / eye, about 7×10 11 vg / eye is about 1×10 12 vg / eye, including any and all increments therebetween. In some embodiments, an effective dose of an AAV2-CMV-rtTA4 vector is approximately 2.66×10 11 vg / eye.

[0268] Embodiments of the method include administering 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 to a subject via one or more suitable routes, including left eye (OS) injection, right eye (OD) injection, or binocular (OU) injection. Administration may comprise one or more injections, for example, administration may comprise 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 comprise 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. Injections comprising AAV vectors comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 may be administered, and injections comprising AAV vectors comprising nucleic acid molecules encoding rtTA may be administered simultaneously or sequentially. For example, an injection comprising an AAV composition comprising an AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 can be administered before, simultaneously with, or after an injection comprising an AAV vector comprising a nucleic acid molecule encoding rtTA.

[0269] Embodiments of the method further comprise 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 prior to administering the AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the AAV vector comprising nucleic acid molecules encoding rtTA.

[0270] The antibiotic may be administered at the time of administration of the AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the AAV vector comprising nucleic acid molecules encoding rtTA. The antibiotic may be administered at least one day after administration of the AAV composition comprising an AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the AAV vector comprising nucleic acid molecules encoding rtTA. The antibiotic may be administered at least 2 days, 3 days, 4 days, 5 days, or more than 5 days after administration of the AAV composition comprising an AAV vector comprising nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the AAV vector comprising nucleic acid molecules encoding rtTA.

[0271] 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 sequences that express OCT4, SOX2, and KLF4, and an AAV genome comprising a nucleic acid sequence encoding transactivator 3 or transactivator 4.

[0272] Preparation method of AAV-OSK vector

[0273] Provided herein are methods for recombinantly preparing AAV. In some embodiments, the method includes introducing one or more vectors described herein into cells under conditions that produce AAV. The cells may include a cell population. The cell population may include any suitable cells as understood in the art, including, for example, HEK293 cells, HEK293T cells, COS cells, CHO cells, BHK cells, HeLa cells, etc. One or more vectors may be introduced into cells using one or more suitable techniques (including, for example, transfection, transduction, and / or infection). Exemplary methods for recombinant production of AAV include transient transfection (e.g., using one or more transfer plasmids containing a first vector, a second vector, and optionally a third vector as described herein), viral infection (e.g., using one or more recombinant helper viruses, such as adenovirus, poxvirus (e.g., vaccinia virus), herpes virus (including HSV, cytomegalovirus, or baculovirus, containing a first vector, a second vector, and optionally a third vector as described herein)), and transfection or infection of a stable producer cell line (e.g., using stable producer cells, such as mammalian or insect cells, containing a Rep nucleotide sequence encoding one or more AAV Rep proteins and / or a Cap nucleotide sequence encoding one or more AAV capsid proteins as described herein, and using an AAV genome as described herein delivered in the form of a plasmid or recombinant helper virus). 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 transcriptional activator 3, such as SEQ ID NOs: 21 or 36. Alternatively, the second vector may comprise one or more nucleic acid sequences that express transcriptional activator 4, such as SEQ ID NO: 28 or 37.

[0274] Further provided herein are methods for producing AAV, comprising modifying a cell to express one or more plasmids. The one or more plasmids may include one or more AAV2Rep-Ca p plasmids, one or more helper plasmids, and one or more transfer plasmids. The one or more transfer plasmids may include: a first transfer plasmid comprising one or more nucleic acids encoding one or more of: OCT4, SOX2, and KLF4; a second transfer plasmid comprising one or more nucleic acids encoding transactivator 3; or a transfer plasmid comprising 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 comprising one or more nucleic acids encoding one or more of: OCT4, SOX2, and KLF4; a second transfer plasmid comprising one or more nucleic acids encoding transactivator 4; or a transfer plasmid comprising one or more nucleic acids encoding one or more of: OCT4, SOX2, KLF4, and transactivator 4.

[0275] definition

[0276] The singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0277] As used herein, the term "about" means a range of values ​​that includes the specified value and that one of ordinary skill in the art would consider to be reasonably similar to the specified value. For example, "about" means within a standard deviation using measurement methods generally accepted in the art. For example, "about" means that the range extends to + / - 10%, + / - 5%, + / - 3%, or + / - 1% of the specified value.

[0278] The term "at least" followed by a number is used herein to indicate the starting point of a range that begins with that number (the range may be a range with or without an upper limit, depending on the variable being defined). For example, "at least 1" means 1 or greater than 1.

[0279] The term "at most" followed by a number is used herein to indicate the endpoint of a range ending in that number (the range may be a range with 1 or 0 as the lower limit, or a range with no lower limit, depending on the variable being defined). For example, "at most 4" means 4 or less, and "at most 40%" means 40% or less. When a range is given in this specification as "(first number) to (second number)" or "(first number) - (second number)", this means that the lower limit of the range is the first number and the upper limit is the second number. For example, 25 to 100 nm means a range with a lower limit of 25 mm and an upper limit of 100 mm.

[0280] In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or percent "identity" refers to two or more sequences or subsequences that are identical, or have a specified percentage of identical amino acid residues or nucleotides, when compared and aligned for maximum correspondence. Methods of sequence alignment for comparison are well known in the art. After alignment, the number of matches is determined by counting the number of positions of identical nucleotides or amino acid residues in the two 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 resulting value by 100. For example, a peptide sequence with 1166 matches is 75.0% identical to the test sequence when aligned with a reference sequence having 1554 amino acids (1166 ÷ 1554 * 100 = 75.0). As the terms are used herein, gaps in the alignment do not reduce the percent sequence identity. Unless otherwise indicated, optimal alignment of sequences for comparison is performed by the global alignment algorithm of Needleman and Wunsch, Mol. Biol. 48:443 (1970), as implemented by EMBOSS Needle (World Wide Web address: 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 those described in 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 was calculated using the Needleman-Wunsch algorithm implementation provided by the U.S. National Library of Medicine (World Wide Web) at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=GlobalAln).

[0281] For example, sequence identity can be determined by the standard method for the similarity that is generally used for comparing two polypeptides or two polynucleotide sequences.Use computer program (such as EMBOSS Needle or BLAST), two polypeptides or two polynucleotide sequences are compared, to realize the best match of their respective residues (along the full length of one or two sequences, or along the predetermined portion of one or two sequences).Described program provides acquiescent opening penalty and acquiescent gap penalty, and the scoring matrix that can be used in combination with computer program, such as PAM 250 (standard scoring matrix; See Dayhoff et al., Atlasof Protein Sequence and Structure, Vol. 5, Supplement 3 (1978)).

[0282] In this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations thereof, such as "comprises" and "comprising," as well as "has" or "having" and "includes" or "including," will be understood to mean the inclusion of stated elements or steps or groups of elements or steps but not the exclusion of any other elements or steps or groups of elements or steps. "Consisting essentially of" or "consists essentially of" means the exclusion of elements or steps that materially affect the basic and novel characteristics of the claimed invention.

[0283] "Subjects" intended for administration include, but are not limited to, humans (i.e., males or females of any age group, such as pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly people)) and / or other non-human animals, such as mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals, such as cattle, pigs, horses, sheep, goats, cats, and / or dogs) and birds (e.g., commercially relevant birds, such as chickens, ducks, geese, and / or turkeys). In certain embodiments, the animal is a mammal. The animal can be male or female and at any stage of development. The non-human animal can be a transgenic animal.

