Reversing aging of the central nervous system

Abstract

Provided herein are engineered nucleic acids (e.g., expression vectors including viral vectors, e.g., lentiviral vectors, adenoviral vectors, AAV vectors, herpes viral vectors, and retroviral vectors) encoding OCT4; KLF4; SOX2; or any combination thereof, which are useful, for example, in inducing cell reprogramming, tissue repair, tissue regeneration, organ regeneration, reversing aging in the central nervous system or ex vivo, or any combination thereof. Also provided herein are recombinant viruses (e.g., lentiviruses, alphaviruses, vaccinia viruses, adenoviruses, herpes viruses, retroviruses, or AAVs) comprising engineered nucleic acids (e.g., engineered nucleic acids), engineered cells, compositions comprising engineered nucleic acids, recombinant viruses, engineered cells, engineered proteins, chemicals capable of activating expression of OCT4; KLF4; SOX2; or any combination thereof, engineered proteins selected from the group consisting of OCT4; KLF4; SOX2; or any combination thereof, antibodies capable of activating expression of OCT4; KLF4; SOX2; or any combination thereof, and methods of treating a disease (e.g., a neurological disease), preventing a disease (e.g., a neurological disease), controlling cell reprogramming (e.g., inducing or inducing and then halting), controlling tissue repair, controlling tissue regeneration, or any combination thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application No. 63 / 328,069, filed April 6, 2022, which is incorporated herein by reference in its entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (H082470401WO00-SEQ-FL.xml; size: 278,541 bytes; created on April 5, 2023) are incorporated herein by reference in their entirety.

[0003] Government support This invention was made with government support under award AG068303 from the National Institutes of Health (NIH). The government has certain rights in this invention. [Background technology]

[0004] In many animals, including vertebrates, vital organs have limited intrinsic regenerative and repair capabilities. Acute injuries and chronic disorders can damage vital organs and tissues, including the brain. However, mature somatic cells often cannot survive these injuries. Even if they survive, they are unable to self-renew and transdifferentiate to replace damaged cells. Furthermore, cells capable of self-renewal may be limited in number, have limited capacity, and are particularly susceptible to damage with age. In contrast to somatic cells from adults, cells from individuals chronologically closer to conception, such as those from embryos and infants, exhibit cellular youthfulness and have a greater ability to withstand injury and stress and heal, renew, and regenerate organs and tissues. Therefore, compositions and methods directed at rejuvenating cells and thereby restoring them from an aged, mature state to a younger, more vigorous state have long been sought for treating certain injuries and diseases, and generally for reversing and preventing aging throughout the organism.

[0005] The body has two types of information: digital and analog. DNA is digital information, and the epigenome is analog information. Analog information does not last as long as digital, and analog information cannot be copied with as much fidelity as digital information. This has implications for how long an organism survives and develops. Aging was once thought to be a process driven by mutations in a cell's genetic material. This has largely been abandoned as an explanation. Aging is now primarily thought to be due to epigenetic changes that cause cells to transcribe the wrong genes at the wrong time, a process that over time leads to greater dysfunction, disease, incurability, and ultimately death. Yamanaka factors (OCT4, SOX2, c-Myc, and KLF4) have previously been shown to induce pluripotency in vitro (Takahashi et al., Cell. 2006 Aug 25;126(4):663-76) and reverse the DNA methylation clock of aging (Horvath, Genome Biol. 2013). Nanog and Lin28 may cooperate with Yamanaka factors to help induce pluripotency. Tet1, NR5A-2, Sall4, and NKX3-1 may substitute for Oct4 (Gao et al., Cell Stem Cell 12, 1-17, April 4, 2013; and Mai et al., Nature Cell Biology 20, 900-908, 2018). However, expression of the original four transcription factors in transgenic mice induces teratomas in vivo, along with other acute toxicities such as intestinal epithelial metaplasia, which can kill the animals within a few days (Abad et al., Nature. 2013 Oct 17;502(7471):340-5). Therefore, a non-toxic and efficient method of cell reprogramming is needed. Summary of the Invention [Means for solving the problem]

[0006] It is hypothesized that the cellular aging process is caused by the loss of both genetic and epigenetic information. Previous studies have hypothesized that aging is primarily caused by the loss of genetic information (most commonly in the form of gene mutations such as substitutions and deletions on the genome of an organism), but the systems, compositions, uses, kits, and methods disclosed herein are based on the unexpected finding that aging of the central nervous system is primarily driven by the loss of specific epigenetic information that is established closer to fertilization and terminal differentiation of specific cells. Epigenetic information, sometimes referred to as a cell's "analog" information, typically takes the form of covalent modifications of DNA, such as 5-methylcytosine (5mC), hydroxymethylcytosine (5hmeC), 5-formylcytosine (fC), 5-carboxylcytosine (caC), and adenine methylation, and of certain proteins, such as lysine acetylation of histone proteins, lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation. Loss of this analog information can result in the dysregulation of essential cellular processes, such as those that maintain cellular identity, causing cells to exhibit traits typically associated with aging, such as senescence.

[0007] Brain aging is often characterized by the progressive loss of neuronal plasticity and function, leading to cognitive decline and increased vulnerability to neurodegeneration.Most clinically approved treatments for neurological disorders attempt to regulate the activity of neurotransmitters by targeting proteins, rather than reversing the cellular causes of aging.For example, memantine is an NMDA receptor antagonist and is approved by the Food and Drug Administration in the United States and Europe for patients with Alzheimer's disease.Other treatments for Alzheimer's disease include cholinesterase inhibitors.However, these inhibitors fail to address the underlying disease.

[0008] Surprisingly, as disclosed herein, it has been found that epigenetic reprogramming by three transcription factors is sufficient to improve neuronal and cognitive function in vivo.The disclosed method, composition, use and kit rejuvenate cells by preventing and reversing the cellular causes of aging in central nervous system (for example, brain).In some embodiments, central nervous system does not include eye.In some embodiments, central nervous system does not include retina, uvea, pupil, lens, cornea and / or sclera.

[0009] The present disclosure stems from the unexpected discovery that, in some embodiments, precise expression of OCT4, SOX2, and KLF4 in the absence of exogenous c-Myc expression can be used to promote reprogramming and tissue regeneration in the central nervous system (e.g., the brain) in vivo without acute toxicity. Surprisingly, in some embodiments, the compositions disclosed herein can penetrate the blood-brain barrier. The results disclosed herein demonstrate that OCT4, SOX2, and KLF4 expression can be used to reverse brain aging in the absence of exogenous c-Myc expression. The expression vectors provided herein, in certain embodiments, allow for precise control and targeting of OCT4, SOX2, and KLF4 (OSK) expression to the central nervous system, delivery of viruses (e.g., adeno-associated viruses (AAVs)) to high viral titers (e.g., 2×10 per preparation), and delivery of HIV-1-associated viruses (e.g., HIV-1-associated viruses (AAVs) to the central nervous system). 12 Particles, 1 x 10 per mL 13 This allows for incorporation of the drug in large amounts (more than 10 ...

[0010] Aspects of the present disclosure provide several systems, compositions, uses, kits, and methods that may be useful for the efficient rejuvenation of cells, tissues, and / or organs of the central nervous system. In some embodiments, the central nervous system does not include the retina. In some embodiments, cells, tissues, and / or organs of the central nervous system are brain cells, brain tissue, and / or the brain. For example, aspects of the present disclosure provide nucleic acids that may be useful for the efficient rejuvenation of the central nervous system by promoting the expression of OCT4, SOX2, and / or KLF4 without inducing c-Myc expression. In some embodiments, such nucleic acids encode inducers (e.g., tetracycline transactivators or reverse tetracycline transactivators) operably linked to the CaMKIIα promoter. In some embodiments, the CaMKIIα promoter is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 149. In some embodiments, the nucleic acid encoding the inducer does not comprise a synapsin-I promoter or a CaMKII-gamma promoter. In some embodiments, the synapsin-I promoter is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 157. In some embodiments, the nucleic acid encoding the inducer is a viral vector. In some embodiments, the viral vector is packaged into an AAV.PHP.eB virus.In some embodiments, the nucleic acid encoding the inducer comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to rtTA2S-M2 (SEQ ID NO: 14), pLVX-rtTA-hOSK-all-in-one (human) (SEQ ID NO: 123), pAAV-CaMKIIα-tTA2 (SEQ ID NO: 124), pAAV-CaMKIIα-rtTA2S-M2 (SEQ ID NO: 125), pAAV-CaMKIIα-rtTA3 (SEQ ID NO: 126), CaMKIIα promoter (SEQ ID NO: 146, 149, or 154); the reverse complement of rtTA Advanced (SEQ ID NO: 128), tTA Advanced (SEQ ID NO: 137), and / or tTA (SEQ ID NO: 158). In some embodiments, the nucleic acid encoding the inducer does not comprise a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to pAAV-ihSyn1-tTA (SEQ ID NO: 127). In some embodiments, the inducer comprises a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to rtTA Advanced (SEQ ID NO: 129), tTA Advanced (SEQ ID NO: 138), rtTA2S-M2 (SEQ ID NO: 15), and / or tTA (SEQ ID NO: 159).

[0011] Aspects of the present disclosure provide nucleic acids that may be useful for inducing the expression of OCT4, KLF4, and / or SOX2 in cells, tissues, or organs of the central nervous system in the absence of inducing c-Myc expression. These nucleic acids may be useful for rejuvenating cells, tissues, or organs of the central nervous system. In some embodiments, the nucleic acid has (a) a nucleic acid sequence encoding OCT4, KLF4, and SOX2 operably linked to a TRE promoter; and (b) a nucleic acid sequence encoding rtTA operably linked to a UbC promoter. In some embodiments, the nucleic acid sequence encoding OCT4, KLF4, and SOX2 further encodes a 2A peptide. In some embodiments, the nucleic acid having (a) and (b) further encodes a neomycin resistance gene and / or includes a WPRE sequence. In some embodiments, the neomycin resistance gene is operably linked to a PGK promoter. In some embodiments, the nucleic acid having (a) and (b) encodes at least one protein sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to a sequence selected from rtTA Advanced (SEQ ID NO: 129); human OCT4 (SEQ ID NO: 41); P2A (SEQ ID NO: 118); human SOX2 (SEQ ID NO: 43); T2A (SEQ ID NO: 9); human KLF4 (SEQ ID NO: 45); and a neomycin resistance gene (SEQ ID NO: 134). In some embodiments, the nucleic acid having (a) and (b) comprises at least one sequence that is at least 70% identical to a sequence selected from the reverse complement of rtTA Advanced (SEQ ID NO: 128); the reverse complement of the UbC promoter (SEQ ID NO: 130); the P tight TRE promoter (SEQ ID NO: 24); human OCT4 (SEQ ID NO: 40); P2A (SEQ ID NO: 119); human SOX2 (SEQ ID NO: 42); T2A (SEQ ID NO: 120); human KLF4 (SEQ ID NO: 131); PGK promoter (SEQ ID NO: 132); neomycin resistance gene (SEQ ID NO: 133); and WPRE (SEQ ID NO: 135).In some embodiments, the nucleic acid having (a) and (b) comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to pLVX-rtTA-hOSK-all-in-one(human) (SEQ ID NO: 123).

[0012] In some embodiments, the nucleic acid encodes tTA, wherein tTA comprises a sequence at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 138. In some embodiments, the nucleic acid encoding tTA is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 137. In some embodiments, the nucleic acid encoding tTA comprises an hGH pA sequence and / or a CMV promoter. In some embodiments, the hGH pA sequence is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 139. In some embodiments, the CMV promoter is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 136. In some embodiments, the nucleic acid encoding tTA comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to pAAV-CMV-tTA(Advanced) (SEQ ID NO: 32).

[0013] In some embodiments, the nucleic acid comprises a nucleic acid encoding OCT4, SOX2, and KLF4 operably linked to a TRE promoter. In some embodiments, the nucleic acid sequence encoding OCT4, SOX2, and KLF4 operably linked to a TRE promoter further encodes a 2A peptide and / or SV40 pA. In some embodiments, the 2A peptide comprises a sequence at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to T2A (SEQ ID NO: 9) or P2A (SEQ ID NO: 118). In some embodiments, the nucleic acid sequence encoding OCT4, SOX2, and KLF4 encodes at least one sequence 70% identical to a sequence selected from mouse OCT4 (SEQ ID NO: 2); human OCT4 (SEQ ID NO: 40); mouse SOX2 (SEQ ID NO: 4); human SOX2 (SEQ ID NO: 42); human KLF4 (SEQ ID NO: 131); and mouse KLF4 (SEQ ID NO: 6). In some embodiments, the TRE promoter operably linked to the nucleic acid sequence encoding OCT4, SOX2, and KLF4 is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO:7. In some embodiments, the nucleic acid sequence encoding OCT4, SOX2, and KLF4 operably linked to the TRE promoter comprises at least one sequence that is 70% identical to a sequence selected from TRE3G (SEQ ID NO: 7); mouse Oct4 (SEQ ID NO: 1); P2A (SEQ ID NO: 144); mouse Klf4 (SEQ ID NO: 145); SV40 pA (SEQ ID NO: 143); mouse Sox2 (SEQ ID NO: 3); and T2A (SEQ ID NO: 120), optionally wherein the sequence is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to pAAV-TRE3G-OSK (mouse) SEQ ID NO: 16.

[0014] In some embodiments, the nucleic acid comprises a nucleic acid encoding an inducer, and the nucleic acid encoding the inducer is operably linked to the CaMKIIα promoter. In some embodiments, the inducer is tTA or rtTA. In some embodiments, the inducer comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to tTA Advanced (SEQ ID NO: 138), rtTA2S-M2 (SEQ ID NO: 15), or rtTA3 (SEQ ID NO: 11). In some embodiments, the CaMKIIα promoter comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 146, SEQ ID NO: 149, or SEQ ID NO: 154. In some embodiments, the nucleic acid encoding the inducer further comprises a WPRE and / or hGH pA sequence. In some embodiments, the WPRE sequence is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 147, 152, or 155. In some embodiments, the hGH pA sequence is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 148, 153, or 156.

[0015] In some embodiments, the nucleic acid encoding the inducer comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to a sequence selected from tTA-Advanced (SEQ ID NO: 137), rtTA2S-M2 (SEQ ID NO: 14), or rtTA3 (SEQ ID NO: 10). In some embodiments, the nucleic acid encoding the inducer comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to a sequence selected from pAAV-CaMKIIα-tTA2 (SEQ ID NO: 124), pAAV-CaMKIIα-rtTA2S-M2 (SEQ ID NO: 125), or pAAV-CaMKIIα-rtTA3 (SEQ ID NO: 126).

[0016] In some embodiments, the nucleic acid does not comprise a synapsin-I promoter operably linked to a nucleic acid sequence encoding an inducer. In some embodiments, the nucleic acid does not comprise a sequence at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 157. In some embodiments, the nucleic acid does not comprise a sequence at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to pAAV-ihSyn1-tTA (SEQ ID NO: 127).

[0017] In some embodiments, OCT4, SOX2, and / or KLF4 are expressed in the central nervous system (e.g., the brain) for at most one month. Without being bound by theory, expression of OCT4, SOX2, and / or KLF4 for two months may fail to rejuvenate the central nervous system.

[0018] A further aspect of the present disclosure provides a recombinant virus (e.g., a lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV) comprising a nucleic acid encoding OCT4, KLF4, and / or SOX2 and / or an inducer disclosed herein for delivery to the central nervous system.

[0019] In yet another aspect, the present disclosure provides a method for controlling (e.g., inducing) cell reprogramming, tissue repair, tissue regeneration, organ regeneration, reversing aging, or any combination thereof. In certain embodiments, one or more expression vectors (e.g., AAV containing expression vectors) are administered to a subject with a neurological disease or to a cell, tissue, or organ of the central nervous system. The subject may have an injury or condition, be suspected of having a condition or injury, or be at risk for a condition or injury. Without being bound by a particular theory, expression of transcription factors OCT4, SOX2, and KLF4 induces cell reprogramming. In some embodiments, when a nucleic acid (e.g., an engineered nucleic acid) encoding OCT4, SOX2, KLF4, or a combination thereof (e.g., an engineered nucleic acid) is operably linked to an inducible promoter, administration of an inducer (e.g., a chemical agent, a protein, a nucleic acid (e.g., an engineered nucleic acid) encoding the inducer) is under appropriate conditions (e.g., in the presence or absence of tetracycline). In certain embodiments, the inducer (e.g., rtTA) can bind to the promoter and drive expression of an operably linked nucleic acid (e.g., an engineered nucleic acid) only when the inducer is bound to tetracycline. In certain embodiments, the inducer (e.g., tTA) cannot bind to the promoter and drive expression of an operably linked nucleic acid (e.g., an engineered nucleic acid) when the inducer is bound to tetracycline. The condition can be a neurological disease (e.g., a disease affecting the brain and / or a neurodegenerative disease), cancer, aging, an age-related disease, an injury, or a disease affecting neurons and / or nerves. In certain embodiments, the cell, tissue, or organ is from the central nervous system, e.g., the brain or spinal cord. In some embodiments, the cell or tissue is a neuronal cell or neural tissue. In some embodiments, the cell is a neuron. In some embodiments, the neuron is an excitatory neuron. In some embodiments, the brain cell is a neuron or a glial cell. In some embodiments, the cell from the central nervous system is a neuron, a glial cell, or a choroid plexus cell.In some embodiments, the glial cell is an astrocyte, an oligodendrocyte, an ependymal cell, or a microglial cell. In some embodiments, the neuron is a sensory neuron, a motor neuron, or an interneuron.

[0020] In certain embodiments, the method further comprises controlling a biological process in the subject's central nervous system. In some embodiments, the methods described herein comprise controlling any biological process, including cell reprogramming, tissue repair, tissue survival, tissue regeneration, tissue growth, tissue function, organ regeneration, organ survival, organ function, or any combination thereof. In some embodiments, the method comprises inducing cell reprogramming, reversing aging, improving tissue function, improving organ function, promoting tissue repair, promoting tissue survival, promoting tissue regeneration, promoting tissue growth, promoting angiogenesis, reducing scar formation, promoting organ regeneration, promoting organ survival, treating disease, or any combination thereof, in vivo or in vitro. For example, the method may induce cell reprogramming, cell survival, organ regeneration, tissue regeneration, or a combination thereof. In certain embodiments, the method comprises inducing and then stopping cell reprogramming, cell survival, tissue regeneration, organ regeneration, aging, or a combination thereof. In certain embodiments, the method reverses aging of a cell, tissue, organ, or subject. In some embodiments, the method does not induce cancer. Cancer formation can include teratoma formation and / or uncontrolled cell growth. In some embodiments, the method does not induce unwanted cell proliferation. In some embodiments, the method does not induce malignant cell growth. In some embodiments, the method does not induce tumor growth or tumor formation. In some embodiments, the method does not induce glaucoma or macular degeneration.

[0021] In some embodiments, the methods described herein reverse the epigenetic clock of cells, tissues, and / or organs from the central nervous system, or any combination thereof. In some embodiments, the epigenetic clock is determined using a DNA methylation-based (DNAm) age estimator. In some embodiments, the method modulates the expression of one or more genes associated with aging. In some embodiments, the method reduces the expression of one or more genes associated with aging. In some embodiments, the method modulates the expression of one or more genes associated with aging. In some embodiments, the one or more genes are genes expressed by cells of the central nervous system. In some embodiments, the one or more genes are genes expressed in the brain or spinal cord. In some embodiments, the one or more genes are expressed in excitatory neurons or glial cells. In some embodiments, the one or more genes are genes expressed by brain cells, such as neurons or glial cells. In some embodiments, the one or more genes are genes expressed by neurons, glial cells, or choroid plexus cells. In some embodiments, the one or more genes are genes expressed by astrocytes, oligodendrocytes, ependymal cells, or microglial cells. In some embodiments, the one or more genes are genes expressed by sensory neurons, motor neurons, or interneurons.

[0022] Another aspect of the present disclosure provides engineered cells produced by any of the methods described herein. In some embodiments, the engineered cells are cells of the central nervous system. In some embodiments, the engineered cells are neuronal cells. The engineered cells of the present disclosure can be generated ex vivo, and the method can further include producing an engineered tissue or an engineered organ. In some embodiments, the method of the present disclosure includes administering the engineered cells, engineered tissue, and / or engineered organ of the present disclosure to a subject in need thereof. In some embodiments, the method further includes treating a neurological disease.

[0023] Aspects of the present disclosure also provide compositions comprising any of the nucleic acids described herein, any of the engineered proteins described herein, any of the chemicals, recombinant viruses, cells, and / or any of the factors, alone or in combination. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition of the present disclosure further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises an engineered nucleic acid encoding OCT4, SOX2, and / or KLF4. In some embodiments, the pharmaceutical composition comprises an engineered nucleic acid (e.g., an engineered nucleic acid) (e.g., an expression vector, including, e.g., a viral vector) encoding an inducer (e.g., rtTA or tTA).

[0024] Aspects of the present disclosure also provide kits comprising any of the nucleic acids, viruses, cells, compositions, and factors disclosed herein and instructions for rejuvenating cells, tissues, or organs from the central nervous system.

[0025] The details of one or more embodiments of the invention are presented herein. Other features, objects, and advantages of the invention will be apparent from the detailed description, examples, figures, and claims.

[0026] All references cited in this application are incorporated herein by reference.

[0027] definition Definitions of certain terms are set forth in more detail below. The present disclosure is not intended to be limited in any way by the exemplary enumeration of substitute forms set forth herein.

[0028] "AAV" or "adeno-associated virus" is a non-enveloped virus that can carry and deliver nucleic acids (e.g., engineered nucleic acids) (e.g., nucleic acids encoding OCT4; KLF4; SOX2; or any combination thereof (e.g., engineered nucleic acids)) and belongs to the Dependoparvovirus genus. In some cases, AAV can deliver nucleic acids encoding inducers. Generally, AAV does not integrate into the genome. The tissue-specific targeting ability of AAV is often determined by the AAV capsid serotype (see Table 1 below for examples of AAV serotypes and their usefulness in tissue-specific delivery). Non-limiting serotypes of AAV include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV PHP.b, and variants thereof. In certain embodiments, the AAV serotype is a variant of AAV9 (e.g., AAV PHP.b). In certain embodiments, the AAV serotype is AAV PHP.eB. In some embodiments, the AAV serotype is not AAV2 or AAV9. In some embodiments, the AAV capsid comprises a targeting moiety for one or more cells of the central nervous system. See, for example, WO2023004367 and WO2022232327. In some embodiments, the AAV capsid is AAV-BI30. See, for example, Krolak et al., Nat Cardiovasc Res. 2022 Apr;1(4):389-400.

[0029] A "recombinant virus" is a virus (e.g., a lentivirus, adenovirus, retrovirus, herpesvirus, human papillomavirus, alphavirus, vaccinia virus, or adeno-associated virus (AAV)) that has been isolated from its natural environment (e.g., from a host cell, tissue, or subject) or artificially produced.

[0030] The term "AAV vector," as used herein, refers to a nucleic acid (e.g., an engineered nucleic acid) that includes AAV inverted terminal repeats (ITRs) flanking an expression cassette (e.g., an expression cassette including nucleic acids (e.g., engineered nucleic acids) encoding OCT4, KLF4, and SOX2, each alone or in combination, or an expression cassette encoding rtTA or tTA). The AAV vector may further include a promoter sequence.

[0031] The terms "administer," "administering," or "administration," as used herein, refer to the introduction into a cell, tissue, organ, and / or subject of any of the compositions described herein, any of the nucleic acids described herein, any of the engineered proteins described herein, any of the chemicals that activate (e.g., induce expression of) OCT4, KLF4, and / or SOX2, any of the chemicals that activate (e.g., induce expression of) one or more transcription factors selected from OCT4; KLF4; SOX2; and any combination thereof, any of the antibodies that activate (e.g., induce expression of) OCT4, KLF4, and / or SOX2, any of the antibodies that activate (e.g., induce expression of) one or more transcription factors selected from OCT4; KLF4; SOX2; and any combination thereof, and / or any of the recombinant viruses described herein (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV), alone or in combination. In some embodiments, a nucleic acid (e.g., an engineered nucleic acid) encoding the inducer, an engineered protein encoding the inducer, a chemical capable of modulating (e.g., activating or inhibiting) the activity of the inducer, and / or a recombinant virus encoding the inducer is also administered to the cell, tissue, organ, and / or subject.any nucleic acid (for example, an engineered nucleic acid) capable of inducing expression of one or more transcription factors selected from OCT4; KLF4; SOX2; and any combination thereof (for example, an expression vector), including any nucleic acid (for example, an engineered nucleic acid) capable of inducing expression of OCT4, KLF4, and / or SOX2; any engineered protein described herein; any chemical that activates OCT4, KLF4, and / or SOX2 (for example, by inducing their expression); any engineered protein encoding OCT4, KLF4, SOX2, or any combination thereof; any chemical that activates OCT4; KLF4; SOX2; or any combination thereof (for example, by inducing their expression); any antibody that activates OCT4, KLF4, and / or SOX2 (for example, by inducing their expression); OCT4; KLF4; SOX2; or Any of the compositions described herein comprising any of the antibodies that activate (e.g., induce the expression of) any combination thereof, and / or any of the recombinant viruses described herein (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV), alone or in combination, can be administered intravenously, intradermally, intra-arterially, intralesionally, intratumorally, intracranially, intra-articularly, intraprostatically, intrapleurally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, intramucosally, pericardially, intraumbilically, intraocularly, orally, topically, locally, systemically, by injection, infusion, continuous infusion, by local perfusion bath directly onto 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 by any combination of the foregoing (e.g., see Remington's See Pharmaceutical Sciences (1990).In some embodiments, compositions comprising a nucleic acid (e.g., an engineered nucleic acid) encoding the inducer, an engineered protein encoding the inducer, a chemical capable of modulating (e.g., activating or inhibiting) the activity of the inducer, and / or a recombinant virus encoding the inducer are also administered to cells, tissues, organs, and / or subjects, and may be administered using any suitable method.

[0032] The terms "epigenome" or "epigenetics" refer to the intracellular modifications and structural changes that control the expression of nucleic acids (e.g., engineered nucleic acids) or genomic information in cells. Epigenome changes occur during and drive embryonic development, disease progression, and aging.

[0033] The term "epigenetic clock" can refer to an age estimator or an innate biological process. In some embodiments, rejuvenating or reversing the epigenetic clock refers to reducing the estimated age of a cell, tissue, organ, or subject. The epigenetic clock can be partially or completely reversed or rejuvenated by any of the methods described herein. In some embodiments, the age estimator is an epigenetic age estimator. For example, the epigenetic age estimator can be a set of CpG dinucleotides that, when used in combination with a mathematical algorithm, can be used to estimate the age of the source of DNA, including a cell, organ, or tissue. In some embodiments, the age estimator is a DNA methylation-based (DNAm) age estimator. In some embodiments, the DNAm age estimator is calculated as an age correlation using the Pearson correlation coefficient r between DNA methylation-based (DNam) age (also known as estimated age) and chronological age. In some embodiments, the DNA methylation-based (DNAm) age estimator is a single-tissue DNA methylation-based age estimator. In some embodiments, the DNA methylation-based age estimator is a multi-tissue DNA methylation-based age estimator. In some embodiments, the DNAm age estimator is DNAm PhenoAge. For example, see Horvath and Raj, Nat Rev Genet. 2018 Jun;19(6):371-384 and Levine et al., Aging (Albany NY). 2018 Apr 18;10(4):573-591.

[0034] "Epigenetic information," as used herein, includes covalent modifications of DNA, such as 5-methylcytosine (5mC), hydroxymethylcytosine (5hmeC), 5-formylcytosine (fC), 5-carboxylcytosine (caC), and adenine methylation, and of certain proteins, such as lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation of histone proteins, and the 3D architecture of the cell, including TADs (topological domains) and compartments. Epigenetic information is sometimes referred to as the "analog" information of the cell.

[0035] "Restoring the expression of at least one gene to a youthful level" means increasing the expression of a down-regulated gene or decreasing the expression of an up-regulated gene that changes during aging. In some embodiments, the at least one gene is at least one gene selected from the group consisting of RE1 silencing transcription factor (REST), (tumor protein P73) TP73, glial fibrillary acidic protein (GFAP), intercellular adhesion molecule 2 (ICAM2), and thioredoxin interacting protein (TXNIP).

[0036] The term "cell," as used herein, is meant to encompass not only an individual cell, but also the particular tissue or organ from which it originates.

[0037] The term "cellular senescence" refers to cells that have exited the cell cycle and exhibit epigenetic markers consistent with senescence or express senescent cell markers (e.g., senescence-associated beta-galactosidase or inflammatory cytokines). Cellular senescence can be partial or complete.

[0038] The term "gene expression" refers to the degree 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.

[0039] The term "cell reprogramming" refers to the process of using reprogramming factors to modulate the epigenome of a cell (e.g., reverse or prevent epigenetic changes in cells that cause dysfunction, weakness, cell death, senescence, or aging). Cell reprogramming can be complete, resulting in a differentiated cell (e.g., a somatic cell) being reprogrammed into a pluripotent stem cell. Cell reprogramming can be incomplete, resulting in a differentiated cell (e.g., a somatic cell) retaining its cellular identity (e.g., a lineage-specific stem cell). Cell reprogramming can be incomplete, e.g., not producing stem cells, resulting in a cell rejuvenating or taking on more youthful attributes (e.g., increased survival, reduced inflammation, or the ability to divide). Cell reprogramming can provide additional cellular functions or prevent cellular aging (e.g., transdifferentiation, or transition to cellular senescence). Cell reprogramming can induce temporary or permanent changes in gene expression. In some embodiments, incomplete cell reprogramming is indicated by the absence of Nanog expression. In some embodiments, cellular reprogramming prevents senescence from occurring.

[0040] The term "rejuvenating cells," as used herein, is meant to encompass preventing or reversing the cellular causes of aging without inducing a pluripotent state. Rejuvenated cells, as used herein, include, for example, central nervous system cells that are not diseased cells.

[0041] "Pluripotent state," as used herein, is meant to encompass a state in which cells express at least one stem cell marker, such as, but not limited to, Esrrb, Nanog, Lin28, TRA-1-60 / TRA-1-81 / TRA-2-54, SSEA1, or SSEA4. Methods for measuring the expression of stem cell markers on cells are known in the art and include the methods described herein.

[0042] The term "transdifferentiation" refers to the process by which one cell type is transformed into another cell type without entering a pluripotent state.Transdifferentiation can also be referred to as lineage reprogramming or lineage conversion.For example, see Cieslar-Pobuda et al., Biochim Biophys Acta Mol Cell Res. 2017 July;1864(7):1359-1369, the entire contents of which are incorporated herein by reference.

[0043] The term "central nervous system" refers to the part of the nervous system that includes the brain, cochlea, spinal cord, medulla, pons, cerebellum, midbrain, diencephalon, and cerebral hemispheres. In some embodiments, the central nervous system includes cranial nerves. In some embodiments, the central nervous system excludes the eye. In some embodiments, the central nervous system excludes the retina, uvea, pupil, lens, cornea, and / or sclera. In some embodiments, a cell or tissue is derived from the central nervous system. Non-limiting examples of cells from the central nervous system include neurons and glial cells. In some embodiments, a neuron is an excitatory neuron. In some embodiments, a cell from the central nervous system is a brain cell. In some embodiments, a brain cell is a neuron or a glial cell. In some embodiments, a cell from the central nervous system is a neuron, a glial cell, or a choroid plexus cell. In some embodiments, a glial cell is an astrocyte, an oligodendrocyte, an ependymal cell, or a microglial cell. In some embodiments, a neuron is a sensory neuron, a motor neuron, or an interneuron.

[0044] The terms "condition," "disease," and "disorder" are used interchangeably. Non-limiting examples of conditions, diseases, and disorders include acute injury, neurodegenerative disease, chronic disease, proliferative disease, cardiovascular disease, genetic disease, inflammatory disease, autoimmune disease, neurological disease, hematological disease, painful condition, psychiatric disorder, metabolic disorder, chronic disease, cancer, aging, age-related diseases, and diseases affecting any tissue of a subject. For example, age-related conditions include heart failure, stroke, heart disease, atherosclerosis, neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, dementia, Friedreich's ataxia, amyotrophic lateral sclerosis, or vascular dementia), cognitive decline, forgetfulness, diabetes, osteoporosis, arthritis, muscle loss, hearing loss (partial or complete), eye-related conditions (e.g., poor vision or retinal disease), glaucoma, progeria-like syndromes (e.g., Hutchinson-Gilford Progeria Syndrome), and cancer. In certain embodiments, the disease is a retinal disease (e.g., macular degeneration). In some embodiments, the age-related condition is aging. As a non-limiting example, glial cell senescence may be the cause of Alzheimer's disease. For example, see Bussian, et al., Nature. 2018 Oct;562(7728):578-582. In some cases, the disease is nerve damage. In some embodiments, the nerve damage is neurapraxia, axonotomesis, or neurotomesis. In some embodiments, the disease is not an optical disease, an ophthalmic disease, or an eye disease.

[0045] In some cases, the condition is nerve injury. In some cases, the condition is central nervous system (CNS) injury. In some cases, the nerve injury is spinal cord injury. In some cases, the nerve injury is neuropraxia, axonotomesis, or neurotomesis.

[0046] In some cases, the condition increases the DNA methylation-based age of a cell, tissue, organ, and / or subject relative to a control. In some embodiments, the cell is a cell of the central nervous system. In some cases, the condition increases the DNA methylation-based age of a cell, tissue, organ, and / or subject by at least 1%, at least 5%, 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%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1,000% relative to a control. In some cases, the control is a cell, tissue, organ, and / or subject that does not have the condition. In some cases, the control is the same cell, tissue, organ, and / or subject prior to having the condition. Without being bound by any particular theory, any of the methods described herein may be useful for reducing the DNA methylation-based age of diseased cells, diseased tissues, diseased organs, and / or subjects having, at risk for, or suspected of having a disease. In some cases, the disease increases the DNA methylation-based age of the cells, tissues, organs, and / or subjects. In some cases, the disease is an injury.

[0047] In some cases, the condition is aging. In some cases, aging is driven by epigenetic noise. For examples, see Oberdoerffer and Sinclair. Nat Rev Mol Cell Biol 8, 692–702, doi:10.1038 / nrm2238 (2007); Oberdoerffer et al. Cell 135, 907–918, doi:10.1016 / j.cell.2008.10.025 (2008). Without being bound by any particular theory, mammalian cells may retain faithful copies of epigenetic information from early in life. Analogous to Shannon's "observer" system in information theory, this is essentially a backup copy of the original signal to allow its reconstruction at the receiving end if information is lost or noise is introduced during transmission. For a description of the observer system, see, for example, Shannon, The Bell System Technical Journal 27, 379-423 (1948).

[0048] As used herein, an "optical disease," "ophthalmologic disease," or "eye disease" refers to a disease or condition of the eye. Non-limiting examples of conditions affecting the eye include ectropion, lagophthalmos, blepharochalasis, ptosis, styes, yellow patches, dermatitis, demodex, leishmaniasis, loiasis, onchocerciasis, pediculosis, (herpes simplex), leprosy, molluscum contagiosum, tuberculosis, strawberry, shingles, impetigo, dacryoadenitis, lacrimation, exophthalmos, conjunctivitis, scleritis, keratitis, corneal ulcer / abrasion, snow blindness / arc eye, Thygeson's punctate superficial keratitis, corneal neovascularization, Fuchs' dystrophy, keratoconus, keratoconjunctivitis sicca, iritis, iritis, uveitis, and sympathetic ophthalmia. , cataract, lens, chorioretinal inflammation, isolated chorioretinal inflammation, chorioretinitis, choroiditis, retinitis, retinal choroiditis, diffuse chorioretinal inflammation, exudative retinopathy, posterior cyclitis, pars planitis, chorioretinal inflammation, Harada's disease, chorioretinal inflammation, choroid, choroidoretinal scar, macular scar, posterior pole (post-inflammatory) (post-traumatic), solar retinopathy, choroidal degeneration, atrophy, sclerosis, angioid streaks, choroidal dystrophy, choroideremia, choroidal, areolar (para-optic disc), gyriform atrophy, choroid, ornithineemia, choroidal hemorrhage, choroidal detachment, chorioretinal, choroidoretinal Inflammatory, infectious and parasitic diseases, chorioretinitis, syphilis, toxoplasmosis, tuberculosis, chorioretinal, retinal detachment, retina, choroid, astigmatism, retinoschisis, hypertensive retinopathy, diabetic retinopathy, retinopathy, retinopathy of prematurity, age-related macular degeneration, macula, macular degeneration, target maculopathy, preretinal membrane, peripheral retinal degeneration, hereditary retinal dystrophies, retinitis pigmentosa, retinal hemorrhage, retinal layers, central serous retinopathy, retinal detachment, retinal disorders, macular edema, macula, retinal disorders, diabetic retinopathy, glaucoma, optic neuropathy, high intraocular pressure, open-angle glaucoma, angle-closure glaucoma, normal-tension glaucoma disorders, open-angle glaucoma, angle-closure glaucoma, floaters, Leber's hereditary optic neuropathy, optic disc drusen, strabismus, ophthalmoplegia, progressive external ophthalmoplegia, esotropia, exotropia, disorders of refraction, accommodation, hyperopia, myopia, astigmatism, anisometropia, presbyopia, ophthalmoplegia, amblyopia, Leber's congenital amaurosis, scotoma, anopsia, color blindness, color blindness / maskun, cone cells, night blindness, blindness, river blindness, microphthalmia / coloboma, optic nerve, brain, spinal cord, ocular redness, Argyll-Robertson pupil, pupil, keratomycosis, xerophthalmia, and aniridia. In some embodiments, the visual disease is an acute or chronic eye injury.The methods disclosed herein exclude the treatment of visual, ophthalmic, or eye diseases.