[0284] sequence

[0285] Table 7: AAV2-TRE-OSK vector sequences

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304] Table 8: pAAV2-CMV-rtTA3VP16 vector sequence

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312] Table 9: AAV2-CMV-rtTA4 V16 vector sequences

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321] Example

[0322] Example 1 - Method of Producing Vectors

[0323] This study aimed to develop a method to manufacture bulk vectors and to evaluate the stability of the prepared batches.

[0324] AAV production protocol

[0325] Material

[0326] cell:

[0327] HEK293T cells were used to produce the vectors described herein. Cells were grown in DMEM medium (Invitrogen, catalog number 11995073) containing 10% fetal bovine serum (FBS) (Invitrogen HI FBS, catalog number 16140), penicillin / streptomycin (In vitrogen, catalog number 15140-122), glutamine (Invitrogen catalog number 25030).

[0328] Plasmids:

[0329] Recombinant AAV2-TRE-OSK plasmid (such as Figure 1The AAV2 Rep-Cap plasmid is composed of the following components: (1) a nucleic acid sequence encoding an AAV2 capsid protein or a fragment thereof, (2) a nucleic acid encoding a functional rep gene, (3) a recombinant AAV transfer vector comprising the AAV2 inverted terminal repeats (SEQ ID NO: 16, SEQ ID NO: 32) flanked by transgenes encoding OCT4, KLF4, and SOX2 (SEQ ID NO: 13), operably linked to an inducible TRE promoter (TR E3G, 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). The AAV2 Rep-Cap plasmid comprises the pAAV-RC plasmid (Stratagene catalog number 240071). In some cases, AAV2 hybrid vectors, such as AAV2 / 1AAV2 / 2, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, and AAV2 / 9, carry capsid proteins from serotypes AAV1, 2, 3, 5, 6, 7, 8, and 9. Helper plasmids include the pHelper plasmid (Stratagene, catalog number 240071) and carry adenovirus-derived genes for introducing helper functions. Figure 1 As shown, the entire AAV2-TRE3G-OSK-SV40pA vector is 7250 base pairs in length, and two inverted terminal repeats (ITRs) are located on both sides of the OSK sequence.

[0330] 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 inducer.

[0331] For the recombinant Tet-On plasmid pAAV2-CMV-rtTA, pAAV2-CMV-rtTA 3VP16 (such as Figure 2 ) was prepared using a similar method as described above, but using a transfer plasmid with a C MV constitutive promoter (SEQ ID NO: 17) operably linked to an inducer, rtTA3 (SEQ ID NO 19), having three vp16 domains at the 3′ end. An alternative recombinant pAAV2-CMV-rtTA, pAAV2-CMV-rtTA4 (V16) (as shown) can be prepared in the same manner. Figure 3 shown).

[0332] PEI solution (1 μg / μl, Polysciences, catalog number 23966-2) was prepared by dissolving PEI powder in H2O, heating to 80°C, cooling to room temperature, neutralizing to pH 7.0, filter sterilizing, aliquoting, and storing at -20°C. Transfection efficiency was tested for each new batch prepared.

[0333] method

[0334] Vector production:

[0335] On day 1, HEK293T cells were seeded into ten 15 cm culture dishes and transfected for 24 hours. Each 15 cm dish was inoculated with 25 mL of culture medium. The cells were split ten times on a 15 cm plate to 70-90% density (standard transfection density) to provide a yield of 5E12 viral genomes (vg). The medium was replaced with 5% FBS to slow growth and reduce purification time, with one plate replaced at a time to prevent cell death.

[0336] On the second day, 1 hour before transfection, the medium was replaced with 20 mL of freshly warmed medium. TM Prepare DNA solution in a tube. The amounts of DNA and reagents for each dish are shown in Table 3 below. Calculate the amount of DNA based on the size of the inverted terminal repeat (ITR) plasmid. Prepare one tube of master mix for five 15 cm dishes. Dilute all plasmids to 1 μg / μl in sterile HO as shown in Table 3 below.

[0337] A 10 mL sample of DMEM (without phenol red, Invitrogen catalog number 31053-036) was prepared. A 785 μl aliquot of PEI solution was added and the culture medium was mixed. It was then incubated at room temperature for 20-30 minutes.

[0338] Table 3 - Reagent Volumes per Plate

[0339]

[0340] Next, 2 mL of the DNA-PEI mixture was added to ten 15 cm culture dishes and the transfected cells were incubated.

[0341] On day 3, remove the culture medium and replace with 25 mL of freshly warmed culture medium. For AAV2 / 2 and AAV2 / 6, use serum-supplemented culture medium. For AAV2 / 1, AAV2 / 5, AAV2 / 7, AAV2 / 8, and AAV2 / 9, use serum-free culture medium.

[0342] On day 5, cells were scraped from their current culture medium using a cell scraper and transferred to a 50 mL tube. The cell suspension was then spun at 1000 relative centrifugal force (rcf) for 5 minutes. The supernatant was then discarded.

[0343] Transfection procedure:

[0344] All cells were combined into one 50 mL tube, washed with PBS, and spun at 1000 rpm for 5 minutes, followed by discarding the supernatant.

[0345] The cell pellet was resuspended in a volume of hypotonic buffer five times the packed cell volume (approximately 25 mL). This was then incubated on ice for 10 minutes. 0.11% by volume of 10X recovery buffer was then added and mixed by pipetting (typically a volume of 3.3 mL).

[0346] The nuclei were then spun at 2000 rcf for 10 minutes, yielding approximately 1 mL of nuclear pellet per ten 15 cm dishes. The pellets were stored at -80°C for further purification.

[0347] Solution preparation:

[0348] 5M NaCl salt solution

[0349] Add 292.2g of NaCl to 200g of deionized (DI) water in a 2L bottle. Shake the mixture and pour it into a large graduated cylinder. Then add deionized water to 1L and pour it back into the 2L bottle. Add a stirring rod and heat and stir the volume until dissolved. Alternatively, autoclave the volume. Then cool the solution and sterilize it through a 0.2μm filter.

[0350] 40% PEG-8000, 2.5M NaCl (5x stock solution)

[0351] In a 1L graduated cylinder, combine 400g of PEG-8000 and 500mL of 5M NaCl. Add deionized water to 1L. This typically requires about 100mL of water. Transfer the solution to a 2L flask and shake well. Add a large stir bar and heat the solution in a 55°C water bath overnight. The next day, sterile filter the solution using a 0.2μm filter. This filtration step typically takes about 30 minutes.

[0352] Harvesting AAV from culture medium and cells:

[0353] Harvest culture medium and cells without using trypsin. Collect culture medium with a pipette and sterile filter with a 0.2 μM filter. Collect cells and spin. Add the remaining supernatant to culture medium that has been sterile filtered with a 0.2 μm filter. Cells are harvested in one of several ways. Collect cells with a cell scraper. Alternatively, "bombard" cells with a volume of 10 mL per 2 plates of PBS that does not contain calcium and magnesium. Add 40% PEG 8000 solution (adjusted to pH 7.4) to a final concentration of 12%. Use approximately 25 mL for every 100 mL of culture medium / cell. Stir the solution in a cold room for 1 hour and let it stand for 3 hours (without spinning) or place it in a cold room overnight. The next day, spin the PEG mixture at 3000xg for 20 minutes. Discard the supernatant. Resuspend the precipitate in less than about 7 mL of 1xPBS. First, suspend the precipitate with about 5 mL, and once almost suspended, add up to 2 mL. Then, a 1:10,000 solution of universal nuclease was added and the mixture was incubated at 37° C. for 45 minutes. Optionally, the mixture was spun at 2415×g for 10 minutes at 4° C. The supernatant was transferred to a new tube.