[0049] In some embodiments, the visual disorder is a corneal abrasion.

[0050] In some embodiments, the visual disease is glaucoma.

[0051] In some embodiments, the visual disease is a corneal disease (e.g., a disease affecting the cornea or corneal cells). In some embodiments, the visual disease is acanthamoeba keratitis, ectropion, lagophthalmos, anisocoria, astigmatism, Bell's palsy, blepharitis, blurred vision, burning eyes, cataracts, macular degeneration, age-related macular degeneration, diabetic eye disease, glaucoma, dry eye, poor vision (e.g., low vision), astigmatism, blepharitis, cataracts, chalazion, conjunctivitis, diabetic retinopathy, dry eye, glaucoma, keratitis, keratoconus, macular degeneration, high intraocular pressure, pinguecula, pterygium, retinitis pigmentosa, or eye cancer (e.g., retinoblastoma, ocular melanoma, ocular lymphoma, medulloepithelioma, conjunctival squamous cell carcinoma). Examples of corneal diseases include, but are not limited to, corneal neovascularization (NV), corneal dystrophy, corneal inflammation, corneal abrasion, and corneal fibrosis. In some embodiments, the visual disease is keratoconus. In some embodiments, the visual disease is macular degeneration. Additional non-limiting examples of eye diseases can be found in the International Statistical Classification of Diseases and Related Health Problems (e.g., VII Diseases of the eye and adnexa).

[0052] The visual disease can affect any part of the eye and / or its adnexa. In some embodiments, the visual disease is a disorder of the eyelid, lacrimal system, and / or orbit. In some embodiments, the visual disease is a disorder of the conjunctiva. In some embodiments, the visual disease is a disorder of the sclera, cornea, iris, and / or ciliary body. In some embodiments, the visual disease is a disorder of the lens. In some embodiments, the visual disease is a disorder of the choroid and / or retina. In some embodiments, the visual disease is glaucoma. In some embodiments, the visual disease is a disorder of the vitreous body and / or the eye. In some embodiments, the visual disease is a disorder of the optic nerve and / or visual pathway. In some embodiments, the visual disease is a disorder of the eye muscles, binocular movement, accommodation, and / or refraction. In some embodiments, the visual disease is a visual disorder and / or blindness. In some embodiments, the visual disease is age-related, e.g., age-related blindness, age-related visual loss, and / or retinal function loss.

[0053] Any suitable method can be used to measure visual function. Non-limiting examples include visual acuity testing, pattern electroretinograms, and pathology. The methods disclosed herein exclude the treatment of visual, ophthalmic, or eye diseases.

[0054] The term "genetic disease" refers to a disease caused by one or more abnormalities in a subject's genome, e.g., a disease present from birth. Genetic diseases can be hereditary and can be inherited from parental genes. Genetic diseases can also be caused by mutations or changes in a subject's DNA and / or RNA. In such cases, a genetic disease is hereditary if it occurs in the germline. Exemplary genetic diseases include Asskog-Scott syndrome, Oze syndrome, achondroplasia, acroostosis dysplasia, addiction, adrenoleukodystrophy, albinism, anephthalmos-macrostomia syndrome, Alagille syndrome, alkaptonuria, alpha-1 antitrypsin deficiency, Alport syndrome, Alzheimer's disease, asthma, autoimmune polyglandular syndrome, androgen insensitivity syndrome, Angelman syndrome, ataxia, ataxia-telangiectasia, atherosclerosis, attention-deficit hypertension, and the like. ADHD, autism, baldness, Batten disease, Beckwith-Wiedemann syndrome, Best disease, bipolar disorder, brachydactyly), breast cancer, Burkitt's lymphoma, chronic myeloid leukemia, Charcot-Marie-Tooth disease, Crohn's disease, cleft lip, Cockayne syndrome, Coffin-Lowry syndrome, colon cancer, congenital adrenal hyperplasia, Cornelia de Lange syndrome, Costello syndrome, Cowden syndrome, craniofrontonasal dysplasia, Crigler-Najjar syndrome, Creutzfeldt-Jakob syndrome Disease, cystic fibrosis, deafness, depression, diabetes, varicose vein dysplasia, DiGeorge syndrome, Down syndrome, dyslexia, Duchenne muscular dystrophy, Dubowitz syndrome, ectodermal dysplasia, Ellis-van Creveld syndrome, Ehlers-Danlos, epidermolysis bullosa, epilepsy, essential tremor, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Friedreich's ataxia, Gaucher disease, glaucoma, glucose-galactose malabsorption, glutaric acid urinary tract disease, gyriform atrophy, Goldberg-Shprintzen syndrome (palatocardiofacial syndrome), Gorlin syndrome, Hailey-Hailey disease, hemihypertrophy, hemochromatosis, hemophilia, hereditary motor and sensory neuropathy (HMSN), hereditary nonpolyposis colorectal cancer (HNPCC), Huntington's disease, immunodeficiency with high IgM, early-onset diabetes, Klinefelter syndrome, Kabuki syndrome, Leigh's disease, long QT syndrome, lung cancer, malignant melanoma, bipolar disorder, Marfan syndrome,Menkes syndrome, miscarriage, mucopolysaccharidoses, multiple endocrine neoplasia, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, myotonic dystrophy, neurofibromatosis, Niemann-Pick disease, Noonan syndrome, obesity, ovarian cancer, pancreatic cancer, Parkinson's disease, paroxysmal nocturnal hemoglobinuria, Pendred syndrome, peroneal muscular atrophy, phenylketonuria (PKU), polycystic kidney disease, Prader-Willi syndrome, primary biliary cirrhosis, prostate cancer, REAR syndrome, Refsum's disease, retinitis pigmentosa, retinoblastoma, Rett syndrome, Sanfilippo syndrome, schizophrenia These conditions include, but are not limited to, erythroderma, severe combined immunodeficiency syndrome, sickle cell anemia, spina bifida, spinal muscular atrophy, spinocerebellar atrophy, sudden adult death syndrome, Tangier disease, Tay-Sachs disease, thrombocytopenic radial deficiency syndrome, Townes-Brocks syndrome, tuberous sclerosis complex, Turner syndrome, Usher syndrome, von Hippel-Lindau syndrome, Waardenburg syndrome, Weaver syndrome, Werner syndrome, Williams syndrome, Wilson disease, xeroderma pigmentosum, progeria syndromes (e.g., Hutchinson-Gilford progeria syndrome), and Zellweger syndrome.

[0055] "Proliferative disease" refers to a disease caused by abnormal growth or progression of cells (Walker, Cambridge Dictionary of Biology; Cambridge University Press: Cambridge, UK, 1990). Proliferative diseases can be associated with 1) pathological proliferation of normally quiescent cells; 2) pathological migration of cells from their normal location (e.g., neoplastic cell metastasis); 3) pathological expression of proteolytic enzymes such as matrix metalloproteinases (e.g., collagenase, gelatinase, and elastase); or 4) pathological angiogenesis, such as in proliferative retinopathies and tumor metastasis. Exemplary proliferative diseases include cancer (i.e., "malignant neoplasms"), benign neoplasms, angiogenesis, inflammatory diseases, and autoimmune diseases.

[0056] The terms "neoplasm" and "tumor" are used interchangeably herein to refer to an abnormal mass of tissue in which the growth of the mass exceeds and is out of step with the growth of normal tissue. Neoplasms or tumors can be "benign" or "malignant" depending on the following characteristics: degree of cellular differentiation (including morphology and functionality), growth rate, local invasion, and metastasis. "Benign neoplasms" are generally well differentiated, characteristically have slower growth rates than malignant neoplasms, and remain localized at the primary site. In addition, benign neoplasms lack the ability to infiltrate, invasive, or metastasize to distant sites. Exemplary benign neoplasms include, but are not limited to, lipomas, chondromas, adenomas, acrochordons, senile hemangiomas, seborrheic keratoses, moles, and sebaceous hyperplasias. In some cases, certain "benign" tumors can later give rise to malignant neoplasms. This can result from additional genetic alterations in a subpopulation of neoplastic cells in the tumor; these tumors are referred to as "premalignant neoplasms." An exemplary premalignant neoplasm is a teratoma. In contrast, "malignant neoplasms" are generally poorly differentiated (anaplastic) and have characteristically rapid growth accompanied by gradual infiltration, invasion, and destruction of surrounding tissue. Furthermore, malignant neoplasms generally have the ability to metastasize to distant sites. The terms "metastasis," "metastatic," or "metastasize" refer to the spread or migration of cancerous cells from a primary or original tumor to another organ or tissue, typically identifiable by the presence of a "secondary tumor" or "secondary cell mass" of the histological type of the primary or original tumor, rather than the organ or tissue in which the secondary (metastatic) tumor is located. For example, prostate cancer that has migrated to bone is said to be metastatic prostate cancer, and includes cancerous prostate cancer cells growing in bone tissue.

[0057]

[0058] The term "Nanog" refers to a DNA-binding homeobox transcription factor associated with embryonic stem (ES) cell proliferation, renewal, and pluripotency. Non-limiting examples of amino acid sequences encoding Nanog include the amino acid sequences of UniProtKB Accession Nos. Q9H9S0, Q4JM65, Q80Z64, and A7Y7W3. In some embodiments, the amino acid sequence encoding Nanog comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to the sequence set forth in UniProtKB Accession Nos. Q9H9S0, Q4JM65, Q80Z64, or A7Y7W3.

[0059] The term "neurological disease" refers to any disorder of the nervous system, including diseases and injuries involving the central nervous system (brain, brainstem, and cerebellum), peripheral nervous system (including cranial nerves), and autonomic nervous system (portions of which are located in both the central and peripheral nervous systems). Neurodegenerative diseases refer to a type of neurological disease marked by loss of nerve cells, including, but not limited to, Alzheimer's disease, Parkinson's disease, dementia, Friedreich's ataxia, amyotrophic lateral sclerosis, tauopathies (including frontotemporal dementia), and Huntington's disease. Examples of neurological diseases include, but are not limited to, vascular dementia, stroke, headache, stupor and coma, dementia, seizures, sleep disorders, trauma, infection, neoplasms, neuro-ophthalmology, movement disorders, demyelinating diseases, spinal cord disorders, and disorders of the peripheral nerves, muscles, and neuromuscular junction. Addiction and psychosis include, but are not limited to, bipolar disorder and schizophrenia, which are also included in the definition of neurological disease. Further examples of neurological disorders include acquired epileptic aphasia; acute disseminated encephalomyelitis; adrenoleukodystrophy; agenesis of the corpus callosum; agnosia; Aicardi syndrome; Alexander disease; Alpers disease; alternating hemiplegia; Alzheimer's disease; amyotrophic lateral sclerosis; anencephaly; Angelman syndrome; angiomatosis; anoxia; aphasia; apraxia; arachnoid cysts; arachnoiditis; Arnold-Chiari malformation; cerebral arteriovenous malformations; Asperger's syndrome; ataxia-telangiectasia; attention-deficit hyperactivity disorder; autism; autonomic dysfunction; back pain; Batten disease; Behçet's disease; Bell's palsy; benign essential blepharospasm; benign focal muscular atrophy; benign intracranial hypertension; Binswanger's disease; blepharospasm; Bloch-Saunders disease. Rutsberger syndrome; brachial plexus injury; brain abscess; brain injury; brain tumor (including glioblastoma multiforme); spinal cord tumor; Brown-Séquard syndrome; Canavan disease; carpal tunnel syndrome (CTS); causalgia; central pain syndrome; central pontine myelinolysis; craniopathy; cerebral aneurysm; cerebral arteriosclerosis; cerebral atrophy; cerebral gigantism; cerebral palsy; Charcot-Marie-Tooth disease; chemotherapy-induced neuropathy and neuropathic pain; Chiari malformation; chorea; chronic inflammatory demyelinating polyneuropathy (CIDP); chronic pain; chronic regional pain syndrome; Coffin-Lowry syndrome; coma, including persistent vegetative state; congenital bilateral facial palsy; corticobasal degeneration; cranial arteritis; craniosynostosis;Creutzfeldt-Jakob disease; cumulative trauma disorder; Cushing's syndrome; cytomegalic inclusion body disease (CIBD); cytomegalovirus infection; dancing eye, dancing foot syndrome; Dandy-Walker syndrome; Dawson's disease; Domorsia syndrome; Dejerine-Klumpke palsy; dementia; dermatomyositis; diabetic neuropathy; diffuse sclerosis; autonomic neuropathy; dysgraphia; dyslexia; dystonia; early infantile epileptic encephalopathy; empty sella syndrome; encephalitis; encephalocele; trigeminal angiomatosis; epilepsy; Erb's palsy; essential tremor; Fabry disease; Earl's syndrome; syncope; familial spastic paraparesis; febrile seizures; Fisher's syndrome; Friedreich's ataxia; frontotemporal dementia and other "tauopathies"; Gaucher's disease; Gerstmann's syndrome; giant cell arteritis; giant cell inclusion disease; globoid cell leukodystrophy; Guillain-Barré syndrome; HTFV-1-associated myelopathy; Hallervorden-Spatz disease; head injury; headache; hemifacial spasm; hereditary spastic paraplegia; hereditary polyneuropathic ataxia; herpes zoster oticus; herpes zoster; Hirayama's syndrome; HIV-associated dementia and neuropathy (including the neurological manifestations of AIDS) see); Holoprosencephaly; Huntington's disease and other polyglutamine repeat disorders; Hydroanencephaly; Hydrocephalus; Hypercortisolism; Hypoxia; Immune-mediated encephalomyelitis; Inclusion body myositis; Incontinentia pigmenti; Infantile form; Phytanic acid storage disease; Infantile Refsum disease; Infantile spasms; Inflammatory myopathy; Intracranial cysts; Intracranial hypertension; Joubert syndrome; Kearns-Sayre syndrome; Kennedy disease; Kinsborn syndrome; Klippel-Feil syndrome; Krabbe disease; Kugelberg-Welander disease; Kuru; Lafora disease; Lambert-Eaton myasthenic syndrome; Landau-Clerc syndrome Huhner syndrome; Lateral medullary (Wallenberg) syndrome; Learning disability; Leigh disease; Lennox-Gastaut syndrome; Lesch-Nyhan syndrome; Leukodystrophy; Dementia with Lewy bodies; Lissencephaly; Locked-in syndrome; Lou Gehrig's disease (also known as motor neuron disease or amyotrophic lateral sclerosis); Lumbar disc disease; Neurological sequelae of Lyme disease; Machado-Joseph disease; Megacephaly; Megalencephaly; Melkerson-Rosenthal syndrome; Meniere's disease; Meningitis; Menkes disease; Metachromatic leukodystrophy; Microcephaly; Migraine; Miller-Fisher syndrome; Petit mal seizures;Mitochondrial myopathy; Moebius syndrome; unilateral muscular atrophy; motor neuron disease; Moyamoya disease; mucopolysaccharidosis; multi-infarct dementia; multifocal motor neuropathy; multiple sclerosis and other demyelinating disorders; multiple system atrophy with orthostatic hypotension; muscular dystrophy; myasthenia gravis; myelinating diffuse sclerosis; infantile myoclonic encephalopathy; myoclonus; myopathy; congenital myotonia; narcolepsy; neurofibromatosis; neuroleptic malignant syndrome; neurological manifestations of AIDS; neurological sequelae of lupus; neuromyotonia; neuronal ceroid lipofuscinosis; neuronal mobility disorders Harm;Niemann-Pick disease;O'Sullivan-McLeod syndrome;Occipital neuralgia;Coccidal spinal insufficiency sequence;Ohtahara syndrome;Olivopontocerebellar atrophy;Opsoclonus-myoclonus;Optic neuritis;Orthostatic hypotension;Overuse syndrome;Paresthesias;Parkinson's disease;Congenital paramyotonia;Paraneoplastic disorders;Seizures;Parry-Romberg syndrome;Pelizaeus-Merzbach disease;Periodic paralysis;Peripheral neuropathy;Painful neuropathy and neuropathic pain;Persistent vegetative state;Pervasive developmental disorder;Photic sneeze reflex;Phytanic acid storage disease;Pick's disease;Pinchner's disease B; Pituitary tumor; Polymyositis; Porencephaly; Post-polio syndrome; Postherpetic neuralgia (PHN); Post-infectious encephalomyelitis; Orthostatic hypotension; Prader-Willi syndrome; Primary lateral sclerosis; Prion disease; Progressive; Facial hemimetrophy; Progressive multifocal leukoencephalopathy; Progressive sclerosing poliodystrophy; Progressive supranuclear palsy; Pseudotumor cerebri; Ramsay-Hunt syndrome (Types I and II); Rasmussen encephalitis; Reflex sympathetic dystrophy syndrome; Refsum's disease; Repetitive movement disorder; Repetitive stress injury; Restless legs syndrome; Retrovirus-associated myelopathy; Rett syndrome; Reye's syndrome; Saint-Vicki's disease Tuss's chorea; Sandhoff's disease; Schilder's disease; Schizencephaly; Septo-optic dysplasia; Shaken baby syndrome; Herpes zoster; Shy-Drager syndrome; Sjogren's syndrome; Sleep apnea; Sotos syndrome; Spasticity; Spina bifida; Spinal cord injury; Spinal tumor; Spinal muscular atrophy; Stiff-person syndrome; Stroke; Sturge-Weber syndrome; Subacute sclerosing panencephalitis; Subarachnoid hemorrhage; Subcortical arteriosclerotic encephalopathy; Sydenham's chorea; Syncope; Syringomyelia; Tardive dyskinesia; Tay-Sachs disease; Temporal arteritis; Tethered cord syndrome; Thomsen's disease; Thoracic outlet syndrome; Trigeminal neuralgia; Todd's palsy;Neurological disorders include Tourette's syndrome, transient ischemic attacks, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paraparesis, tuberous sclerosis, vascular dementia (multi-infarct dementia), vasculitis, including temporal arteritis, von Hippel-Lindau disease (VHL), Wallenberg syndrome, Werdnig-Hoffmann disease, West syndrome, whiplash syndrome, Williams syndrome, Wilson's disease, and Zellweger syndrome. In some embodiments, the neurological disorder affects the central nervous system, including the brain and spinal cord. In some embodiments, the neurological disorder affects neurons. In some embodiments, the neurological disorder affects nerves. In some embodiments, the neurological disorder affects the spinal cord. In some embodiments, the neurological disorder is a brain disease (i.e., a disease that affects the brain). In some embodiments, the brain disease is Alzheimer's disease.