[0354] Concentrate AAV using ultracentrifugation:

[0355] Solution preparation

[0356] Prepare a 1M MgCl2 stock solution at 1000X and use it to prepare MK buffer. Prepare a 2.5M KCl stock solution at 1000X and use it to prepare MK buffer.

[0357] A 1 M solution of NaCl / phosphate buffered saline (PBS) MK buffer was prepared by dissolving 58.4 g of NaCl in 1 mL of 1 M MgCl2 and 1 mL of 2.5 M KCl. Next, 1× 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 through a 0.22 μm filter and stored at 4° C. The final concentrations of the solution were 1 M NaCl, 1 mM MgCl 2 , and 2.5 mM KCl.

[0358] Prepare 1×PBS-MK buffer by dissolving 1 mL of 1M MgCl2 and 1 mL of 2.5M KCl in 2×500 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 final concentration of the solution was 1 mM MgCl2 and 2.5 mM KCl.

[0359] Prepare a 0.001% Pluronic-F68 (formulation buffer) solution by adding 500 μL of sterile 1000X Pluronic F-68 (1% solution) to 500 mL of 1X DPBS (Gibco, TC stock). Store the solution at 4°C for up to one month or aliquot and store at -80°C for up to one year.

[0360] By adding 25g of sorbitol to 500μL of sterile 1000X PLURONIC TM F-68 (1% solution) was added to 500 mL of 1× DPBS (Gibco, TC stock solution) to prepare 0.001% PLURONIC TM - F68 + 5% sorbitol (freezing buffer) solution. The solution can be stored at 4°C for up to one month, or aliquoted and stored at -80°C for up to one year.

[0361] 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 1×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 1×PBS-MK buffer. A 60% iodixanol solution was prepared by mixing 150 mL of 60% iodixanol with 675 μL of phenol red.

[0362] Table 4: Iodixanol Solution

[0363]

[0364] Note that all solutions were sterile filtered using a 0.2 μm filter.

[0365] To generate a super gradient, add the NUCLASE supernatant to a Beckman optically sealed tube. If the volumes are unequal, add 1 M NaCl, PBS-MK mixture to a final volume of approximately 7 mL.

[0366] Fill the tube from the bottom using a 10 mL syringe and a long hypodermic needle. Reuse the same syringe for each solution, but change the needle after each sampling to prevent cross-contamination between AAV preparations. 5 mL is the minimum volume for any layer.

[0367] Balance the tubes to 5-10 μg. Balance the tubes in pairs. Sort them first so that the most similar tubes are paired, and then add PBS to the lighter tube in each pair. Add PBS by touching the tip to the side of the tube to prevent droplets from disturbing the layers. Cap and load the tubes into the ultracentrifuge.

[0368] 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 piercing the bottom of the tubes with an 18-gauge needle. Before piercing with the needle, the black stopper at the top was removed to prevent air bubbles from forming, which could disrupt the layers. Most 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 a significant color change occurred or the solution became turbid.

[0369] Separation of protein fractions on gel

[0370] Denature the sample with 4× LDS containing 2.5% β-mercaptoethanol at 70°C for 10 minutes. To prepare a 20 μL aliquot, mix 10 μL of sample with 5 μL of H₂O and 5 μL of LDS containing 2.5% β-mercaptoethanol. If necessary, prepare additional running buffer (200 mL of 10× Tris-Glycine SDS buffer, 1800 mL of Millipure water).

[0371] The samples are then loaded onto a Tris Glycine gel. Use either a 4-12% or 4-20% gradient gel. For AAV capsids, either is suitable.

[0372] Assemble the gel cassette and load 20 μL of sample per well into the gel wells. Run the gel at 225 V for 30-45 minutes until the blue dye reaches the bottom of the gel.

[0373] Then use SYPRO TM The gel was stained with SYPRO Red and imaged. TM Prepare staining solution (7.5% acetic acid, SYPRO TM Red is 5000×. This is sufficient for one gel. The gel was stained for 1 hour at room temperature while covered on a slow shaker. The staining solution was then removed and a dye-free acetic acid solution was added. The gel was incubated for 1 to 5 minutes to destain. The gel was imaged on a gel mount with an EtBr setting (UV).

[0374] The good fractions were then combined and washed with 1×PBS containing 0.001% F68. In some cases, approximately 20 mL of PBS+F68 was added to bring the final volume to slightly less than 30 mL to dilute the iodixanol and facilitate passage through the filter.

[0375] The sample was spun at 4700 g for 5 minutes. When all material had flowed through, the sample was washed again 3 times, 15 mL each time, to prepare a clear final solution. The sample was then dispensed into labeled tubes (virus name, payload, date) and stored at 4°C for up to 1 week. In order to extend the storage time, the final eluate was prepared with 1× PBS containing 0.001% F68 and 5% sorbitol. The sample was frozen at -80°C.

[0376] Determination of viral titer by qPCR

[0377] Viral titer can be measured by various molecular biology techniques, including quantitative PCR (qPCR). Viral titer can be reported as genome copies per ml (GC) or vector genomes per ml (VG). Each ml of GC is equivalent to each ml of VG.

[0378] AAV2-TRE-OSK virus was analyzed by qPCR to quantify the titer using the following primers: TRE3G F: AACGTATCTACAGTTACTCCCTATC and TRE3G R: GGTAGGAAGTGGTACGGAAAG. The titer of the batch used was found to be 1.53 × 10 12 vg / mL. Injecting 200 mL of AAV2-TRE-OSK into the eye is equivalent to approximately 2.66×10 11 vg.

[0379] The pAAV2-CMV-rtTA3VP16 virus was analyzed by qPCR to quantify the titer using the following primers: WPRE F: CACTGACAATTCCGTGGTGT and WPRE R: GAGATCCGACTCGTCTGAGG. The titer of the batch used was found to be 1.33 × 10 13 vg / mL. Injecting 20 mL of pAAV2-CMV-rtTA3VP16 into the eye is equivalent to approximately 2.11×10 11 vg.

[0380] Samples were prepared by aliquoting 12.5 μl of Master Mix into test tubes. The ThermoFisher Fast Advanced TAQMAN TMMaster Mix or IDT Primetime Master Mix. To each tube, add 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 H2O for a total volume of 25 μl.

[0381] Example 2 - Methods of treating NAION in non-human primates

[0382] The present study aimed to determine whether epigenetic reprogramming could improve RGC function and restore visual function (pERG) in a non-human primate (NHP) NAION (non-arteritic anterior ischemic optic neuropathy) model. In a photothrombotic experimental model of non-arteritic anterior ischemic optic neuropathy (NAION), 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. The vector system was intravitreally (IVT) administered to African green monkeys and induced by systemic administration of a fluorophore for laser excitation at the optic nerve head (ONH). Figure 5 Schematic depicting the study design of the NHP NAION study.

[0383] method

[0384] Subject recruitment:

[0385] Monkeys with normal slit lamp and fundus examinations, color fundus photographs (CFPs), optical coherence tomography (OCT), confocal scanning laser ophthalmoscopy (cSLO), pattern electroretinogram (pERG), and pattern visual evoked potential (pVEP) were enrolled in the study. For baseline screening and all subsequent procedures, anesthesia was achieved with intramuscular ketamine (8 mg / kg) and xylazine (1.6 mg / kg), and pupil dilation was achieved with topical 10% phenylephrine and / or 1% cyclopentolate.

[0386] Test object:

[0387] The subjects were administered a composition comprising a two-vector system comprising an AAV2-TRE-OSK vector and a pAAV2-CMV-rtTA3VP16 vector. The AAV2-TRE-OSK vector was prepared into approximately 1.53×10 12 vg / mL and administered to subjects at a dose of 200 μL per dose. The pAAV2-CMV-rtTA3VP16 vector was prepared to a concentration of approximately 1.33×10 13The two vectors were administered at a concentration of 10:1 v / v, or approximately a 1:1 viral genome ratio (vg / vg).

[0388] The pAAV2-CMV-rtTA3VP16 vector contains a CMV promoter that controls the expression of a reverse tetracycline-controlled transcriptional activator (rtTA) fused to three copies of viral protein 16 (VP16). The encoded rtTA3VP16 protein contains the amino acid substitutions V9I, G12S, F67S, and R171K, as well as three VP16 domains. References to pAAV2-CMV-rtTA3(VP16), AAV2-CMV-rtTA3VP16, AAV2-CMV-rtTA3(VP16), and pAAV2-rtTA3-Kan all refer to the same pAAV2-CMV-rtTA3VP16.

[0389] Intravitreal administration:

[0390] According to treatment assignment, the eyes received a single intravitreal (IVT) injection of the test article or vehicle (Table 5). For IVT administration, one drop of 0.5% proparacaine hydrochloride was first placed in the eye, followed by a lid speculum and 5% povidone-iodine solution, and then rinsed with sterile 0.9% saline. A 31-gauge 0.375-inch needle was inserted from the inferotemporal direction approximately 2.5 mm posterior to the limbus at the level of the ora serrata, and a 220 μL injection was administered into the central vitreous. Following the injection, 1% topical atropine and a topical triple antibiotic ointment of neomycin, polymyxin, and bacitracin (or equivalent) were applied.

[0391] Table 5 - Treatment conditions

[0392]

[0393] Doxycycline administration

[0394] Monkeys received oral doxycycline (5 mg / kg) in their food (banana chips) starting one day before dosing and continuing until the end of the study (Table 6).

[0395] Table 6 - Dosing regimen for monkey studies

[0396]

[0397] X = events for all treatment groups; Gp1A = events for Group 1A; Gp1B = events for Group 1B; Gp2 = events for Group 2; Gp3 = events for Group 3.

[0398] *Baseline pre-screening evaluations will include assessment of up to 24 monkeys to reach the target number of study enrollments.

[0399] **Daily oral doxycycline dosing will begin on Day -21 and continue through Day 42 for all animals.

[0400] ***Inspection will be completed within +- 4 days.

[0401] §Vitreous puncture just before drug administration

[0402] Immunosuppression

[0403] Starting on day 0, monkeys received methylprednisolone (40 mg, IM) weekly (Table 6). If inflammation exceeding 2+ anterior and / or vitreous cells was observed on subsequent examinations, subconjunctival dexamethasone (100 mL, 40 mg / mL) was administered and repeated based on clinical observations. If inflammation did not respond to dexamethasone, methylprednisolone was increased to 80 mg IM weekly. Steroids were administered consistently to all treated animals.

[0404] NAION induction

[0405] Monkeys were divided into two cohorts based on examination and weight criteria. After mydriasis was achieved and a saphenous vein catheter was placed, rose bengal (2.5 mg / kg; 0.1 ml / kg, 25 mg / ml) was administered intravenously (IV) and laser spot application to the ONH was initiated 25 seconds later. Laser spot application was performed with an Iridex Oculight TX 532 nm laser using a 0.9× contact laser lens. Four spots were placed in the four quadrants of the ONH, with each 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 layer of the ONH, the second to the inferior temporal layer, the third to the inferior nasal layer, and the fourth to the superior nasal layer. Each spot was placed to avoid retinal vein and artery branches. Post-laser CFP imaging was performed immediately to record the response to the laser photothrombosis procedure.

[0406] Intraocular pressure measurement

[0407] Intraocular pressure (IOP) was measured binocularly (OU) or in both eyes using a TonoVet tonometer set to the dog (d) calibration setting. Three measurements were performed on each eye at each ophthalmological examination time point (Table 6), and the mean IOP was defined.

[0408] Vitreous humor collection

[0409] At the designated time points (Table 6), vitreous humor (approximately 170 μL) was collected using a 1.0 mL syringe OU equipped with a 27-gauge needle, which was inserted into the mid-vitreous body at the level of the ora serrata through a temporal scleral puncture. Vitreous humor aliquots were transferred to pre-labeled cryovials, quickly frozen, and stored and shipped for analysis.

[0410] Full-field electroretinogram (ffERG)

[0411] At the specified time points (Table 6), slit lamp biomicroscopy and retinoscopy were performed on both eyes (OU) using the LKC UTAS full-field ERG device and emWin software (version 9.8.0). After sedation and topical instillation of 10% phenylephrine and 1% cyclopentanol, the eyes were immediately allowed to dark adapt for 30 minutes. Before stimulus exposure, additional mydriatic agents were applied. ffERG was performed OD first, then OS (or OS first, then OD in random order). After unilateral sequential testing with the placement of a DTL electrode (or Burian-Allen contact lens electrode), a subcutaneous reference electrode on the ipsilateral outer canthus, and a ground electrode on the upper limb, a dark white flash in the bowl of the LEDGanzfeld stimulator triggered a dark rod-specific response. Under dark conditions, mixed rod and cone responses were obtained using a standard bright white flash. In order to assess the photopic function of cone photoreceptors, the monkeys were allowed to adapt to the ambient light in the room for 10 minutes, and then a stroboscopic white light stimulus was presented to the dilated eye at maximum flash intensity. The following ISCEV toxicology study stimulus standards were used:

[0412] Scotopic vision: 0.158 cd-s m 2 (-12dB) stimulation (measured rod-driven response of open bipolar cells, b-wave)

[0413] Dark vision 2.51cd-s m 2 (0 dB) stimulation (rod- ​​and cone-driven responses of both photoreceptors, the a-wave, and the response of bipolar cells, the b-wave)

[0414] · Bright vision 2.51cd-s m 2 (0 dB) stimulation (the cone-driven response of both photoreceptors, the a-wave, and the responses of the on and off bipolar cells, the b-wave)

[0415] 2.51cd-s m 2 Photopic 30 Hz flicker stimulation (cone-driven response) under (0 dB) stimulation

[0416] Stimulus induction is indicated by a marker. The time-integrated luminance of the stimulus and background is recorded as absolute values. Monkeys underwent scotopic testing before photopic testing and received stimulus exposures in an order of increasing stimulus intensity for a given adaptation. Single stimulus exposure preceded flickering stimulus exposure to avoid bleaching and loss of adaptation. Waveforms were analyzed for a- and b-wave amplitudes and latencies. The b-wave amplitude was measured from the a-wave trough to the b-wave peak, or, if the a-wave was absent, from the prestimulus baseline to the b-wave peak. The a-wave amplitude was measured from the prestimulus baseline to the a-wave peak. A nonhuman primate ophthalmologic scoring system was used to assess ocular pathological changes in response to NAION induction and therapeutic interventions, with summary scores generated based on the examination components. The incidence of papilledema and flame hemorrhages was also assessed.

[0417] Imaging

[0418] Color anterior segment and fundus photography were performed using a Topcon TRC-50EX retinal camera equipped with Canon 6D digital imaging hardware and New Vision Fundus Image Analysis System software.

[0419] Optical coherence tomography (OCT) and confocal scanning laser ophthalmoscopy (cSLO)

[0420] 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 referenced to baseline images for subsequent imaging functions. Whole-body OCT volume scans of the optic nerve and the entire macula were performed through the posterior pole. Images were qualitatively evaluated by quantitative analysis of retinal thickness. For posterior pole scans centered on the macula, ganglion cell layer (GCL) and retinal nerve fiber layer (RNFL) thickness maps were generated, and thickness data were exported to a spreadsheet. Peripapillary retinal nerve fiber layer thickness (pRNFL) was determined for each quadrant (superior, inferior, temporal, and nasal). HEYEX raw data files were generated for further analysis.