[0060] "Painful conditions" include neuropathic pain (e.g., peripheral neuropathic pain), central pain, deafferentation pain, chronic pain (e.g., chronic nociceptive pain and other forms of chronic pain, e.g., post-operative pain, e.g., pain occurring after hip, knee, or other replacement surgery), pre-operative pain, stimulation of nociceptors (nociceptive pain), acute pain (e.g., phantom and transient acute pain), non-inflammatory pain, inflammatory pain, cancer-related pain, wound pain, burn pain, post-operative pain, pain associated with a medical procedure, pain resulting from pruritus Pain relief includes, but is not limited to, pain associated with bladder pain syndrome, premenstrual dysphoric disorder and / or premenstrual syndrome, pain associated with chronic fatigue syndrome, pain associated with preterm labor, pain associated with withdrawal from drug addiction, joint pain, arthritic pain (e.g., pain associated with crystalline arthritis, osteoarthritis, psoriatic arthritis, gouty arthritis, reactive arthritis, rheumatoid arthritis, or Reiter's arthritis), lumbosacral pain, musculoskeletal pain, headache, migraine, muscle pain, lower back pain, neck pain, dental pain, dental / maxillofacial pain, visceral pain, etc. One or more of the painful conditions contemplated herein may include a mixture of various types of pain as provided above and herein (e.g., nociceptive pain, inflammatory pain, neuropathic pain, etc.). In some embodiments, a particular type of pain may predominate. In other embodiments, the painful condition includes two or more types of pain, without one predominating. A clinician skilled in the art can determine the dosage to achieve a therapeutically effective amount for a particular subject based on the painful condition.

[0061] The term "mental disorder" refers to a disease of the mind and includes diseases and disorders listed in the Diagnostic and Statistical Manual of Mental Disorders - Fourth Edition (DSM-IV), published by the American Psychiatric Association, Washington, DC (1994). Mental disorders include anxiety disorders (e.g., acute stress disorder (agoraphobia), generalized anxiety disorder, obsessive-compulsive disorder, panic disorder, post-traumatic stress disorder, separation anxiety disorder, social phobia, and specific phobia), childhood disorders (e.g., attention-deficit / hyperactivity disorder, conduct disorder, and oppositional defiant disorder), eating disorders (e.g., anorexia nervosa and bulimia nervosa), mood disorders (e.g., depression, bipolar disorder, cyclothymic disorder, dysthymic disorder, and major depressive disorder), personality disorders (e.g., antisocial personality disorder, avoidant personality disorder, borderline personality disorder, dependent personality disorder, histrionic personality disorder, narcissistic personality disorder, obsessive-compulsive disorder, and obsessive-compulsive disorder). These conditions include, but are not limited to, personality disorders (e.g., brief psychotic disorder, delusional disorder, schizoaffective disorder, schizophreniform disorder, schizophrenia, and shared psychotic disorder), substance-related disorders (e.g., alcohol dependence, amphetamine dependence, cannabis dependence, cocaine dependence, hallucinogen dependence, inhalant dependence, nicotine dependence, opioid dependence, phencyclidine dependence, and sedative dependence), adjustment disorders, autism, delirium, dementia, multi-infarct dementia, learning and memory disorders (e.g., amnesia and age-related forgetfulness), and Tourette's disorder.

[0062] In some embodiments, disease is characterized by cell dysfunction.For example, disease can be mitochondrial disease.Non-limiting mitochondrial diseases include Friedreich's ataxia, Alper's disease, Barth syndrome, beta-oxidation disorder, carnitine deficiency, CPT1 deficiency and mitochondrial DNA depletion.Cell dysfunction can include mitochondrial dysfunction, RNA replication dysfunction, DNA replication dysfunction, translation dysfunction and / or protein folding dysfunction.

[0063] In some embodiments, the disease or condition is caused by a wound, bleeding, injury (e.g., a broken bone, a gunshot wound, an incision, scarring during surgery (e.g., a Cesarean section)). In some embodiments, the disease or condition is caused by a spinal cord injury. In some embodiments, the disease or condition is caused by a brain injury.

[0064] "Cellular causes of aging," as used herein, encompasses the loss or alteration of epigenetic information.

[0065] The term "c-Myc" or "Myc" refers to a phosphorylated nuclear protein associated with cell cycle progression. C-Myc can form a heterodimer with the transcription factor MAX, and the heterodimer can bind to an E-box consensus sequence on a nucleic acid (e.g., an engineered nucleic acid) to regulate the transcription of a target gene. In certain embodiments, the nucleotide sequence encoding c-Myc comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to the sequence set forth in the NCBI RefSeq database under accession numbers 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 expression of OCT4; KLF4; SOX2; or any combination thereof in the absence of inducing c-Myc expression or in the absence of activating c-Myc. The absence of inducing c-Myc expression may refer to the absence of substantial induction of c-Myc expression above endogenous levels of c-Myc expression in a cell, tissue, subject, or any combination thereof. The absence of substantial induction of c-Myc expression compared to endogenous levels of c-Myc expression in a cell, tissue, subject, or any combination thereof can refer to increasing c-Myc expression by less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or any value therebetween, compared to endogenous levels of c-Myc expression in a cell, tissue, subject, or any combination thereof. The absence of activating c-Myc expression can refer to the absence of substantial activation of c-Myc (e.g., activity) above endogenous c-Myc activity in a cell, tissue, subject, or any combination thereof.The absence of substantial induction of c-Myc activity compared to endogenous c-Myc activity in a cell, tissue, subject, or any combination thereof may refer to increasing c-Myc activity by less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or any value therebetween compared to endogenous c-Myc activity in a cell, tissue, subject, or any combination thereof.

[0066] The terms "effective amount" and "therapeutically effective amount," as used herein, refer to an amount or concentration of a compound of the present invention that, when administered to a subject, is effective to at least partially treat a condition from which the subject suffers.

[0067] As used herein, a protein that is "functional" or "active" is one that retains its biological activity (e.g., can act as a transcription factor or as an inducer). Conversely, a protein that is non-functional or inactive is one that is unable to perform one or more of its wild-type functions.

[0068] The term "gene" refers to a nucleic acid (e.g., an engineered nucleic acid) fragment that expresses a protein and includes regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. A "native gene" refers to a gene found in nature with its own regulatory sequences. A "chimeric gene" or "chimeric construct" refers to any gene or construct that is not a native gene, containing regulatory and coding sequences that are not found together in nature. Thus, a chimeric gene or chimeric construct can contain regulatory and coding sequences from different sources, or regulatory 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 on the genome of an organism. A "foreign" gene refers to a gene not normally found in a host organism but that is introduced into a host organism by gene transfer. A foreign gene can include a native gene inserted into a non-native organism, or a chimeric gene. A "transgene" is a gene that has been introduced into the genome by a transformation procedure.

[0069] "Homolog" or "homologous" refers to a sequence (e.g., a nucleic acid (e.g., an engineered nucleic acid) or amino acid sequence) that shares 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% identity). Homologous sequences include, but are not limited to, paralogous or orthologous sequences. Paralogous sequences result from gene duplication within the genome of a species, while orthologous sequences diverge after a speciation event. Functional homologs retain one or more biological activities of the wild-type protein. In certain embodiments, a functional homolog of OCT4, KLF4, or SOX2 retains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the biological activity (e.g., transcription factor activity) of its wild-type counterpart.

[0070] "KLF4," which may also be referred to as Kruppel-like factor 4, EZF, or GKLF, is a zinc finger transcription factor. KLF4 is associated with the regulation of differentiation and proliferation and can interact with coactivators, including members of the p300-CBP coactivator family. As used herein, the KLF4 transcription factor, its homologs (e.g., functional homologs), or variants can be derived from any species, including humans. In certain embodiments, a nucleic acid (e.g., an engineered nucleic acid) encoding human KLF4 comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a nucleic acid (e.g., an engineered nucleic acid) listed in the NCBI RefSeq database under accession numbers NM_004235.5, NM_001314052.1, SEQ ID NO: 131, or SEQ ID NO: 145. 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, SEQ ID NO:44, SEQ ID NO:131, or SEQ ID NO:145. SEQ ID NOs:5 and 145 are non-limiting examples of nucleotide sequences encoding KLF4 from Mus musculus. SEQ ID NO:44 is a non-limiting example of a nucleotide sequence encoding human KLF4. In certain embodiments, KLF4 comprises an amino acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to NP_001300981.1 or NP_004226.3. In certain embodiments, KLF4 comprises an amino acid sequence that is at least 70% (eg, at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:6.In certain embodiments, KLF4 comprises an amino acid sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 45. SEQ ID NO: 6 is a non-limiting example of an amino acid sequence encoding KLF4 from Mus musculus. SEQ ID NO: 45 is a non-limiting example of an amino acid sequence encoding human KLF4. Nucleic acids (e.g., engineered nucleic acids) encoding other KLF4 transcription factors (e.g., from other species) can be found in publicly available databases, including GenBank.

[0071] "Inverted terminal repeats" or "ITRs" are nucleic acid (e.g., engineered nucleic acid) sequences that are reverse-complementary to each other. Generally, in AAV vectors, ITRs are found on both sides of a cassette (e.g., an expression cassette containing a nucleic acid (e.g., engineered nucleic acid) encoding OCT4; KLF4; SOX2; or any combination thereof). In some cases, the cassette encodes an inducer. AAV ITRs are found in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV.PHP.b ( For example: In some embodiments, the AAV ITRs are from an AAV that targets the central nervous system (e.g., AAV.PHP.b( For example: In some embodiments, the AAV serotype is not AAV2 or AAV9. In some embodiments, the AAV serotype corresponds to an AAV capsid that targets the central nervous system.

[0072] The terms "nucleic acid," "polynucleotide," "nucleotide sequence," "nucleic acid (e.g., engineered nucleic acid) molecule," "nucleic acid (e.g., engineered nucleic acid) sequence," and "oligonucleotide" refer to a series of nucleotide bases (also called "nucleotides") on DNA and RNA, and refer to any chain of two or more nucleotides. The terms "nucleic acid" or "nucleic acid (e.g., engineered nucleic acid) sequence," "nucleic acid (e.g., engineered nucleic acid) molecule," "nucleic acid (e.g., engineered nucleic acid) fragment," or "polynucleotide" can be used interchangeably with "gene," "mRNA encoded by a gene," and "cDNA."

[0073] Nucleic acids (e.g., engineered nucleic acids) can be single-stranded or double-stranded, chimeric mixtures, or derivatives or modified versions thereof. Oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, the molecule's stability, its hybridization parameters, etc. Nucleotide sequences typically carry genetic information, including information used by cellular machinery to make proteins and enzymes. These terms encompass double- or single-stranded genomic and cDNA, RNA, both synthetic and engineered polynucleotides, and both sense and antisense polynucleotides. This encompasses single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA, and RNA-RNA hybrids, as well as "protein nucleic acids (e.g., engineered nucleic acids)" (PNAs) formed by conjugating bases to an amino acid backbone. This also encompasses nucleic acids (e.g., engineered nucleic acids) containing carbohydrates or lipids. Exemplary DNA includes single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), plasmid DNA (pDNA), genomic DNA (gDNA), complementary DNA (cDNA), antisense DNA, chloroplast DNA (ctDNA or cpDNA), microsatellite DNA, mitochondrial DNA (mtDNA or mDNA), kinetoplast DNA (kDNA), provirus, lysogen, repetitive DNA, satellite DNA, and viral DNA.Exemplary RNAs include single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), short interfering RNA (siRNA), messenger RNA (mRNA), precursor messenger RNA (pre-mRNA), short hairpin RNA or short hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), transfer RNA (tRNA), antisense RNA (asRNA), heterogeneous nuclear RNA (hnRNA), coding RNA, non-coding RNA (ncRNA), long non-coding RNA (long ncRNA or lncRNA), satellite RNA, viral satellite RNA, signal recognition particle RNA, small cytoplasmic RNA, small nuclear RNA (snRNA), ribosomal RNA (rRNA), Piwi-interacting RNA (piRNA), polyinosinic acid, ribozymes, flexizymes, small nucleolar RNA (snoRNA), splice leader RNA, viral RNA, and viral satellite RNA.

[0074] The nucleic acids described herein (e.g., engineered nucleic acids) can be synthesized by standard methods known in the art, for example, by using an automatic DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, etc.). For example, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al., Nucl. Acids Res., 16, 3209, (1988), and methylphosphonate oligonucleotides can be prepared by using controlled pore glass polymer supports (Sarin et al., Proc. Natl Acad. Sci. USA 85, 7448-7451, (1988)). Several methods have been developed for delivering antisense DNA or RNA to cells, for example, antisense molecules can be directly injected into tissue sites, or modified antisense molecules (antisense linked to peptides or antibodies that specifically bind to receptors or antigens expressed on the surface of target cells) designed to target desired cells can be administered systemically. Alternatively, RNA molecules can be produced by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors incorporating suitable RNA polymerase promoters, such as the T7 or SP6 polymerase promoter. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be stably introduced into cell lines. However, in many cases, it is difficult to achieve intracellular concentrations of antisense sufficient to suppress translation of endogenous mRNA. Therefore, a preferred approach utilizes recombinant DNA constructs in which antisense oligonucleotides are placed under the control of a strong promoter. Use of such constructs to transfect patient target cells will result in the transcription of sufficient amounts of single-stranded RNA that will form complementary base pairs with endogenous target gene transcripts, thereby preventing translation of the target gene mRNA.For example, a vector can be introduced in vivo so that it can be taken up by cells and direct the transcription of antisense RNA. As long as it can be transcribed to produce the desired antisense RNA, such a vector can remain episomal or be integrated into a chromosome. Such a vector can be constructed by standard recombinant DNA technology methods in the art. The vector can be a plasmid, virus, or other vector known in the art that is used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be driven by any promoter known in the art to function 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 directly introduced into a tissue site.

[0075] Nucleic acids (e.g., engineered nucleic acids) can be flanked by natural regulatory (expression control) sequences or can be linked to heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5' and 3' non-coding regions, etc. Nucleic acids (e.g., engineered nucleic acids) can also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, "caps," substitution of one or more of the naturally occurring nucleotides with analogs, and internucleotide modifications, such as, for example, with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Polynucleotides may contain one or more additional covalently linked moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalating agents (e.g., acridine, psoralen, etc.), chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. Polynucleotides may be derivatized by forming methyl or ethyl phosphotriesters or alkyl phosphoramidate linkages. Furthermore, the polynucleotides herein may also be modified with labels that can provide detectable signals either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, epitope tags, isotopes (e.g., radioisotopes), biotin, etc.

[0076] A "recombinant nucleic acid (e.g., engineered nucleic acid) molecule" or "engineered nucleic acid" is a nucleic acid (e.g., engineered nucleic acid) molecule that has undergone molecular biological manipulation, i.e., a non-naturally occurring nucleic acid (e.g., engineered nucleic acid) molecule or a genetically engineered nucleic acid (e.g., engineered nucleic acid) molecule. Furthermore, the term "recombinant DNA molecule" or "engineered nucleic acid" refers to a nucleic acid (e.g., engineered nucleic acid) sequence that does not occur in nature or that can be made by the artificial combination of two otherwise separate fragments of nucleic acid (e.g., engineered nucleic acid) sequence, i.e., by ligating together pieces of DNA that are not normally contiguous. "Recombinantly produced" refers to an artificial combination, often achieved either by chemical synthesis means or by the artificial manipulation of isolated fragments of nucleic acid (e.g., engineered nucleic acid). For example, by genetic engineering techniques using restriction enzymes, ligases, for example, by similar recombinant techniques described by Sambrook et al., Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, NY; (1989) or Ausubel et al., Current Protocols in Molecular Biology, Current Protocols (1989), and DNA Cloning: A Practical Approach, Volumes I and II (ed. D.N. Glover) IREL Press, Oxford, (1985), each of which is incorporated herein by reference.

[0077] Such manipulations can typically be performed to introduce or remove sequence recognition sites, replacing codons with redundant codons or conservative amino acids that encode them. Alternatively, they can be performed to join together nucleic acid (e.g., engineered nucleic acid) fragments of desired functions to create a single gene entity containing a desired combination of functions not found in nature. In many cases, restriction enzyme recognition sites are 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 be incorporated by design.

[0078] "OCT4," which may also be referred to as octamer-binding transcription factor 4, OCT3, OCT3 / 4, POU5F1, or POU class 5 homeobox 1, is a transcription factor associated with embryonic development and cell fate determination. Similar to other OCT transcription factors, OCT4 is characterized by a bipartite DNA-binding domain called the POU domain. As used herein, the OCT4 transcription factor, its homolog, or variant may be derived from any species, including humans. In certain embodiments, the nucleic acid (e.g., an engineered nucleic acid) encoding human OCT4 is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a nucleic acid (e.g., an engineered nucleic acid) set forth in NCBI RefSeq under accession numbers NM_002701, NM_203289, NM_001173531, NM_001285986, or NM_001285987. In certain embodiments, the nucleic acid (e.g., an engineered nucleic acid) encoding OCT4 comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to the nucleic acid (e.g., 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 human OCT4 comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to the nucleic acid (e.g., 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) set forth in NCBI RefSeq under accession numbers NP_001167002.1, NP_001272915.1, NP_001272916.1, NP_002692.2, or NP_976034.4. In certain embodiments, OCT4 comprises an amino acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 2. SEQ ID NO: 2 is a non-limiting example of an amino acid sequence encoding OCT4 from Mus musculus. In certain embodiments, OCT4 comprises an amino acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 41. SEQ ID NO: 41 is a non-limiting example of an amino acid sequence encoding human OCT4. Other OCT4 transcription factors (e.g., from other species) are known, and nucleic acids (e.g., engineered nucleic acids) encoding OCT4 transcription factors can be found in publicly available databases, including GenBank.