[0421] Graphical ERG and VEP

[0422] At the designated time points (Table 6), pattern ERG (pERG) and pattern VEP (pVEP) recordings were performed in an OU manner by positioning the monkey with a DTL electrode and an additional active electrode placed in the occipital protuberance at the midline of the posterior skull, together with a ground electrode placed in the arm. An alternating black and white checkerboard pattern was projected at 2 Hz with a luminance of 100 cd / m2 and a contrast of 80% within the 45% visual field generated by the VERIS multifocal ERG instrument using a VERIS retinal projection stimulator. The grid size was varied in logarithmic steps, and the recordings for each grid size were repeated twice, and the latency and amplitude were determined. The pVEP amplitude and pERG N95 amplitude were determined. Figure 6 Shown are representative pERG measurements illustrating the induction of NAOIN following laser treatment of the NHP optic nerve head.

[0423] Clinical observation

[0424] General health was assessed by cage-side observation twice daily, beginning one week before dosing and until the end of the study.

[0425] Detailed clinical examination

[0426] Detailed clinical observations and physical examinations were performed at designated time points (Table 6). Respiratory rate, heart rate, blood pressure, auscultation, and integumentary integrity were also assessed. Body temperature was measured using a digital rectal thermometer.

[0427] weight

[0428] Body weights were collected between ophthalmic examinations (Table 6).

[0429] Differential CBC

[0430] Blood (0.5 mL) was transferred directly into K2EDTA lavender top vacutainers (Greiner MiniCollectEDTA tubes Ref. 450475) and kept on ice until differential CBC analysis on the Hemavet analyzer.

[0431] DNA methylation blood

[0432] Blood was transferred directly to a 0.5 mL microcentrifuge tube anticoagulated with K2EDTA. The samples were then lysed and DNA isolated on QIAMP MiniElute columns according to the manufacturer's instructions (Qiagen). The DNA samples were analyzed to determine DNA methylation, a measure of biological age.

[0433] serum

[0434] At the designated time points (Table 6), blood (3 ml) was transferred directly to serum separator tubes (red top) and allowed to stand at room temperature for 30 to 60 minutes, followed by centrifugation at 3000 rpm for 10 minutes at 4° C. Two usable serum aliquots (approximately 0.5 mL × 2) were carefully transferred to labeled 1.8 mL cryovials and stored below −70° C. and shipped to designated laboratories for nAb analysis.

[0435] Enucleation and eye disposal

[0436] 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 the animals were euthanized with sodium pentobarbital (100 mg / kg IV). After placing a suture mark at the 12 o'clock position, the eyeball (OU) was removed. Excess tissue on the eyeball connected to the optic nerve was trimmed off and placed in Davidson's fixative for 24 hours at room temperature, while approximately 300 μl of Davidson's fixative was injected into the eyeball. The eyeball was transferred to phosphate buffered saline (PBS) containing 0.05% sodium azide, stored in a container maintained at 4°C, and transported to a designated histology laboratory for histological and immunohistochemical processing and analysis by a board-certified veterinary pathologist. Additional histological quantitative scoring was performed under the guidance of qualitative findings.

[0437] Axon quantification

[0438] Axon quantification is accomplished using a workflow consisting of three computer programs. The first program identifies the entire optic nerve from the slide background, improving the speed and accuracy of the next program. The second program distinguishes nerve bundles from connective tissue, improving the accuracy of the final application by eliminating erroneous counts of axon-like connective tissue elements. The final program locates and classifies axons into three categories: healthy, semi-healthy, and degenerated. The final program also generates numerical data, such as the number and area of ​​each axon category, allowing for calculation of density and / or ratio endpoints.

[0439] pERG results

[0440] All NHPs (African green monkeys; n=20) underwent NAION-like lesions induced on day 0 by intravenous injection of Rose Bengal, followed by laser treatment of the ONH of the OS eye. Pre-treated NHPs (n=6) received intravitreal (IVT) injections of OSK (doxycycline-induced AAV2-OSK; AAV2-TRE-OSK + pAAV2-CMV-rtTA3VP16 ratio 1:1) or vehicle (n=4) into the OS eye on day 28. Post-treatment NHPs received IVT injections of OSK (n=6) or vehicle (n=4) into the OS eye on day +1. Throughout the experiment, all NHPs received daily oral doxycycline. Both eyes were examined at baseline and then weekly until day +35 (corresponding to day 42) after laser treatment to perform multiple imaging and functional measurements of retinal ganglion cells (RGCs).

[0441] OSK pretreatment 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 Post-OSK treatment showed significant recovery of pERG function 5 weeks after laser treatment (p50-n95 amplitude: 4.60±0.24 vs. 2.89±0.79, P=0.039) ( Figure 8 ).

[0442] To further analyze the effects of OSK treatment on NAION, a correlation analysis was performed comparing the extent of early optic disc edema with the extent of pERG deficits at week 5 after laser treatment. Optic disc edema was calculated by measuring the optic disc size at day 8 and subtracting the baseline optic disc size. Optic disc edema indicates early laser-induced 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 absolute amplitude at baseline. pERG deficits indicate long-term damage to retinal ganglion cells (RGCs). In vehicle-treated NHPs, the extent of early optic disc edema correlated with the extent of pERG deficits at week 5 after laser treatment, suggesting a long-term effect of the initial laser damage ( Figure 9 and Figure 10A Surprisingly, OSK-treated NHPs showed a reduced correlation between the degree of early optic disc edema and pERG deficits at 5 weeks after laser treatment, suggesting reduced RGC damage ( Figure 10B ).

[0443] A similar correlation analysis was performed at the end of the experiment to compare the extent of early optic disc edema with the extent of axonal density damage at the end of the study. In vehicle-treated NHPs, the extent of early optic disc edema correlated with the extent of axonal density damage at the end of the study, suggesting a long-term effect of the initial laser injury ( Figure 11 and Figure 12A Surprisingly, at the end of the study, OSK-treated NHPs showed a reduced correlation between the degree of early optic disc edema and axonal density damage, suggesting prevention or reversal of axonal damage ( Figure 12B ).

[0444] Table 10: Summary of NHP-related data (axon density and pERG)

[0445] Axon density pERG Vehicle (all) n=7 <![CDATA[R 2 =0.76]]> <![CDATA[R 2 =0.71]]> OSK (all) n = 12 <![CDATA[R 2 =0.23]]> <![CDATA[R 2 =0.19]]> OSK (prevention) n=6 <![CDATA[R 2 =0.18]]> <![CDATA[R 2 =0.27 <!-- 76 -->]]> OSK (rescue) n=6 <![CDATA[R 2 =0.33]]> <![CDATA[R 2 =0.09]]>

[0446] In the NHP of vehicle treatment, the degree of early optic disc edema is relevant to the axon density degree at the end of the study, showing the long-term impact of initial laser damage. In the NHP of OSK treatment, by preventing and / or reversing axon damage, the predictability of axon density at the end of the study is lower (correlation decreases) for the degree of early optic disc edema. In the NHP of vehicle treatment, the pERG effect amplitude at the end of the study is relevant to the degree of early optic disc edema, showing the long-term impact of initial laser damage. In the NHP of OSK treatment, by preventing and / or reversing axon damage, the predictability of pERG effect amplitude at the end of the study is lower (correlation decreases) for the degree of early optic disc edema.

[0447] Example 3 - Clinical Trial

[0448] This clinical trial will evaluate the effects of a single dose of ER-100, a dual-vector AAV2 administered intravitreally (IVT) containing a 1:1 ratio of AAV2-CMV-rtTA-L to AAV2-TRE-OSK, in patients with glaucoma or NAION. ER-100 is administered along with oral doxycycline, a widely used antibiotic, to activate OSK expression.

[0449] Men and women aged at least 50 years with clinical findings consistent with NAION or men and women aged at least 35 years with clinical findings consistent with glaucoma will be enrolled. The doxycycline-responsive two-vector system will be administered intravitreally. Participants will receive oral doxycycline starting on the day of viral administration and continuing until day 56 of the clinical trial.

[0450] This is a first-in-human (FIH) multicenter study that will include a dose escalation phase (Phase 1a) followed by an expansion phase (Phase 1b). ER-100 will be administered to one eye in an open-label manner. Systemic doxycycline will be used for 8 weeks, followed by a placebo doxycycline for an additional 6 weeks, so that participants will not know when doxycycline is discontinued. Participants will be blinded to the placebo doxycycline.

[0451] 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 injection (IVT) in patients with optic neuropathy (glaucoma, NAION). The following endpoints will be monitored: the incidence of treatment-emergent adverse events (TEAEs), the incidence of dose-limiting toxicities at different dose levels as defined by the glaucoma and NAION regimens, changes from baseline in safety laboratory tests, and negative changes from baseline in ocular assessments. These endpoints will be measured over time with doxycycline (up to Day 56) and over time with placebo (Days 57-84) or with no treatment (after Day 84).

[0452] The secondary objective of this clinical trial is to explore the efficacy of a single IVT injection of ER-100 in patients with optic neuropathy (glaucoma, NAION). The following endpoints will be measured and compared to changes 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 perimetry, 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. Any anatomical changes in the optic nerve head and retina observed via color fundus examination / photography will also be described. These endpoints will be measured during the time period of doxycycline use (up to Day 56) and during the time period of placebo use (Days 57-84) or without treatment (after Day 84).

[0453] Another secondary objective of this clinical trial is to characterize the immune response, viral shedding, and kinetics of ER-100. The following endpoints will be monitored during the doxycycline timeframe (up to day 56) and the placebo timeframe (days 57-84) or when not treated (after day 84): neutralizing antibody levels to AAV2; measures based on ELISPOT assays (including IFγ, rtTA-L, OSK, capsid protein); anti-drug antibody levels to rtTA-L and OSK; ER-100 viral shedding will be 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 (up to day 56) and the placebo timeframe (days 57-84) or when not treated (after day 84).

[0454] Results are expected to show that expression of OSK in RGCs of NAOIN subjects will result in increased pERG amplitude, improved visual acuity, reduced visual field defects, and overall improved ocular function.

[0455] List of Examples I

[0456] Item 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.

[0457] Item 2. The method of Item 1, further comprising administering to the subject a nucleic acid molecule comprising a nucleic acid sequence encoding a reverse tetracycline-controlled transactivator (rtTA).

[0458] Item 3. The method of Item 1 or Item 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.

[0459] Item 4. The method of Item 3, wherein the inducible promoter comprises a tetracycline response element (TRE).

[0460] Item 5. The method of Item 4, wherein the tetracycline antibiotic is doxycycline.

[0461] Item 6. The method of Item 4, wherein the inducible promoter is a TRE2 promoter.

[0462] Item 7. The method of any one of Items 2-6, wherein rtTA is rtTA3 or rtTA4.

[0463] Item 8. The method of any one of Items 2-7, wherein the nucleic acid molecule comprising a nucleic acid sequence encoding rtTA is operably linked to a CMV promoter.

[0464] Item 9. The method as described in any of the preceding items, 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.

[0465] Item 10. The method of Item 9, wherein the AAV vector is serotype 2 (AAV2).

[0466] Item 11. The method according to any one of the preceding items, 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.

[0467] Item 12. The method according to any one of the preceding items, 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.

[0468] Item 13. The method of Item 12, wherein the self-cleaving peptide is a 2A peptide.

[0469] Item 14. The method of any one of the preceding items, wherein the nucleic acid molecules encoding OCT4, SOX2, and KLF4 are flanked by inverted terminal repeats (ITRs), and wherein the distance between ITRs is 4.7 kb or less.

[0470] Item 15. The method of any of the preceding items, further comprising administering an induction agent to the subject.

[0471] Item 16. The method of any one of Items 2-15, wherein the nucleic acid molecule encoding reverse tetracycline-controlled transactivator (rtTA) is an AAV vector that does not contain nucleic acid molecules encoding OCT4, SOX2, and KLF4.

[0472] Item 17. The method of any one of the preceding items, wherein the nucleic acid molecule encoding OCT4, SOX2, and KLF4 comprises nucleic acid elements in the following order:

[0473] a) first inverted terminal repeat (ITR) sequence (SEQ ID NO: 16);

[0474] b) TRE promoter sequence (SEQ ID NO: 7);

[0475] c) a nucleic acid sequence encoding OCT4 (SEQ ID NO: 1);

[0476] d) a nucleic acid sequence encoding P2A (SEQ ID NO: 8);

[0477] e) a nucleic acid sequence encoding SOX2 (SEQ ID NO: 3);

[0478] f) a nucleic acid sequence encoding T2A (SEQ ID NO: 10);

[0479] g) a nucleic acid sequence encoding KLF4 (SEQ ID NO: 5);

[0480] h) an SV-40 derived terminator sequence (SEQ ID NO: 12); and

[0481] i) a second inverted terminal repeat (ITR) sequence (SEQ ID NO: 32).

[0482] Item 18. The method of any one of the preceding items, wherein the nucleic acid sequence encoding OCT4 comprises SEQ ID NO: 1.

[0483] Item 19. The method of any one of the preceding items, wherein the nucleic acid sequence encoding SOX2 comprises SEQ ID NO: 3.

[0484] Item 20. The method of any one of the preceding items, wherein KLF4 is a human KLF4 protein.

[0485] Item 21. The method of any one of the preceding items, wherein the nucleic acid sequence encoding KLF4 comprises SEQ ID NO:5.

[0486] Item 22. The method of Item 17, wherein the nucleic acid sequence encoding P2A comprises SEQ ID NO:8.

[0487] Item 23. The method of Item 17, wherein P2A comprises the amino acid sequence of SEQ ID NO:9.

[0488] Item 24. The method of Item 17, wherein T2A comprises the amino acid sequence of SEQ ID NO: 11.

[0489] Item 25. The method of Item 17, wherein the nucleic acid sequence encoding T2A is GAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAA AATCCCGGCCCA (SEQ ID NO: 10).

[0490] Item 26. The method of Item 17, wherein the TRE promoter sequence is SEQ ID NO: 7.

[0491] Item 27. The method of Item 17, wherein the SV-40 derived terminator sequence is SEQ ID NO:12.

[0492] Item 28. The method of Item 17, wherein the first ITR sequence is SEQ ID NO: 16.

[0493] Item 29. The method of Item 17, wherein the nucleic acid molecule encoding OCT4, SOX2, and KLF4 comprises SEQ ID NO:13.

[0494] Item 30. The method of Item 17, wherein the nucleic acid molecule encoding OCT4, SOX2, and KLF4 comprises SEQ ID NO:14.

[0495] Item 31. The method of any one of Items 2-30, wherein the nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the nucleic acid molecule encoding rtTA are administered sequentially or simultaneously.

[0496] Item 32. The method of any of the preceding items, wherein nucleic acid molecules encoding OCT4, SOX2, and KLF4 are administered intravitreally.