[0079] The term "promoter" refers to a control region of a nucleic acid (e.g., an engineered nucleic acid) sequence at which the initiation and rate of transcription of the remainder of the nucleic acid (e.g., an engineered nucleic acid) sequence are controlled. A promoter may also contain subregions to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. A promoter may be constitutive, inducible, activatable, repressible, tissue-specific, or any combination thereof. A promoter drives the expression or transcription of the nucleic acid (e.g., an engineered nucleic acid) sequence it controls. As used herein, a promoter is considered "operably linked" when it is in the correct functional location and orientation relative to the nucleic acid (e.g., an engineered nucleic acid) sequence it controls to control ("drive") the initiation of transcription of that sequence, the expression of that sequence, or a combination thereof. A promoter may include a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) to any promoter sequence disclosed herein.

[0080] The promoter can promote ubiquitous or tissue-specific expression of an operably linked nucleic acid sequence (e.g., an engineered nucleic acid) from any species, including humans. In some embodiments, the promoter is a eukaryotic promoter. Non-limiting examples of eukaryotic promoters include TDH3, PGK1, PKC1, TDH2, PYK1, TPI1, AT1, CMV, EF1 alpha, SV40, PGK1 (human or mouse), Ubc, human beta-actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIIα, GAL1, GAL10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, and U6, as would be known to one skilled in the art (see, for example, Addgene's website: blog.addgene.org / plasmids-101-the-promoter-region).

[0081] Non-limiting examples of ubiquitous promoters include tetracycline-responsive promoters (under appropriate conditions), CMV (e.g., SEQ ID NO: 48 or 136), EF1 alpha, SV40 promoter, PGK1 (SEQ ID NO: 132), Ubc (SEQ ID NO: 130), CAG, human beta-actin gene promoter, RSV promoter (e.g., SEQ ID NO: 47), EFS promoter (e.g., SEQ ID NO: 49), and promoters containing an upstream activating sequence (UAS). In certain embodiments, the promoter is a mammalian promoter.

[0082] Non-limiting examples of tissue-specific promoters include central nervous system-specific (e.g., brain-specific, neuron-specific, microglia-specific, or astrocyte-specific), liver-specific, muscle-specific, lung-specific, heart-specific, bone-specific, gut-specific, skin-specific promoters, brain-specific promoters, and eye-specific promoters. For example, a muscle-specific promoter is the desmin promoter (e.g., a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 29). Non-limiting examples of eye-specific promoters include the human GRK1 (rhodopsin kinase) promoter (e.g., SEQ ID NO: 50), the human CRX (cone-rod homeobox transcription factor) promoter (e.g., SEQ ID NO: 51), and the human NRL promoter (neural retina leucine zipper transcription factor enhancer upstream of the human TK terminal promoter). In some embodiments, the tissue-specific promoter does not include an eye-specific promoter. In some embodiments, the tissue-specific promoter is a promoter capable of inducing gene expression in the central nervous system. In some embodiments, the tissue-specific promoter is a promoter capable of directing gene expression in neural cells, hi some embodiments, the tissue-specific promoter is a promoter capable of directing gene expression in the brain.

[0083] Non-limiting examples of central nervous system-specific promoters include the Tα1α-tubulin promoter, CaMKIIα, neuron-specific enolase (NSE) promoter, synapsin I (SYN) promoter, nestin (NES) promoter, GFAP promoter, F4 / 80 promoter, and Cx3cr1 promoter. In some embodiments, the neuron-specific promoter is selected from the group consisting of the Tα1α-tubulin promoter, CaMKIIα, NSE, SYN, and NES. In some embodiments, the astrocyte-specific promoter is the GFAP promoter. In some embodiments, the microglia-specific promoter is F4 / 80 or Cx3cr1. In some embodiments, the brain-specific promoter is the Tα1α-tubulin promoter, CaMKIIα, neuron-specific enolase (NSE) promoter, synapsin I (SYN) promoter, nestin (NES) promoter, GFAP promoter, F4 / 80 promoter, or Cx3cr1 promoter. In some embodiments, the brain-specific promoter does not include the synapsin I (SYN) promoter sequence.

[0084] In some embodiments, the tissue-specific promoter comprises a CaMKIIα promoter. In some embodiments, the CaMKIIα promoter 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: 146, 149, or 154.

[0085] In some embodiments, the tissue-specific promoter does not include a synapsin-I promoter, does not include a CaMKII-gamma promoter, or a combination thereof. In some embodiments, the expression vector does not include 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: 157 encoding the synapsin-I promoter.

[0086] In some embodiments, promoter is specific to senescent cells.For example, promoter can specifically induce the expression of operably linked nucleic acid in senescent cells but not in non-senescent cells.For example, p16 promoter can be used to promote the expression of operably linked nucleic acid in senescent cells.

[0087] In some embodiments, the promoters of the present disclosure are suitable for use in AAV vectors. See, e.g., U.S. Patent Application Publication No. 2018 / 0155789, which is incorporated by reference in its entirety.

[0088] Non-limiting examples of constitutive promoters include CaMKIIα, synapsin-I, CP1, CMV, EF1 alpha, SV40, PGK1, Ubc, human beta-actin, beta-tubulin, CAG, Ac5, Rosa26 promoter, COL1A1 promoter, polyhedrin, TEF1, GDS, CaM3 5S, Ubi, H1, U6, red opsin promoter (red promoter), rhodopsin promoter (rho promoter), cone arrestin promoter (car promoter), and rhodopsin kinase promoter (rk promoter). The Ubc promoter may comprise a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 18. In some cases, the constitutive promoter is the Rosa26 promoter. In some cases, the constitutive promoter is the COL1A1 promoter. The red opsin promoter may comprise a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 101. The rho promoter may comprise a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 102. The cone arrestin promoter may comprise a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 103. The rhodopsin kinase promoter may comprise a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 104. Tissue-specific promoters can be used to drive the 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 the expression of rtTA or rTA. In some embodiments, tissue-specific promoters are used to drive the expression of OCT4, KLF4, and SOX2.In some embodiments, the tissue-specific promoter is not a muscle-specific promoter. In some embodiments, the tissue-specific promoter is not an eye-specific promoter. In some embodiments, the tissue-specific promoter does not comprise a sequence at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to a sequence selected from SEQ ID NOs: 101-104.

[0089] In some embodiments, the promoter operably linked to the engineered nucleic acid, including, for example, a nucleic acid encoding rtTA, tTA, OCT4, KLF4, SOX2, or any combination thereof, does not comprise an eye-specific promoter. In some embodiments, the promoter operably linked to the engineered nucleic acid, including, for example, a nucleic acid encoding rtTA, tTA, OCT4, KLF4, SOX2, or any combination thereof, does not comprise a synapsin-I promoter, does not comprise a CaMKII-gamma promoter, or a combination thereof. In some embodiments, the promoter operably linked to a sequence encoding a transcription factor (e.g., OCT4, SOX2, KLF4, or any combination thereof) and / or an inducer does not comprise a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 157.

[0090] An "inducible promoter" is one that is characterized by initiating or enhancing transcriptional activity when in the presence of, affected by, or contacted with an inducer. The inducer can be endogenous or can be a normally exogenous condition, compound, factor, or protein that contacts an engineered nucleic acid (e.g., an engineered nucleic acid) in such a way that it is active in inducing transcriptional activity from the inducible promoter. In certain embodiments, the inducer is a tetracycline-sensitive protein (e.g., tTA or rtTA, TetR family regulators).

[0091] Inducible promoters for use in accordance with the present disclosure include any inducible promoter described herein or known to one of skill in the art. Examples of inducible promoters include, without limitation, chemically / biochemically regulated and physically regulated promoters, e.g., alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters), and other tetracycline-responsive promoter systems, including tetracycline repressor protein (TetR, e.g., SEQ ID NO: 26, or TetRKRAB, e.g., SEQ ID NO: 27), tetracycline operator sequence (tetO) and tetracycline transactivator fusion protein (tTA), and tetracycline operator sequence (tetO) and reverse tetracycline transactivator fusion protein (tTA). Examples of promoters include promoters based on the integrin (rtTA), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptor, and promoters from the steroid / retinoid / thyroid 25 receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (a protein that binds and sequesters metal ions) genes from yeast, mouse, and human), pathogenesis-regulated promoters (e.g., those induced by salicylic acid, ethylene, or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters), pH-regulated promoters, and light-regulated promoters. A non-limiting example of an induction system using a light-regulated promoter is provided by Wang et al., Nat. Methods. 2012 Feb 12;9(3):266-9.

[0092] In certain embodiments, the inducible promoter comprises a tetracycline (Tet) responsive element. For example, the inducible 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). For example, a TRE (e.g., TRE2) promoter can comprise a nucleic acid (e.g., engineered nucleic acid) sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 23. For example, a TRE (e.g., P tight) promoter can comprise a nucleic acid (e.g., engineered nucleic acid) sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 24.

[0093] Additional non-limiting examples of inducible promoters include mifepristone-responsive promoters (e.g., the GAL4-E1b promoter) and coumermycin-responsive promoters. See, e.g., Zhao et al., Hum Gene Ther. 2003 Nov 20;14(17):1619-29.

[0094] As used herein, a "reverse tetracycline transactivator" ("rtTA") is an inducer that can bind to a TRE promoter (e.g., a TRE3G, TRE2, or P tight promoter) in the presence of tetracycline (e.g., doxycycline) and drive expression of a transgene operably linked to the TRE promoter. rtTAs generally comprise a mutant tetracycline repressor DNA-binding protein (TetR) and a transactivation domain (see, e.g., Gossen et al., Science. 1995 Jun 23;268(5218):1766-9, and any of the transactivation domains listed therein). The mutant TetR domain can bind to a TRE promoter when bound to tetracycline. See, e.g., International Publication No. WO 2020 / 069339, published April 2, 2020, entitled "Mutant Reverse Tetracycline Transactivators for Expression of Genes," which is incorporated herein by reference in its entirety.

[0095] "SRY box 2" or "SOX2" is a member of the SRY-related HMG box (SOX) family of transcription factors. SOX2 has been implicated in promoting embryonic development. Members of the SOX (SRY-related HMG box) family of transcription factors are characterized by a high mobility group 5 (HMG) box DNA sequence. This HMG box is a DNA-binding domain that is highly conserved among eukaryotic species. As used herein, the SOX2 transcription factor, its homolog, or variant may be derived from any species, including humans. In certain embodiments, a nucleic acid (e.g., an engineered nucleic acid) encoding SOX2 comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a nucleic acid (e.g., an engineered nucleic acid) set forth in NCBI RefSeq under accession number NM_011443.4. In certain embodiments, a nucleic acid (e.g., an engineered nucleic acid) encoding human SOX2 comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a nucleic acid (e.g., an engineered nucleic acid) set forth in NCBI RefSeq under accession number NM_003106.4. In certain embodiments, SOX2 comprises a nucleic acid (e.g., an engineered nucleic acid) sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:3 or SEQ ID NO:42. SEQ ID NO:3 is a non-limiting example of a nucleotide sequence encoding SOX2 from Mus musculus. SEQ ID NO:42 is a non-limiting example of a nucleotide sequence encoding human SOX2. In certain embodiments, 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 the amino acid sequence set forth in NCBI RefSeq under 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. Other SOX2 transcription factors (e.g., from other species) are known, and nucleic acids (e.g., engineered nucleic acids) encoding SOX2 transcription factors can be found in publicly available databases, including GenBank.

[0096] A "multicistronic vector" is a vector encoding more than one amino acid sequence (e.g., a vector encoding OCT4 and KLF4, OCT4 and SOX2, KLF4 and SOX2, or OCT4, SOX2, and KL4 (OSK)). Multicistronic vectors allow the expression of multiple amino acid sequences from a nucleic acid (e.g., engineered nucleic acid) sequence. The nucleic acid (e.g., engineered nucleic acid) sequences encoding each transcription factor (e.g., OCT4, KLF4, or SOX2) can be connected or separated to produce unconnected proteins. For example, an internal ribosome entry site (IRES) or polypeptide cleavage signal can be placed between the nucleic acid (e.g., engineered nucleic acid) sequences encoding each transcription factor on the vector. Exemplary polypeptide cleavage signals include 2A peptides (e.g., T2A, P2A, E2A, and F2A). The 2A peptide can comprise a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 9 or 118. In some embodiments, the expression vector of the present disclosure is a multicistronic expression vector.

[0097] "Reversing aging" or "reversing ageing," as used herein, refers to altering physical characteristics associated with aging. All animals typically undergo a period of growth and maturation, followed by a period of progressive and irreversible physiological decline, culminating in death. The length of time from birth to death is known as the organism's lifespan, and each organism has a characteristic life expectancy. Aging is the physical manifestation of underlying changes over time, measured by a percentage of life expectancy.

[0098] "Subjects" to which administration is contemplated include, but are not limited to, humans (i.e., male or female of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults)) and / or other non-human animals, such as mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals, e.g., cows, pigs, horses, sheep, goats, cats, and / or dogs) and birds (e.g., commercially relevant birds, e.g., 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.

[0099] Those skilled in the art will recognize that the biological age of pediatric or adult subjects can vary depending on animal type.As a non-limiting example, adult mice can be 1 year old, while adult humans can be over 21 years old.In some embodiments, pediatric subjects are less than 21 years old, less than 20 years old, less than 15 years old, less than 10 years old, less than 9 years old, less than 8 years old, less than 7 years old, less than 6 years old, less than 5 years old, less than 4 years old, less than 3 years old, less than 2 years old, less than 1 year old, less than 10 months old, less than 9 months old, less than 8 months old, less than 7 months old, less than 6 months old, less than 5 months old, less than 4 months old, less than 2 months old, or less than 1 month old. In some embodiments, the adult subject is at least 3 weeks old, 1 month old, at least 2 months old, at least 3 months old, at least 4 months old, at least 5 months old, at least 6 months old, at least 7 months old, at least 8 months old, at least 9 months old, at least 10 months old, at least 11 months old, at least 1 year old, at least 2 years old, at least 3 years old, at least 5 years old, at least 10 years old, at least 15 years old, at least 20 years old, at least 25 years old, at least 30 years old, at least 40 years old, at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, at least 80 years old, at least 90 years old, or at least 100 years old. In some embodiments, the middle-aged adult subject is between 1 and 6 months old, between 6 and 12 months old, between 1 and 5 years old, between 5 and 10 years old, between 10 and 20 years old, between 20 and 30 years old, between 30 and 50 years old, between 50 and 60 years old, between 40 and 60 years old, between 40 and 50 years old, or between 45 and 65 years old.In some embodiments, an older adult subject is at least 1 month old, at least 2 months old, at least 3 months old, at least 4 months old, at least 5 months old, at least 6 months old, at least 7 months old, at least 8 months old, at least 9 months old, at least 10 months old, at least 11 months old, at least 1 year old, at least 2 years old, at least 3 years old, at least 5 years old, at least 10 years old, at least 15 years old, at least 20 years old, at least 25 years old, at least 30 years old, at least 40 years old, at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, at least 80 years old, at least 90 years old, or at least 100 years old.

[0100] A "terminator" or "terminator sequence," as used herein, is a nucleic acid (e.g., engineered nucleic acid) sequence that causes transcription to stop. A terminator can be unidirectional or bidirectional. It is composed of a DNA sequence involved in the specific termination of an RNA transcript by an RNA polymerase. A terminator sequence prevents transcriptional activation of a downstream nucleic acid (e.g., engineered nucleic acid) sequence by an upstream promoter. Thus, in certain embodiments, a terminator that terminates the production of an RNA transcript is contemplated.

[0101] The most commonly used type of terminator is a forward terminator. Normally, when placed downstream of a nucleic acid (e.g., an engineered nucleic acid) sequence to be transcribed, a forward transcription terminator will cause transcription to cease. In some embodiments, a bidirectional transcription terminator can be used, which normally causes transcription to terminate on both the forward and reverse strands. In some embodiments, a reverse transcription terminator can be used, which normally terminates transcription of only the reverse strand.

[0102] Non-limiting examples of mammalian terminator sequences include the bovine growth hormone terminator, and viral termination sequences, such as the SV40 terminator, spy, yejM, secG-leuU, thrLABC, rrnB T1, hisLGDCBHAFI, metZWV, rrnC, xapR, aspA, and arcA terminators. In certain embodiments, the terminator sequence is SV40 and comprises a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:8 or 143. In certain embodiments, the terminator sequence is hGH pA 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: 139, 148, 153, 156, or 161.

[0103] As used herein, the "Tet-Off" system is a type of inducible system that can suppress expression of a specific transgene in the presence of tetracycline (e.g., doxycycline (DOX)). Conversely, the Tet-Off system can induce expression of a specific transgene in the absence of tetracycline (e.g., doxycycline or DOX). In certain embodiments, the Tet-Off system comprises a tetracycline-responsive promoter operably linked to a transgene (e.g., encoding OCT4; KLF4; SOX2; or any combination thereof) and a tetracycline-regulated transactivator (tTA). In some embodiments, the inducer is tTA. The transgene with a tetracycline-responsive promoter (e.g., TRE3G, Ptight, or TRE2) and the tetracycline-regulated transactivator can be encoded on the same vector or on separate vectors. See, for example, International Publication No. WO 2020 / 069339, entitled "Mutant Reverse Tetracycline Transactivators for Expression of Genes," which was published on April 2, 2020, and is incorporated herein by reference in its entirety. In certain embodiments, the inducer is a tetracycline-regulated transactivator (tTA) (e.g., any of the tTAs disclosed herein). In certain embodiments, the inducer is a tTA and comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to any of the tTAs disclosed herein. In certain embodiments, the inducer is a tTA 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: 138 or 159. In certain embodiments, the inducer is tTA and is encoded by a sequence 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: 137 or 158.Without being bound by any particular theory, tetracycline (e.g., doxycycline) may bind to tTA and prevent it from binding to its corresponding promoter (e.g., a promoter containing a tetracycline-responsive element (TRE)) and driving expression of an operably linked nucleic acid. Without being bound by any particular theory, the nucleic acid encoding the inducer (e.g., the engineered nucleic acid) may not be on the same vector as any of the nucleic acids encoding OCT4, KLF4, and SOX2 (e.g., the engineered nucleic acid) in order to reduce the size of the viral vector and improve viral titer.