[0497] Item 33. The method of any one of Items 2-32, wherein the nucleic acid molecule encoding rtTA is administered intravitreally.

[0498] Item 34. The method of any one of Items 2-31, wherein the nucleic acid molecules encoding OCT4, SOX2, and KLF4 and the nucleic acid molecule encoding rtTA are administered at a ratio of about 1:1.

[0499] Item 35. The method of any one of Items 16-34, wherein the AAV vector 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).

[0500] Item 36. A method as described in any of the preceding items, wherein the nucleic acid molecule encoding OCT4, SOX2 and KLF4 is an AAV2-TRE-OSK vector comprising a nucleic acid molecule containing SEQ ID NO: 35, and the nucleic acid molecule encoding rtTA is a pAAV2-CMV-rtTA3VP16 vector comprising a nucleic acid molecule containing SEQ ID NO: 36 or an AAV2-CMV-rtTA4 vector comprising a nucleic acid molecule containing SEQ ID NO: 37.

[0501] Item 37. The method of Item 16, wherein the nucleic acid molecule encoding the reverse transcriptase tetracycline-controlled transactivator (rtTA) is an AAV vector comprising a nucleic acid element of the following sequence:

[0502] a) first inverted terminal repeat (ITR) sequence (SEQ ID NO: 22);

[0503] b) CMV promoter sequence (SEQ ID NO: 17);

[0504] c) CMV enhancer sequence (SEQ ID NO: 18);

[0505] d) a nucleic acid sequence encoding rtTA3 (SEQ ID NO: 19);

[0506] e) WPRE sequence (SEQ ID NO: 23), and

[0507] f) a second inverted terminal repeat (ITR) sequence (SEQ ID NO: 33).

[0508] Item 38. The method of Item 16, wherein the nucleic acid molecule encoding the reverse transcriptase tetracycline-controlled transactivator (rtTA) is an AAV vector comprising a nucleic acid element of the following sequence:

[0509] a) first inverted terminal repeat (ITR) sequence (SEQ ID NO: 29);

[0510] b) CMV promoter sequence (SEQ ID NO: 24);

[0511] c) CMV enhancer sequence (SEQ ID NO: 25);

[0512] d) a nucleic acid sequence encoding rtTA4 (SEQ ID NO: 26);

[0513] e) WPRE sequence (SEQ ID NO: 31)

[0514] f) SV40 sequence (SEQ ID NO 30), and

[0515] g) a second inverted terminal repeat (ITR) sequence (SEQ ID NO: 34).

[0516] Item 39. The method of Item 36, wherein the effective amount of the AAV2-TRE-OSK vector is about 1×10 9 vg / eye is about 1×10 14 vg / eye range.

[0517] Item 40. The method of Item 36, wherein the effective amount of the AAV2-CMV-rtTA3 vector is about 1×10 9 vg / eye is about 1×10 14 vg / eye range.

[0518] Item 41. The method of Item 36, wherein the effective amount of the AAV2-CMV-rtTA4 vector is about 1×10 9 vg / eye is about 1×10 14 vg / eye range.

[0519] Item 42. The method of any one of the preceding items, wherein the nucleic acid molecules encoding OCT4, SOX2, and KLF4 are administered to the subject by left eye (OS) injection, right eye (OD) injection, or binocular (OU) injection.

[0520] Item 43. The method of any one of Items 2-42, wherein the nucleic acid molecule encoding rtTA is administered to the subject by left eye (OS) injection, right eye (OD) injection, or binocular (OU) injection.

[0521] Item 44. The method of any of the preceding items, further comprising administering to the subject an effective amount of an antibiotic.

[0522] Item 45. The method of Item 44, wherein the antibiotic is administered at least one day prior to administration of the nucleic acid molecule encoding rtTA.

[0523] Item 46. The method of Item 44, wherein an antibiotic is administered when the nucleic acid molecule encoding rtTA is administered.

[0524] Item 47. The method of Item 44, wherein the antibiotic is administered at least one day after administration of the nucleic acid molecule encoding rtTA.

[0525] Item 48. A method for recombinantly producing AAV, the method comprising introducing a vector into a cell under conditions that produce AAV, wherein the vector comprises one or more nucleic acid sequences encoding a) OCT4, SOX2, and KLF4.

[0526] Item 49. The method of Item 48, wherein the cells comprise a population of HEK293T cells.

[0527] Item 50. A method of producing AAV, comprising modifying a cell to express one or more plasmids, the 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.

[0528] Item 51. The method of Item 50, wherein the cells comprise a population of HEK293T cells.

[0529] Item 52. A method 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.

[0530] Item 53. The method of claim 52, wherein the composition does not reprogram cells, tissues, or organs of the subject to a pluripotent state.

[0531] Item 54. The method of Item 52, wherein the composition does not induce c-Myc expression in the subject.

[0532] Item 55. The method of Item 52, wherein the polynucleotide comprises DNA, RNA, or a combination thereof.

[0533] Item 56. The method of Item 55, wherein the DNA comprises plasmid DNA.

[0534] Item 57. The method of Item 55, wherein the RNA comprises mRNA.

[0535] Item 58. The method of Item 52, wherein the polynucleotide comprises an inducible promoter.

[0536] Item 59. The method of Item 58, wherein the inducible promoter comprises a tetracycline response element (TRE).

[0537] Item 60. The method of Item 52, further comprising administering an inducing agent to the subject to induce expression of OCT4, SOX2, and KLF4 in the subject.

[0538] Item 61. The method of Item 60, wherein the inducer comprises a tetracycline antibiotic.

[0539] Item 62. The method of Item 61, wherein the tetracycline antibiotic is doxycycline.

[0540] Item 63. The method of Item 60, wherein the inducing agent comprises a reverse tetracycline-controlled transactivator (rtTA) or a polynucleotide encoding rtTA.

[0541] Item 64. The method of Item 63, wherein the polynucleotide encoding rtTA is located in an expression vector.

[0542] Item 65. The method of Item 60, wherein the composition and the inducer are administered sequentially or simultaneously.

[0543] Item 66. The method of Item 60, wherein the composition and the inducer are administered in a ratio of about 1:1.

[0544] Item 67. The method of Item 52, wherein the expression vector further comprises a polynucleotide encoding a self-cleaving peptide.

[0545] Item 68. The method of Item 67, wherein the self-cleaving peptide is a 2A peptide.

[0546] Item 69. The method of Item 52, wherein the polynucleotide comprises an inverted terminal repeat (ITR).

[0547] Item 70. The method of Item 52, wherein the expression vector is a viral expression vector, wherein the viral vector is a lentiviral, retroviral, adenoviral, alphaviral, vaccinia virus, or adeno-associated virus (AAV) vector.

[0548] Item 71. The method of Item 70, wherein the AAV vector is serotype 2 (AAV2).

[0549] Item 72. The method of Item 52, wherein the polynucleotide comprises nucleic acid elements in the following order:

[0550] a. first inverted terminal repeat (ITR) sequence;

[0551] b. TRE3G promoter sequence;

[0552] c.OCT4 sequence;

[0553] d. P2A cleavage sequence;

[0554] e. SOX2 sequence;

[0555] f.T2A cleavage sequence;

[0556] g.KLF4 sequence;

[0557] h. SV-40 derived terminator sequence; and

[0558] i. Second inverted terminal repeat (ITR) sequence.

[0559] Item 73. The method of Item 52, wherein

[0560] i) OCT4 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 2;

[0561] ii) SOX2 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 4; and / or

[0562] iii) KLF4 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:6.