[0104] As used herein, the "Tet-On" system is a type of inducible system that can induce expression of a particular transgene in the presence of tetracycline (e.g., doxycycline (DOX)). In certain embodiments, the Tet-On system comprises a tetracycline-responsive promoter operably linked to a transgene (e.g., encoding OCT4; KLF4; SOX2; or any combination thereof) and a reverse tetracycline-regulated transactivator (rtTA). For example, the rtTA can be rtTA3, rtTA4, rtTA Advanced, rtTA2S-M2, or a variant thereof. In certain embodiments, the nucleic acid encoding rtTA3 (e.g., an engineered nucleic acid) comprises a sequence at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%) identical to SEQ ID NO: 10. In certain embodiments, the amino acid sequence 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%, at least 98%, at least 99%, or at least 100%) identical to (SEQ ID NO:11). In certain embodiments, a nucleic acid (e.g., an engineered 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:12. In certain embodiments, the amino acid sequence 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:13).In certain embodiments, a nucleic acid (e.g., an engineered nucleic acid) encoding rtTA Advanced 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: 128. In certain embodiments, an amino acid sequence encoding rtTA Advanced 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: 129). In certain embodiments, a nucleic acid (e.g., an engineered nucleic acid) encoding rtTA2S-M2 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: 14. In certain embodiments, the amino acid sequence encoding rtTA2S-M2 comprises a sequence at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100%) identical to (SEQ ID NO: 15). The tetracycline-responsive promoter (e.g., a promoter comprising a TRE including TRE3G, P tight, and TRE2) and the expression cassette encoding the reverse tetracycline-regulated transactivator can be encoded on the same vector or on separate vectors. See, for example, International Publication No. WO 2020 / 069339, entitled "Mutant Reverse Tetracycline Transactivators for Expression of Genes," which was published on April 2, 2020, and is incorporated herein by reference in its entirety.

[0105] The term "tissue" refers to any biological tissue (including a group of cells, a body part, or an organ) or part thereof of a subject, including blood vessels and / or lymphatic vessels, which are the targets for delivery of the compounds, particles, and / or compositions of the present invention. The tissue may be abnormal, damaged, or unhealthy tissue that may need to be treated. The tissue may also be normal or healthy tissue that is at higher risk than normal of becoming abnormal or unhealthy, which may need to be prevented. In certain embodiments, the tissue is healthy but is not considered optimal for function or survival in its current or future state. For example, in agricultural practices, environmental conditions, including weather and growing conditions (e.g., nutrients), may benefit from any of the methods described herein. In certain embodiments, the tissue is the central nervous system, e.g., the brain. In some embodiments, the cell or tissue is a neuronal cell or nervous tissue, and in some embodiments, the cell is a neuron. In some embodiments, the neuron is an excitatory neuron. In some embodiments, the tissue is not ocular tissue.

[0106] The term "tetracycline repressor" or "TetR" refers to a protein that can bind to a Tet-O sequence (e.g., a Tet-O sequence of a TRE, e.g., the Tet-O sequence may comprise SEQ ID NO: 19) in the absence of tetracycline (e.g., doxycycline) and prevent the binding of rtTA (e.g., rtTA3, rtTA4, or a variant thereof) in the absence of tetracycline (e.g., doxycycline). TetR prevents gene expression from a promoter comprising a TRE in the absence of tetracycline (e.g., doxycycline). In the presence of tetracycline, TetR cannot bind to a promoter comprising a TRE, and TetR cannot prevent transcription. Non-limiting examples of TetR include tetR (e.g., SEQ ID NO: 26) and tetRKRAB (e.g., SEQ ID NO: 28). In some embodiments, the TetR is a TetR fusion (e.g., TRSID, which can be created by fusing TetR to the mSIN30-interacting domain (SID) of Mad1). See, e.g., Zhang et al., J Biol Chem. 2001 Nov 30;276(48):45168-74.

[0107] As used herein, a "TRE promoter" refers to a promoter that includes a tetracycline responsive element (TRE). As used herein, a TRE includes 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. A non-limiting example of a Tet-O sequence is a sequence that is at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 19. In some embodiments, the TRE promoter further includes a minimal promoter located downstream of the tet-O sequence. A minimal promoter is a promoter that includes the minimum 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 that includes a Tet-O). By way of 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:20. For example, the TRE promoter can be a TRE3G promoter (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. In some embodiments, the TRE promoter is a TRE2 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:23. In some embodiments, the TRE promoter is a P tight 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:24.

[0108] The term "tissue repair" in the context of damaged tissue refers to the restoration of tissue architecture, function, or a combination thereof following tissue injury. Tissue repair encompasses tissue regeneration, cell growth, tissue replacement, and / or rewiring (reprogramming) of existing tissue.

[0109] The term "tissue regeneration" refers to the generation of new tissue or cells within a tissue that are of the same type as the desired tissue (e.g., the same type as the damaged tissue or cells). In some embodiments, the methods provided herein promote organ regeneration.

[0110] The term "tissue replacement" refers to the production of a different type of tissue compared to the target tissue (eg, connective tissue to replace damaged tissue).

[0111] As used herein, the terms "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder, or one or more symptoms thereof, as described herein. In certain embodiments, treatment may be administered after one or more symptoms have occurred. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms, or may be administered to treat another damaging factor (e.g., in light of a history of symptoms, genetic or other predisposition, disease therapy, or any combination thereof). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

[0112] 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 (e.g., engineered nucleic acids) sequences include frameshift mutations, nucleotide insertions, and nucleotide deletions.

[0113] The term "WPRE" refers to the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). The WPRE creates a tertiary structure on a nucleic acid (e.g., an expression vector) and can enhance 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: 21, 135, 147, 152, 155, or 160.

[0114] These and other exemplary substitutes are described in more detail in the detailed description, examples, and claims. The present invention is not intended to be limited in any way by the above exemplary enumeration of substitutes. [Brief explanation of the drawings]

[0115] [Figure 1A-1B] Figures 1A-1F show that OCT4, SOX2, and KLF4 (OSK) reprogramming increases electrical firing activity in iPSC-derived neurons using a tet-inducible all-in-one OSK lentiviral vector. Figure 1A is a schematic diagram of the all-in-one OSK lentiviral vector. Figure 1B is a schematic diagram of the I-PpoI lentiviral vector. [Figure 1C-1D] Figure 1C shows the experimental outline for reprogramming iPSC-derived neurons. Figure 1D shows Western blots showing the expression of OCT4, SOX2, and KLF4 after doxycycline treatment. [Figure 1E] FIG. 1E shows electrophysiology measurements using a multi-electrode array. [Figure 1F] Figure 1F shows quantification of electrical firing spikes in control and reprogrammed neurons. TAM, 4-hydroxytamoxifen. Dox, doxycycline.

[0116] [Figure 2A-2B]Figures 2A-2F show that OSK reprogramming improves cognitive function in middle-aged and elderly mice. Figure 2A shows the experimental outline of OSK delivery to the brain and behavioral testing. Figures 2B-2C show the results of a novel object recognition assay in middle-aged experimental mice aged 11 months (Figure 2B). [Figure 2C-2D] Figures 2C-2D show the Morris water maze results at the ages of 12 months (Figure 2C) and 30 months (Figure 2D). [Figures 2E-2F] Figures 2E-2F show the results of expressing OSK for 2 months. Figure 2E shows that 2 months of OSK treatment failed to improve cognitive function in 12-month-old mice. Figure 2F shows that 2 months of OSK treatment failed to improve cognitive function in 30-month-old mice.

[0117] [Figure 3] Figures 3A-3C show CaMKIIα-tTA and CaMKIIα-rtTA AAV vectors for restricting gene expression to excitatory neurons. Figure 3A is a schematic of the CaMKIIα-tTA construct. Figure 3B is a schematic of the CaMKIIα-rtTA construct. Figure 3C shows the body weight of AAV-injected mice.

[0118] [Figure 4A-4B] Figures 4A-4D show that reprogramming using OCT4, KLF4, and SOX2 (OKS) improves cognitive function in Alzheimer's disease mice. Figure 4A shows the design of inducible neuron-specific OKS (iNOKS) transgenic mice. Figure 4B shows a schematic illustrating the experimental design for OKS induction in 5xFAD mice and behavioral assays of cognitive function. [Figure 4C-4D] Figure 4C shows immunofluorescence analysis of OCT4, KLF4, and SOX2 expression in the hippocampus of iNOKS mice after 4 weeks of doxycycline treatment. Figure 4D shows the results of the water T-maze, demonstrating that reprogrammed 5xFAD mice (FAD-iNOKS) tended to require fewer days to reach the preset learning criterion than FAD control mice.

[0119] [Figure 5] Figure 5 shows RNA-seq analysis of hippocampal samples. Gene set enrichment analysis (GSEA) indicated that genes involved in learning and memory function were upregulated in hippocampal samples 1 month after doxycycline withdrawal (post-OSK) but not immediately after 4 weeks of doxycycline treatment (OSK). Most of these gene sets are downregulated with aging or during 2 or 6 weeks of p25 induction. p25 induction reference data is from Gjoneska et al. Nature. 2015 Feb 19;518(7539):365-9. The gene sets for the GSEA analysis in Figure 5 are from the Molecular Signatures Database (MSigDB).

[0120] [Figure 6A] Figures 6A-6C show single-nucleus multiomics, indicating that developmentally relevant genomic loci are hotspots of hypo- and hypermethylation induced by OSK reprogramming. Figure 6A shows hierarchical clustering based on single-nucleus transcriptome data. [Figure 6B] Figure 6B shows a heat map showing the expression of OSK in each cluster. [Figure 6C] FIG. 6C shows the enrichment of hypo- and hypermethylation induced by OSK reprogramming at developmentally relevant genomic loci, which are underlined. DETAILED DESCRIPTION OF THE INVENTION

[0121] Detailed Description of Certain Embodiments of the Invention The present disclosure is based, at least in part, on the unexpected results demonstrating that the expression of OCT4, SOX2, and KLF4 in the absence of exogenous c-Myc expression can be used to promote in vivo partial reprogramming and tissue regeneration.Surprisingly, using neural cells as model tissue, as described herein, it has been determined that in some embodiments, a combination of OCT4, SOX2, and KLF4 (OSK) can be used to reset the youthful gene expression pattern and epigenetic age of cells of the central nervous system, improving the cognitive function of animals.

[0122] The disclosed methods, compositions, uses, and kits are based, in part, on the surprising and unexpected discovery that spatially and temporally specific induction of OCT4, SOX2, and KLF4 expression in the absence of induction of c-Myc expression can rejuvenate cells of the central nervous system (e.g., brain, spinal cord) that are not in the retina without reprogramming the cells to a pluripotent state.

[0123] Aspects of the present disclosure provide several systems, compositions, uses, kits, and methods that may be useful for the efficient rejuvenation of cells, tissues, and / or organs of the central nervous system. In some embodiments, the central nervous system does not include the eye (e.g., the retina, uvea, pupil, lens, cornea, and / or sclera). In some embodiments, the cells, tissues, and / or organs of the central nervous system are brain cells, brain tissue, and / or the brain. For example, aspects of the present disclosure provide nucleic acids that may be useful for the efficient rejuvenation of the central nervous system by promoting the expression of OCT4, SOX2, and / or KLF4 without inducing c-Myc expression. In some embodiments, such nucleic acids encode inducers (e.g., tetracycline transactivators or reverse tetracycline transactivators) operably linked to the CaMKIIα promoter. In some embodiments, the CaMKIIα promoter is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 149. In some embodiments, the nucleic acid encoding the inducer does not comprise a synapsin-I promoter or a CaMKII-gamma promoter. In some embodiments, the synapsin-I promoter is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 157. In some embodiments, the nucleic acid encoding the inducer is a viral vector. In some embodiments, the viral vector is packaged into an AAV.PHP.eB virus.In some embodiments, the nucleic acid encoding the inducer comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to rtTA2S-M2 (SEQ ID NO: 14), pLVX-rtTA-hOSK-all-in-one (human) (SEQ ID NO: 123), pAAV-CaMKIIα-tTA2 (SEQ ID NO: 124), pAAV-CaMKIIα-rtTA2S-M2 (SEQ ID NO: 125), pAAV-CaMKIIα-rtTA3 (SEQ ID NO: 126), CaMKIIα promoter (SEQ ID NO: 146, 149, or 154); the reverse complement of rtTA Advanced (SEQ ID NO: 128), tTA Advanced (SEQ ID NO: 137), and / or tTA (SEQ ID NO: 158). In some embodiments, the nucleic acid encoding the inducer does not comprise a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to pAAV-ihSyn1-tTA (SEQ ID NO: 127). In some embodiments, the inducer comprises a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to rtTA Advanced (SEQ ID NO: 129), tTA Advanced (SEQ ID NO: 138), rtTA2S-M2 (SEQ ID NO: 15), and / or tTA (SEQ ID NO: 159).

[0124] Aspects of the present disclosure provide nucleic acids that may be useful for inducing the expression of OCT4, KLF4, and / or SOX2 in cells, tissues, or organs of the central nervous system in the absence of inducing c-Myc expression. These nucleic acids may be useful for rejuvenating cells, tissues, or organs of the central nervous system. In some embodiments, the nucleic acid has (a) a nucleic acid sequence encoding OCT4, KLF4, and SOX2 operably linked to a TRE promoter; and (b) a nucleic acid sequence encoding rtTA operably linked to a UbC promoter. In some embodiments, the nucleic acid sequence encoding OCT4, KLF4, and SOX2 further encodes a 2A peptide. In some embodiments, the nucleic acid having (a) and (b) further encodes a neomycin resistance gene and / or includes a WPRE sequence. In some embodiments, the neomycin resistance gene is operably linked to a PGK promoter. In some embodiments, the nucleic acid having (a) and (b) encodes at least one protein sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to a sequence selected from rtTA Advanced (SEQ ID NO: 129); human OCT4 (SEQ ID NO: 41); P2A (SEQ ID NO: 118); human SOX2 (SEQ ID NO: 43); T2A (SEQ ID NO: 9); human KLF4 (SEQ ID NO: 45); and a neomycin resistance gene (SEQ ID NO: 134). In some embodiments, the nucleic acid having (a) and (b) comprises at least one sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to a sequence selected from the reverse complement of rtTA Advanced (SEQ ID NO: 128); the reverse complement of the UbC promoter (SEQ ID NO: 130); the P tight TRE promoter (SEQ ID NO: 24); human OCT4 (SEQ ID NO: 40); P2A (SEQ ID NO: 119); human SOX2 (SEQ ID NO: 42); T2A (SEQ ID NO: 120); human KLF4 (SEQ ID NO: 131); PGK promoter (SEQ ID NO: 132); neomycin resistance gene (SEQ ID NO: 133); and WPRE (SEQ ID NO: 135).In some embodiments, the nucleic acid having (a) and (b) comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to pLVX-rtTA-hOSK-all-in-one(human) (SEQ ID NO: 123).

[0125] In some embodiments, the nucleic acid encodes tTA, wherein tTA comprises a sequence at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 138. In some embodiments, the nucleic acid encoding tTA is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 137. In some embodiments, the nucleic acid encoding tTA comprises an hGH pA sequence and / or a CMV promoter. In some embodiments, the hGH pA sequence is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 139. In some embodiments, the CMV promoter is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 136. In some embodiments, the nucleic acid encoding tTA comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to pAAV-CMV-tTA(Advanced) (SEQ ID NO: 32).

[0126] In some embodiments, the nucleic acid comprises a nucleic acid encoding OCT4, SOX2, and KLF4 operably linked to a TRE promoter. In some embodiments, the nucleic acid sequence encoding OCT4, SOX2, and KLF4 operably linked to a TRE promoter further encodes a 2A peptide and / or SV40 pA. In some embodiments, the 2A peptide comprises a sequence at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to T2A (SEQ ID NO: 9) or P2A (SEQ ID NO: 118). In some embodiments, the nucleic acid sequence encoding OCT4, SOX2, and KLF4 encodes at least one sequence 70% identical to a sequence selected from mouse OCT4 (SEQ ID NO: 2); human OCT4 (SEQ ID NO: 40); mouse SOX2 (SEQ ID NO: 4); human SOX2 (SEQ ID NO: 42); human KLF4 (SEQ ID NO: 131); and mouse KLF4 (SEQ ID NO: 6). In some embodiments, the TRE promoter operably linked to the nucleic acid sequence encoding OCT4, SOX2, and KLF4 is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO:7. In some embodiments, the nucleic acid sequence encoding OCT4, SOX2, and KLF4 operably linked to the TRE promoter comprises at least one sequence that is 70% identical to a sequence selected from TRE3G (SEQ ID NO: 7); mouse Oct4 (SEQ ID NO: 1); P2A (SEQ ID NO: 144); mouse Klf4 (SEQ ID NO: 145); SV40 pA (SEQ ID NO: 143); mouse Sox2 (SEQ ID NO: 3); and T2A (SEQ ID NO: 120), optionally wherein the sequence is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to pAAV-TRE3G-OSK (mouse) SEQ ID NO: 16.

[0127] In some embodiments, the nucleic acid comprises a nucleic acid encoding an inducer, and the nucleic acid encoding the inducer is operably linked to the CaMKIIα promoter. In some embodiments, the inducer is tTA or rtTA. In some embodiments, the inducer comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to tTA Advanced (SEQ ID NO: 138), rtTA2S-M2 (SEQ ID NO: 15), or rtTA3 (SEQ ID NO: 11). In some embodiments, the CaMKIIα promoter comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 146, SEQ ID NO: 149, or SEQ ID NO: 154. In some embodiments, the nucleic acid encoding the inducer further comprises a WPRE and / or hGH pA sequence. In some embodiments, the WPRE sequence is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 147, 152, or 155. In some embodiments, the hGH pA sequence is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 148, 153, or 156.

[0128] In some embodiments, the nucleic acid encoding the inducer comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to a sequence selected from tTA-Advanced (SEQ ID NO: 137), rtTA2S-M2 (SEQ ID NO: 14), or rtTA3 (SEQ ID NO: 10). In some embodiments, the nucleic acid encoding the inducer comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to a sequence selected from pAAV-CaMKIIα-tTA2 (SEQ ID NO: 124), pAAV-CaMKIIα-rtTA2S-M2 (SEQ ID NO: 125), or pAAV-CaMKIIα-rtTA3 (SEQ ID NO: 126).

[0129] In some embodiments, the nucleic acid does not comprise a synapsin-I promoter operably linked to a nucleic acid sequence encoding an inducer. In some embodiments, the nucleic acid does not comprise a sequence at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 157. In some embodiments, the nucleic acid does not comprise a sequence at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to pAAV-ihSyn1-tTA (SEQ ID NO: 127).

[0130] In certain embodiments, expression of one or more of the genes is transient (e.g., using an inducible promoter to control gene expression). Expression of one or more of the genes (e.g., OCT4, SOX2, KLF4, or a combination thereof) can be regulated by modulating the activity of an inducer. As a non-limiting example, tetracycline transactivator (tTA) can induce expression from a tetracycline-responsive promoter in the absence of tetracycline. When tetracycline is added, tTA can no longer bind to the promoter and induce expression. As another non-limiting example, reverse tetracycline transactivator (rtTA) can induce expression from a tetracycline-responsive promoter in the presence of tetracycline. When tetracycline is removed, rtTA can no longer bind to the promoter and induce expression. The vectors encoding OCT4, SOX2, and KLF4 (OSK) described herein increased neuronal electrical firing and improved cognitive function in vivo. Thus, expression of these three genes may be useful for regenerating central nervous system tissues and organs, repairing central nervous system tissues and organs, reversing central nervous system aging, treating neurodegenerative diseases and conditions, and / or reprogramming cells of the central nervous system.

[0131] In some embodiments, OCT4, SOX2, and / or KLF4 are expressed in the central nervous system for at most one month. Without being bound by any particular theory, expression of OCT4, SOX2, and / or KLF4 for two months may fail to rejuvenate the central nervous system.

[0132] In some embodiments, the results disclosed herein suggest that expression of OCT4, SOX2, and KLF4 can allow diseased cells in the central nervous system (e.g., the brain) to revert to a healthier state without inducing complete reprogramming. Without being bound by any particular theory, the results disclosed herein suggest that cells maintain a backup epigenome that can be restored using the methods described herein.

[0133] Thus, in various embodiments, the methods and uses of the present invention rejuvenate cells by restoring their cellular identity by reversing or preventing the effects of one or more dysregulated developmental pathways. For example, in certain embodiments, the methods include: (i) increasing the abundance of at least one of histone H2A, histone H2B, histone H3, histone H4, or any combination thereof in a cell; (ii) increasing the abundance of at least one of CHAF1a, CHAF1b, HP1α, NuRD, or any combination thereof in the cells; (iii) increasing at least one heterochromatic mark in the cell, such as, for example, H3K9me3, H3K27me3, or any combination thereof; or decreasing one heterochromatic mark, such as, for example, H4K20me3, or the euchromatic mark H3K4me3; (iv) increasing / decreasing DNA methylation of at least one age-related CpG site in the cells toward a younger level; (v) increasing the abundance of cellular lamin B1; (vi) increasing acetylation of histone H3 at lysine 27 (H3K27ac), increasing acetylation of histone H3 at lysine 56 (H3K56ac), or any combination thereof in a cell; (vii) reducing cellular acetylation of histone H3 at lysine 122 (H3K122Ac) or histone H4 at lysine 16 (H4K16ac), or any combination thereof; (viii) decreasing the abundance of IL6, Ccl2, Ccl20, Apob, p16, LINE-1 repeats, Sat III repeats, Alu elements, IAPs, or any combination thereof; (ix) restoring the balance between euchromatic epigenetic marks, such as H3K4me3, and heterochromatic epigenetic marks, such as H3K9me3 or H3K27me3; (x) induces the formation of euchromatin; (xi) restoring youthful levels of at least one repressive heterochromatin epigenetic mark; and / or (xii) restoring expression of at least one gene to a youthful level. In some embodiments, the youthful level is the expression level of the gene in a subject that is between 1 and 5, 1 and 10, 1 and 20, 1 and 30, or 1 and 40 years old.

[0134] In some embodiments, the at least one gene restored to youthful levels is at least one gene selected from the group consisting of RE1 silencing transcription factor (REST), (tumor protein P73) TP73, glial fibrillary acidic protein (GFAP), intercellular adhesion molecule 2 (ICAM2), and thioredoxin interacting protein (TXNIP).

[0135] In some embodiments, the methods disclosed herein alleviate one or more hallmarks of aging, including genomic instability, telomere attrition, epigenetic modulation, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. See, for example, Kennedy et al., Cell. 2013 Jun 6; 153(6): 1194-1217.

[0136] In some embodiments, the methods disclosed herein increase the expression of one or more of the following gene sets: associative learning, excitatory synapse assembly, central nervous system neuron axonogenesis, central nervous system neurogenesis, memory, control of GABAergic synaptic transmission, control of postsynaptic organization, learning, control of neurogenesis, central nervous system neuron differentiation, and / or central nervous system synapse maturation. For example, see the Molecular Signatures Database (MSigDB) for gene sets. In some embodiments, the gene set is determined using Gene Ontology Terms for Biological Processes (GOBPs). In some embodiments, the methods disclosed herein increase the expression of one or more of these gene sets by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% relative to a control. In some embodiments, the control is the level of expression of one or more gene sets in the absence of inducing OCT4, KLF4, and SOX2 expression. In some embodiments, gene expression is measured at least 4 weeks after the induction of OCT4, KLF4, and SOX2 expression has ceased. In some embodiments, gene expression is measured at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, or at least 50 weeks after induction of OCT4, KLF4, and SOX2 expression has ceased.

[0137] In some embodiments, the methods disclosed herein increase hypomethylated CpG sites and / or hypermethylated CpG sites in developmental loci relative to a control. Hypomethylated CpG sites are CpG sites with reduced methylation relative to a control. Hypermethylated CpG sites are CpG sites with increased methylation relative to a control. In some embodiments, hypomethylated CpG sites are increased in developmental loci related to positive regulation of epidermal development, midgut development, negative regulation of chromatin organization, negative regulation of histone methylation, camera eye lens development, negative regulation of adipocyte differentiation, negative regulation of histone modification, lung morphogenesis, DNA methylation of cytosine, and / or neural progenitor cell proliferation. In some embodiments, the control is the level of hypomethylation and / or hypermethylation in one or more developmental loci in the absence of inducing OCT4, KLF4, and SOX2 expression. In some embodiments, hypermethylated CpG sites are increased in developmental loci associated with skeletal muscle cell differentiation, cardiac looping, cardiac left-right asymmetry determination, uterine morphogenesis, regulation of the Notch signaling pathway, embryonic cranial skeletal morphogenesis, animal organogenesis, tricuspid valve morphogenesis, lens fiber cell differentiation, and / or positive regulation of the BMP signaling pathway. In some embodiments, the control is the level of hypomethylation and / or hypermethylation at one or more developmental loci in the absence of inducing OCT4, KLF4, and SOX2 expression.

[0138] In some embodiments, the methods disclosed herein increase hypomethylated and / or hypermethylated CpG sites at developmentally relevant loci by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% relative to a control. In some embodiments, the control is the level of hypomethylation and / or hypermethylation at one or more developmentally relevant loci in the absence of inducing OCT4, KLF4, and SOX2 expression.

[0139] Without being bound by any particular theory, more specifically, the disclosed methods, compositions, uses, and kits rejuvenate cells in the central nervous system (e.g., the brain) by restoring epigenetic information lost due to the aging process, injury, or disease. The disclosed methods, compositions, uses, and kits include the transcription factors OCT4, SOX2, and KLF4. OCT4, SOX2, and KLF4 are three of the four "Yamanaka factors," the fourth being c-Myc. Yamanaka factors have previously been used to reprogram cells to a pluripotent state. However, induction of expression of the four transcription factors in transgenic mice resulted in the formation of teratomas in vivo, along with other acute toxicities, such as metaplasia in the intestinal epithelium, which can kill the animals within a few days. Furthermore, the fact that the four Yamanaka factors are typically used to fully reprogram cells to a pluripotent state, in which cells lose their pre-established cellular identity, can be dangerous for in vivo applications in which the cellular identity of target cells must be maintained for the integrity of tissues and / or organs. In contrast, in some embodiments, the methods described herein allow for controlled reprogramming and do not result in global changes in demethylation. In some embodiments, the methods described herein do not require complete dedifferentiation of cells.

[0140] Cellular reprogramming allows the generation of multiple cell types from existing somatic cells. Yamanaka factors (OCT4, SOX2, KLF4, and c-Myc, collectively known as OSKM) have been shown to induce pluripotency in differentiated cells, but administration of these factors can induce teratomas or other cancers in vivo (Takahashi et al., Cell 2006 Aug 25;126(4):663-76; Abad et al., Nature 2013 Oct 17;502(7471):340-5). As a result of these safety concerns, the use of Yamanaka factors has largely been limited to in vitro applications. Furthermore, existing methods of gene therapy are plagued by inefficient and inconsistent gene transduction of target cells. The engineered nucleic acids, recombinant viruses containing the same, pharmaceutical compositions thereof, and kits provided herein overcome many of these limitations.

[0141] Engineered Nucleic Acids The engineered nucleic acids of the present disclosure may encode OCT4, SOX2, KLF4, and their homologs or variants (e.g., functional variants), alone or in combination, and / or may include nucleic acids encoding inducers, which may be useful for rejuvenating cells, tissues, and / or organs of the central nervous system. For example, the cells, tissues, and / or organs may be in a subject in need thereof. In some cases, the central nervous system excludes the retina. In some embodiments, the central nervous system excludes the eye (e.g., the retina, uvea, pupil, lens, cornea, and / or sclera). In some embodiments, the cells, tissues, and / or organs are brain cells, brain tissue, and / or the brain. In some embodiments, the subject in need thereof has a neurological disease. In certain embodiments, the engineered nucleic acid (e.g., the engineered nucleic acid) does not encode c-Myc. In certain embodiments, the engineered nucleic acid (e.g., the engineered nucleic acid) does not encode functional c-Myc because it lacks the c-Myc sequence. Assays for determining transcription factor (e.g., OCT4, SOX2, KLF4, or any combination thereof) activity are known in the art and include cell-based and in vitro transcription assays. Transcription factor expression can also be determined using other methods, including enzyme-linked immunosorbent assay (ELISA), Western blot, and RNA quantification (e.g., using reverse transcription polymerase chain reaction).

[0142] Transcription factors (e.g., OCT4, SOX2, KLF4, or homologs or variants thereof, including mammalian OCT4, mammalian SOX2, and mammalian KLF4) can be encoded by a single nucleic acid, or a single nucleic acid (e.g., an engineered nucleic acid) can encode two or more transcription factors (e.g., each operably linked to a different promoter, or both operably linked to the same promoter). For example, in certain embodiments, a nucleic acid (e.g., an engineered nucleic acid) can encode OCT4; SOX2; KLF4; OCT4 and SOX2; OCT4 and KLF4; SOX2 and KLF4; or OCT4, SOX2, and KLF4, in any order.

[0143] In certain embodiments, the engineered nucleic acid (e.g., the engineered nucleic acid) encodes an inducer (e.g., tTA or rtTA). In certain embodiments, the nucleic acid (e.g., the engineered nucleic acid) can encode one or more transcription factors (e.g., one, two, or three transcription factors) and an inducer. In certain embodiments, the inducer is encoded by a separate nucleic acid (e.g., the engineered nucleic acid) that does not also encode a transcription factor (e.g., OCT4, SOX2, or KLF4). In certain embodiments, the inducer is encoded by a nucleic acid (e.g., the engineered nucleic acid) that also encodes a transcription factor (e.g., OCT4, SOX2, and / or KLF4). In certain embodiments, the inducer is encoded by a nucleic acid (e.g., an engineered nucleic acid) that also encodes one or more transcription factors selected from the group consisting of OCT4; SOX2; KLF4; and any combination thereof (e.g., OCT4; SOX2; KLF4; OCT4 and SOX2; OCT4 and KLF4; SOX2 and KLF4; or OCT4, SOX2, and KLF4).

[0144] The transcription factors (e.g., OCT4, SOX2, KLF4, or any combination thereof) or inducers described herein can contain one or more amino acid substitutions.Variant can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, for example, those found in references that compile such methods, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel et al., eds., John Wiley & Sons, Inc., New York.Conservative amino acid substitutions include substitutions between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0145] In certain embodiments, the engineered nucleic acids of the present disclosure include RNA (e.g., mRNA) and / or DNA. In some embodiments, the RNA and / or DNA are further modified. By way of non-limiting example, the nucleic acid (e.g., engineered nucleic acid) of the present disclosure can be modified RNA (e.g., mRNA) encoding OCT4, KLF4, SOX2, derivatives, or any combination thereof. For example, see Warren et al., Cell Stem Cell. 2010 Nov 5;7(5):618-30. For example, the engineered nucleic acids (e.g., RNA or DNA, including mRNA) of the present disclosure can be formulated into nanoparticles for delivery. For example, see Dong et al., Nano Lett. 2016 Feb 10;16(2):842-8. In some embodiments, the nanoparticles comprise acetylated galactose. For example, see Lozano-Torres et al., J Am Chem Soc. 2017 Jul 5;139(26):8808-8811. In some embodiments, the engineered nucleic acid (e.g., RNA or DNA, including mRNA) is electroporated or transfected into cells. In certain embodiments, the engineered nucleic acid is delivered as naked nucleic acid (e.g., naked DNA or naked RNA).

[0146] In some embodiments, the engineered nucleic acid formulated into nanoparticles for delivery is not an AAV vector. Suitable vector backbones for nanoparticle formulation include, but are not limited to, NANOPLASMID™ vectors and NTC "8" series mammalian expression vectors. Non-limiting examples of vector backbones for nanoparticle formulation include NTC9385R and NTC8685. Without being bound by theory, NTC "8" series mammalian expression vectors may be useful because they are generally cleared by cells within a few weeks. NTC "8" series mammalian expression vectors contain a CMV promoter, which may be operably linked to sequences encoding OCT4, KLF4, SOX2, or a combination thereof. Without being bound by theory, NANOPLASMID™ vectors are less immunogenic than other vectors and may express at higher levels and for longer periods of time. This may be useful for long-term expression of operably linked nucleic acids. In some embodiments, NANOPLASMID™ vectors may be useful for long-term expression of OCT4, KLF4, SOX2, or combinations thereof.

[0147] Without being bound by any particular theory, modified RNA (e.g., mRNA) has the advantage of minimal activation of the innate immune response and limited cytotoxicity, which may allow for strong and sustained protein expression. In some embodiments, the RNA (e.g., mRNA) contains modifications including full substitution of either cytidine with 5-methylcytidine (5mC) or uridine with pseudouridine (psi).

[0148] In some embodiments, OCT4, KLF4, and / or SOX2 expression can be activated using a CRISPR activation system. In some embodiments, the expression of one or more transcription factors selected from the group consisting of OCT4, KLF4, SOX2, and combinations thereof can be activated using a CRISPR activation system. For example, see Liao et al., Cell. 2017 Dec 14;171(7):1495-1507.e15; Liu et al., 2018, Cell Stem Cell 22, 1-10 February 1, 2018. Generally, the CRISPR activation system includes an enzymatically dead Cas9 nuclease (or nuclease-deficient Cas9 (dCas9)) fused to a transcription activation complex (e.g., including VP64, P65, Rta, and / or MPH). Non-limiting examples of sequences encoding VP64, P65, Rta, and / or MPH are provided below. VP64, P65, Rta, or MPH can be encoded by a sequence that contains a sequence that is at least 70% identical (e.g., 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) to any of the VP64, P65, Rta, and / or MPH sequences described herein. This Cas9 fusion protein can be referred to as a CRISPR activator. A guide RNA targeting the promoter and / or enhancer region of a gene of interest is used in the CRISPR activation system to target the dCas9 transcription activation complex and drive expression of the endogenous gene.

[0149] In some embodiments, expression of OCT4; KLF4; SOX2; or any combination thereof can be activated using a transcription activator-like effector nuclease (TALEN) or zinc finger nuclease (ZFN) system.

[0150] The engineered nucleic acids of the present disclosure may encode sgRNAs for targeting the promoter and / or enhancer regions of the endogenous loci of OCT4, SOX2, and / or KLF4 in a cell. The engineered nucleic acids of the present disclosure may encode sgRNAs for targeting the promoter and / or enhancer regions of the endogenous loci of one or more transcription factors selected from OCT4; SOX2; KLF4; and any combination thereof in a cell. In some embodiments, the engineered nucleic acid (e.g., an expression vector) further encodes dCas9 (dead Cas9) and a transcription activation complex (e.g., VP64, P65, Rta, MPH). In some embodiments, the dCas9 (dead Cas9) and a transcription activation complex (e.g., VP64, P65, Rta, MPH) are administered to a cell on an engineered nucleic acid (e.g., an expression vector). In some embodiments, the vector encoding the sgRNA and / or dCas9 (dead Cas9) and transcription activation complex (e.g., VP64, P65, Rta, MPH) is a viral vector (e.g., AAV vector). In some embodiments, the dCas9 (dead Cas9) and transcription activation complex (e.g., VP64, P65, Rta, MPH) are introduced into cells as proteins.

[0151] In some embodiments, guide RNAs targeting the enhancer and / or promoter regions of OCT4, SOX2, and / or KLF4 are formulated into nanoparticles and injected together with dCas9-VP64 protein. In some embodiments, guide RNAs targeting the enhancer and / or promoter regions of OCT4, SOX2, KLF4, or any combination thereof are formulated into nanoparticles and injected together with dCas9-VP64 protein. In some embodiments, guide RNAs and / or nucleic acids encoding dCas9 (dead Cas9) and transcription activation complexes (e.g., VP64, P65, Rta, MPH) are administered as naked nucleic acids (e.g., naked DNA formulated into nanoparticles). In some embodiments, the guide RNA and / or nucleic acid encoding dCas9 (dead Cas9) and a transcription activation complex (e.g., VP64, P65, Rta, MPH) is delivered via a recombinant virus (e.g., a lentivirus, adenovirus, retrovirus, herpesvirus, human papillomavirus, alphavirus, vaccinia virus, or adeno-associated virus (AAV)).