[0563] 74. The method according to item 52, wherein

[0564] i) the polynucleotide comprises a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 1;

[0565] ii) the polynucleotide comprises a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 3; and / or

[0566] iii) the polynucleotide comprises a nucleic acid sequence that is at least 75% identical to SEQ ID NO:5.

[0567] Item 75. The method of Item 52, wherein the composition is administered intravitreally.

[0568] Item 76. The method of Item 52, wherein the composition is administered to the subject by left eye (OS) injection, right eye (OD) injection, or both eyes (OU) injection.

[0569] Item 77. The method of Item 52, wherein administration of the composition improves retinal ganglion cell (RGC) function and / or restores visual function in the subject.

[0570] Item 78. The method of Item 52, wherein the prevention or treatment of NAION is measured by electroretinography (pERG).

[0571] Item 79. A method 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, the expression vector comprising a polynucleotide encoding three transcription factors, wherein the three transcription factors consist of OCT4, SOX2, and KLF4.

[0572] List of Examples II

[0573] Item 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, wherein the gene therapy vector comprises 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 Kruppel-like factor 4 (KLF4), operably linked to at least one promoter (or operably linked to a recombination site, blunt-end ligation, or homologous site for genome editing to place the one or more nucleic acid molecules under the control of an endogenous promoter).

[0574] Item 2. The method of Item 1, wherein the ischemic optic neuropathy is non-arteritic anterior ischemic optic neuropathy (NAION).

[0575] Item 3. The method of Item 1, wherein the ischemic optic neuropathy is arteritic anterior ischemic optic neuropathy (A-AION).

[0576] Item 4. The method of Item 1, wherein the ischemic optic neuropathy is posterior ischemic optic neuropathy (PION).

[0577] Item 5. The method of any one of Items 1 to 4, wherein the vector is administered intravitreally.

[0578] Item 6. The method of any one of Items 1 to 5, wherein when administered to the eye in vivo, the method of administering delivers the one or more nucleic acid molecules to retinal ganglion cells.

[0579] Item 7. A method as described in any one of Items 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.

[0580] Item 8. The method of any one of Items 1 to 7, wherein the vector is a viral vector.

[0581] Item 9. The method of any one of Items 1 to 8, wherein the carrier is a liposome or a lipid nanoparticle (LNP).

[0582] Item 10. The method of Item 8, wherein the viral vector is an adeno-associated viral (AAV) vector, optionally an oculotrophic AAV vector.

[0583] Item 11. The method of Item 10, wherein the AAV vector is an AAV serotype 2 (AAV2) vector or a variant thereof.

[0584] Item 12. The method of Item 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, optionally an AAV serotype specific for the eye listed in Table 2.

[0585] Item 13. The method of any one of Items 10 to 12, wherein the one or more nucleic acid molecules is an AAV genome comprising flanking inverted terminal repeats (ITRs).

[0586] Item 14. The method of Item 9, wherein the carrier is an LNP comprising an ionizable lipid, a helper lipid, a sterol, and a poly(ethylene glycol)-lipid (PEG-lipid).

[0587] Item 15. The method of any one of Items 1 to 14, wherein the promoter is an inducible promoter.

[0588] Item 16. The method of Item 15, wherein the inducible promoter is a tetracycline response element (TRE) promoter, optionally a Tet-On promoter, optionally a TRE3G promoter.

[0589] Item 17. The method of Item 16, wherein the method further comprises sequentially or concurrently 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 control the TRE promoter.

[0590] Item 18. The method of Item 17, wherein rtTA is selected from the r tTA listed in Table 1, optionally with MSRLDKSKIINSALELLNGVGIEGLTTRKLAQKLGV EQPTLYWHVKNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPL LKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG (SEQ ID NO:20) an rtTA having at least 80%, at least 90%, at least 85% or 100% identity, and optionally comprising one or more amino acid substitutions selected from V9I, G12S, F67S, G72V, G72P and R171K, optionally comprising the amino acid acidic substitutions G12S, F67S and R171K or the amino acid substitutions V9I, G12S, F67S and R171K.

[0591] Item 19. The method of any one of Items 16 to 18, wherein the method further comprises administering a tetracycline inducer, optionally doxycycline or tetracycline.

[0592] Item 20. The method of any one of Items 1 to 19, wherein the vector is a polycistronic vector and the one or more nucleic acid molecules is a nucleic acid molecule comprising open reading frames encoding OCT4, SOX2, and KLF4.

[0593] Item 21. The method of Item 20, wherein the open reading frame does not encode other proteins.

[0594] Item 22. The method of Item 20 or Item 21, wherein OCT4, SOX2, and KLF4 are linked by a self-cleaving peptide (optionally a 2A peptide).

[0595] Item 23. The method of any one of Items 20 to 22, wherein the open reading frame encodes OCT4, SOX2, and KLF4 in 5′ to 3′ order.

[0596] Item 24. The method of any one of Items 15 to 19, wherein the method comprises administering an inducing agent to the subject for a period of time sufficient to rejuvenate the retinal ganglion cells but not induce pluripotency in the cells.

[0597] Item 25. The method of any one of Items 1 to 25, wherein OCT4 is at least 80%, at least 90%, at least 85% or 100% identical to SEQ ID NO: 2; SOX2 is at least 80%, at least 90%, at least 85% or 100% identical to SEQ ID NO: 4, and / or KLF4 is at least 80%, at least 90%, at least 85% or 100% identical to SEQ ID NO: 6.

[0598] Item 26. The method of any one of Items 1 to 25, wherein the method improves retinal ganglion cell (RGC) function and / or restores visual function in the subject.

[0599] Item 27. The method of any one of Items 1 to 26, wherein the prevention or treatment of NAION is measured by electroretinography (pERG).

[0600] Item 28. A gene therapy vector for use in the method according to any one of Items 1 to 27.

[0601] ***

[0602] Incorporated by reference

[0603] The entire disclosure of each patent and scientific article cited herein is incorporated by reference for all purposes.

[0604] Reference in this specification to any previous publication (or information obtained therefrom) or any known matter is not and should not be taken as an acknowledgment or endorsement or as implying in any form that the previous publication (or information obtained therefrom) or known matter forms part of the common general knowledge in the field to which this specification relates.

[0605] Equivalent solutions

[0606] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The above embodiments are therefore to be considered in all respects as illustrative rather than restrictive 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 equivalence of the claims are intended to be encompassed therein.

Claims

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 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 Kruppel-like factor 4 (KLF4), Operably linked to at least one promoter (or operably linked to a recombination site, blunt-end ligation, or homology site for genome editing to place the one or more nucleic acid molecules under the control of an endogenous promoter).

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

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

4. 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 when administered to the eye in vivo, the method of administration delivers the one or more nucleic acid molecules to retinal ganglion cells.

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 of any one of claims 1 to 7, wherein the vector is a viral vector.

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

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

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

12. The method of claim 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, optionally an AAV serotype specific for the eye listed in Table 2.

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

14. The method of claim 9, wherein the carrier is a LNP comprising 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. The method of claim 15, wherein the inducible promoter is a tetracycline response element (TRE) promoter, optionally a Tet-On promoter, optionally a TRE3G promoter.

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

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

19. The method of any one of claims 16 to 18, wherein the method further comprises administering a tetracycline inducer, optionally doxycycline or tetracycline.

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

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

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

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

24. The method of any one of claims 15 to 19, wherein the method comprises administering the inducing agent to the subject for a period of time sufficient to rejuvenate retinal ganglion cells but not induce multipotency in the cells.

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

6.

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. The method of any one of claims 1 to 26, wherein preventing or treating NAION is measured by electroretinography (pERG).

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

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