[0152] Non-limiting example sequences of guide RNAs targeting the endogenous OCT4 or SOX2 locus are provided in Liu et al., Cell Stem Cell. 2018 Feb 1;22(2):252-261.e4. Non-limiting examples of guide RNAs targeting OCT4, SOX2, and / or KLF4 are also provided in Weltner et al., Nat Commun. 2018 Jul 6;9(1):2643.

[0153] Without being bound by any particular theory, the use of the CRISPR-CAS9 system to activate endogenous expression of OCT4, KLF4, and / or SOX2 in the absence of c-Myc expression may eliminate potential toxicities associated with exogenous and / or supraphysiological gene expression.

[0154] Nucleic acids (e.g., engineered nucleic acids) encoding transcription factors (OCT4, SOX2, KLF4, or any combination thereof) or encoding inducers can be introduced into expression vectors using conventional cloning techniques. Suitable expression vectors include vectors having a promoter (e.g., a constitutive or inducible promoter, including a TRE promoter) operably linked to nucleic acids (e.g., engineered nucleic acids) encoding OCT4, SOX2, KLF4, or any combination thereof, and a terminator sequence (e.g., the SV40 sequence described herein). In some embodiments, the nucleic acid (e.g., engineered nucleic acid) encodes a promoter operably linked to a nucleic acid encoding an inducer. In some embodiments, the vector includes a WPRE sequence. Expression vectors containing the elements necessary for expression are commercially available and known to those skilled in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012).

[0155] The vectors of the present invention may further contain marker sequences for use in identifying cells that have been transformed or transfected with the vector, or that have not been reprogrammed. Markers include, for example, genes encoding proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds (e.g., ampicillin resistance genes, kanamycin resistance genes, neomycin resistance genes, tetracycline resistance genes, and chloramphenicol resistance genes), genes encoding enzymes with activities detectable by standard assays known in the art (e.g., β-galactosidase, senescence-associated β-galactosidase, luciferase, or alkaline phosphatase), and genes that visibly affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques (e.g., green fluorescent protein). In some embodiments, as used herein, vectors are capable of autonomous replication and expression of structural gene products present on the DNA fragments to which they are operably linked. In some embodiments, the vector encoding the neomycin resistance gene 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: 133. In some embodiments, the neomycin resistance gene 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: 134.

[0156] In some embodiments, the expression vector comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to a sequence selected from SEQ ID NOs: 123-127.

[0157] In certain embodiments, the expression vector comprises an inducible promoter (e.g., a tetracycline-responsive promoter) operably linked to a sequence encoding a transcription factor (e.g., OCT4, SOX2, KLF4, or any combination thereof).

[0158] In certain embodiments, the promoter operably linked to a sequence encoding a transcription factor (e.g., OCT4, SOX2, KLF4, or any combination thereof) and / or encoding an inducer is a tissue- or cell-type-specific promoter (e.g., a brain-specific, liver-specific, muscle-specific, neuron-specific, glial cell-specific, endothelial cell-specific, lung-specific, heart-specific, bone-specific, gut-specific, skin-specific, or eye-specific promoter). By way of example, the muscle-specific promoter can be the desmin promoter (e.g., a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:29). In some embodiments, the eye-specific promoter can be a promoter at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a sequence selected from SEQ ID NOs:101-104. In some embodiments, the tissue-specific promoter is not an eye-specific promoter.

[0159] In some embodiments, the tissue-specific promoter is a central nervous system-specific promoter. In some embodiments, the tissue-specific promoter is a neuron-specific promoter. In some embodiments, the neuron-specific promoter is a CaMKIIα promoter. In some embodiments, the brain-specific promoter is a CaMKIIα promoter. In some embodiments, the CaMKIIα promoter 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: 146, 149, or 154.

[0160] In some embodiments, the promoter operably linked to the sequence encoding a transcription factor (e.g., OCT4, SOX2, KLF4, or any combination thereof) and / or the sequence encoding an inducer does not comprise an eye-specific promoter. In some embodiments, the promoter operably linked to the sequence encoding a transcription factor (e.g., OCT4, SOX2, KLF4, or any combination thereof) and / or the sequence encoding an inducer does not comprise a synapsin-I promoter, does not comprise a CaMKII-gamma promoter, or a combination thereof. In some embodiments, the promoter operably linked to the sequence encoding a transcription factor (e.g., OCT4, SOX2, KLF4, or any combination thereof) does not comprise a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) to SEQ ID NO: 157.

[0161] In certain embodiments, the promoter operably linked to a sequence encoding a transcription factor (e.g., OCT4, SOX2, KLF4, or any combination thereof) and / or encoding an inducer is age- or senescence-specific (e.g., an age- or senescence-specific promoter that can be the p16 promoter, or a Cas9-directed transcription factor that binds to methylated DNA that accumulates with senescence).

[0162] In certain embodiments, the expression vector comprises a constitutive promoter operably linked to a nucleic acid (e.g., an engineered nucleic acid) encoding OCT4, SOX2, KLF4, or any combination thereof. In some embodiments, such vectors can be inactivated using a clustered regularly interspaced short palindromic repeats (CRISPR) / guide RNA system. For example, the guide RNA can be complementary to the vector and can target Cas9 nuclease to the vector. In some embodiments, the guide RNA is complementary to a transgene (e.g., a transgene encoding OCT4, KLF4, SOX2, or a combination thereof) in any of the expression vectors described herein. Cas9 can then create a double-strand break in the vector and / or mutate the vector, rendering it inactive.

[0163] In certain embodiments, the promoter operably linked to the sequence encoding the inducer is a constitutive promoter (e.g., CMV, EF1 alpha, SV40 promoter, PGK1, UBC, CAG, human beta-actin gene promoter, or UAS). In certain embodiments, the promoter operably linked to the sequence encoding the inducer is a tissue-specific promoter (e.g., a brain-specific, liver-specific, muscle-specific, neuron-specific, lung-specific, heart-specific, bone-specific, gut-specific, skin-specific promoter, or eye-specific promoter). For example, the muscle-specific promoter can be the desmin promoter (e.g., a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 29).

[0164] The nucleic acid (e.g., engineered nucleic acid) (e.g., expression vector) may further comprise a separator sequence (e.g., an IRES or polypeptide cleavage signal). Exemplary polypeptide cleavage signals include 2A peptides (e.g., T2A, P2A, E2A, and F2A). The 2A 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: 9 or 118. For nucleic acids (e.g., engineered nucleic acids) (e.g., expression vectors) encoding more than one transcription factor (e.g., OCT4, SOX2, KLF4, or any combination thereof), each transcription factor may be operably linked to a different promoter or the same promoter. The transcription factors may be separated on the nucleic acid (e.g., by a peptide separator sequence). Expression of the nucleic acid (e.g., engineered nucleic acid) results in distinct amino acid sequences encoding each transcription factor.

[0165] In certain embodiments, expression vectors of the present disclosure (e.g., expression vectors encoding OCT4, KLF4, SOX2, or combinations thereof) may further comprise 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.).

[0166] In certain embodiments, expression vectors encoding inducers of the present disclosure may further comprise 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.).

[0167] In some embodiments, the expression vector encoding the neomycin resistance gene 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: 133. In some embodiments, the neomycin resistance gene 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: 134.

[0168] In certain embodiments, the expression vector (e.g., encoding OCT4, SOX2, KLF4, or any combination thereof) is present on a viral vector (e.g., an AAV vector). In certain embodiments, the expression vector encoding the inducer is present on a viral vector (e.g., an AAV vector). As used herein, an AAV vector generally comprises ITRs flanking an expression cassette (e.g., a nucleic acid (e.g., an engineered nucleic acid) comprising a promoter sequence operably linked to a sequence encoding OCT4, SOX2, KLF4, or any combination thereof, and a terminator, a nucleic acid (e.g., an engineered nucleic acid) comprising a promoter sequence operably linked to a sequence encoding an inducer, or a combination thereof).

[0169] In certain embodiments, the number of base pairs between two ITRs on an AAV vector of the disclosure is less than 5 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 certain embodiments, an AAV vector having a distance of less than 4.7 kb between two ITRs can be packaged into virus at a titer of at least 0.5 x 10 particle forming units per ml (pfu / ml), at least 1 x 10 pfu / ml, at least 5 x 10 pfu / ml, at least 1 x 10 pfu / ml, at least 5 x 10 pfu / ml, at least 1 x 10 pfu / ml, at least 2 x 10 pfu / ml, at least 3 x 10 pfu / ml, at least 4 x 10 pfu / ml, at least 5 x 10 pfu / ml, at least 6 x 10 pfu / ml, at least 7 x 10 pfu / ml, at least 8 x 10 pfu / ml, at least 9 x 10 pfu / ml, or at least 1 x 10 pfu / ml.

[0170] In certain embodiments, an expression vector of the disclosure is at least 1 kilobase (kb) (e.g., at least 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 50 kb, or 100 kb). In certain embodiments, an expression vector of the disclosure is less than 10 kb (e.g., less than 9 kb, less than 8 kb, less than 7 kb, less than 6 kb, less than 5 kb, less than 4 kb, less than 3 kb, less than 2 kb, or less than 1 kb).

[0171] Without being bound by theory, an expression vector (e.g., an AAV vector) encoding OCT4, SOX2, and KLF4 under a single promoter results in more efficient transduction of all three transcription factors in vivo compared to separate nucleic acids (e.g., engineered nucleic acids) encoding one or two of the transcription factors. In certain embodiments, the infection efficiency of a recombinant virus (e.g., lentivirus, alphavirus, vaccinia virus, retrovirus, adenovirus, herpesvirus, human papillomavirus, or AAV) carrying a vector of the present disclosure in a cell (e.g., an animal cell, including a mammalian cell) is at least 20% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, or 100%).

[0172] In some embodiments, the engineered nucleic acids described herein comprise sequences that are at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a sequence disclosed herein.

[0173] In some embodiments, the 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 SEQ ID NO: 16, SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 121, SEQ ID NO: 123, or SEQ ID NO: 163. In some embodiments, the engineered nucleic acid comprises SEQ ID NO: 16, SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 121, SEQ ID NO: 123, or SEQ ID NO: 163. In some embodiments, the engineered nucleic acid (e.g., an expression vector) consists of SEQ ID NO: 16, SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 121, SEQ ID NO: 123, or SEQ ID NO: 163. See also, for example, WO 2020 / 069373, entitled "Cellular Reprogramming to Reverse Aging and Promote Organ and Tissue regeneration," which was published on April 2, 2020, and is incorporated herein by reference in its entirety.

[0174] In some embodiments, the engineered nucleic acid encoding the inducer comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, or 100%) identical to SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 137, SEQ ID NO: 158, SEQ ID NO: 17, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 146, 149, or 154, SEQ ID NO: 135, 147, 152, 155, or 160, SEQ ID NO: 139, 148, 153, 156, or 161, or SEQ ID NO: 126. In some embodiments, the engineered nucleic acid encoding the inducer does not 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: 127. In some embodiments, the engineered nucleic acid encoding the inducer comprises SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 137, SEQ ID NO: 158, SEQ ID NO: 17, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 146, 149, or 154, SEQ ID NO: 135, 147, 152, 155, or 160, SEQ ID NO: 139, 148, 153, 156, or 161, or SEQ ID NO: 126. In some embodiments, the engineered nucleic acid encoding the inducer does not comprise SEQ ID NO: 127. In some embodiments, the expression vector encoding the inducer consists of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, or SEQ ID NO: 126. See also, for example, WO 2020 / 069339, entitled "Mutant Reverse Tetracycline Transactivators for Expression of Genes." It was published on April 2, 2020, and is incorporated herein by reference in its entirety.

[0175] One aspect of the present disclosure provides a vector (e.g., expression vector) comprising, alone or in combination, a first nucleic acid encoding OCT4 (e.g., engineered nucleic acid), a second nucleic acid encoding SOX2 (e.g., engineered nucleic acid), and a third nucleic acid encoding KLF4 (e.g., engineered nucleic acid), in the absence of an exogenous nucleic acid (e.g., engineered nucleic acid) capable of expressing c-Myc, for use in the central nervous system. In certain embodiments, the vector (e.g., expression vector) comprises a first nucleic acid encoding OCT4 (e.g., engineered nucleic acid), a second nucleic acid encoding SOX2 (e.g., engineered nucleic acid), a third nucleic acid encoding KLF4 (e.g., engineered nucleic acid), or any combination thereof. In certain embodiments, the first, second, and third nucleic acids (e.g., engineered nucleic acids) are present on separate expression vectors. In certain embodiments, two of the first, second, and third nucleic acids (e.g., engineered nucleic acids) are present on the same expression vector. In some embodiments, all three nucleic acids (e.g., engineered nucleic acids) are present on the same expression vector. In certain embodiments, the 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: 2 or 41. In certain embodiments, the 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: 4 or 43. In certain embodiments, the 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: 6 or 45. In certain embodiments, one or more of OCT4, SOX2, and KLF4 are human proteins.In certain embodiments, one or more of OCT4, SOX2, and KLF4 are non-human proteins (e.g., from other mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cows, pigs, horses, sheep, goats, cats, and / or dogs), and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and / or turkeys). When more than one of OCT4, SOX2, and KLF4 is on a single vector, they can be in any order. The terms "first," "second," and "third" imply the order of the genes on the vector. In some embodiments, the vector encodes OCT4, then SOX2, then KLF4. In some embodiments, the vector encodes OCT4, then KLF4, then SOX2. In some embodiments, the vector encodes KLF4, then OCT4, then SOX2. In some embodiments, the vector encodes KLF4, then SOX2, then OCT4. In some embodiments, the vector encodes SOX2, then KLF4, then OCT4. In some embodiments, the vector encodes SOX2, then OCT4, then KLF4.

[0176] The expression vector of the present disclosure may further include an inducible promoter. The expression vector may have only one inducible promoter. In such cases, the expression of OCT4, SOX2, and KLF4 is under the control of the same inducible promoter. In some cases, the expression vector includes more than one inducible promoter. The inducible promoter may include a tetracycline-responsive element (TRE) (e.g., a TRE3G promoter, a TRE2 promoter, or a P tight promoter), a mifepristone-responsive promoter (e.g., a GAL4-E1b promoter), or a coumermycin-responsive promoter. For example, a TRE (e.g., a TRE3G) promoter may include 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: 7. For example, a TRE (e.g., TRE2) promoter can include 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: 23. For example, a TRE (e.g., P tight) promoter can include 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: 24. See, for example, International Publication No. WO 2020 / 069339, entitled "Mutant Reverse Tetracycline Transactivators for Expression of Genes," which was published on April 2, 2020, and is incorporated herein by reference in its entirety.

[0177] In certain embodiments, the inducer can induce expression of a first (e.g., OCT4), a second (e.g., SOX2), a third (e.g., KLF4) nucleic acid (e.g., an engineered nucleic acid), or any combination thereof, from an inducible promoter in the presence of tetracycline (e.g., doxycycline). In certain embodiments, the inducer is a reverse tetracycline-regulated transactivator (rtTA) (e.g., rtTA3, rtTA4, rtTA Advanced, rtTA2S-M2, or any rtTA disclosed herein). In certain embodiments, the inducer is an rtTA and comprises a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to any rtTA disclosed herein. In certain embodiments, the rtTA is rtTA3, which comprises an amino acid sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 11. In certain embodiments, the rtTA is rtTA Advanced, which comprises an amino acid sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 129. In certain embodiments, the rtTA is rtTA4, which comprises a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 13. In certain embodiments, the rtTA is rtTA2S-M2 and comprises a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 15. In certain embodiments, the inducer is a tetracycline-regulated transactivator (tTA) (e.g., any tTA disclosed herein). In certain embodiments, the inducer is a tTA and comprises a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to any tTA disclosed herein.In certain embodiments, the inducer is tTA 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: 138 or 159. In certain embodiments, the inducer is tTA and is encoded by a sequence that 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: 137 or 158.

[0178] In certain embodiments, the inducer is capable of inducing expression of a first nucleic acid (e.g., an engineered nucleic acid) (e.g., OCT4), a second nucleic acid (e.g., an engineered 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). In certain embodiments, the inducer is a tetracycline-regulated transactivator (tTA).

[0179] In certain embodiments, the expression vector of the present disclosure comprises a promoter that is specific to the central nervous system. In some embodiments, the expression vector of the present disclosure comprises a neuron-specific promoter. In some embodiments, the expression vector of the present disclosure comprises a brain-specific promoter. In some embodiments, the expression vector comprises a CaMKIIα promoter. In some embodiments, the CaMKIIα promoter 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: 146, 149, or 154.

[0180] In some embodiments, the expression vector of the present disclosure does not comprise an eye-specific promoter. In some embodiments, the expression vector does not comprise a synapsin-I promoter, does not comprise a CaMKII-gamma promoter, or a combination thereof. In some embodiments, the expression vector does not comprise a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) to SEQ ID NO: 157.

[0181] In certain embodiments, an expression vector of the present disclosure comprises 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: 8 or 143.

[0182] In certain embodiments, the expression vectors of the present disclosure include a separator sequence, which can be useful for generating two distinct amino acid sequences from a single transcript. The separator sequence can encode a self-cleaving peptide (e.g., a 2A peptide including 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: 9 or 118). In certain embodiments, the separator sequence is an internal ribosome entry site (IRES).

[0183] In certain embodiments, the expression vector is a viral vector (e.g., a lentivirus, retrovirus, or adeno-associated virus (AAV) vector). The AAV vectors of the present disclosure generally contain inverted terminal repeats (ITRs) flanking a transgene of interest (e.g., a nucleic acid encoding OCT4, SOX2, KLF4, an inducer, or a combination thereof (e.g., an engineered nucleic acid)). 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).

[0184] In some embodiments, an expression vector (e.g., a viral vector) encoding OCT4, KLF4, and SOX2 comprises a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:121, SEQ ID NO:123, or SEQ ID NO:163. Viral vectors include adeno-associated viral (AAV) vectors, retroviral vectors, lentiviral vectors, and herpesviral vectors. In another aspect, the present disclosure provides a recombinant virus (e.g., lentivirus, adenovirus, retrovirus, herpesvirus, human papillomavirus, alphavirus, vaccinia virus, or adeno-associated virus (AAV)) comprising any of the expression vectors described herein. 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, OCT4, KLF4, and SOX2, or SOX2 and KLF4. In certain embodiments, the recombinant virus encodes OCT4, KLF4, and SOX2. In certain embodiments, the four or more transcription factors encode four or more transcription factors (e.g., OCT4, SOX2, KLF4, and another transcription factor).

[0185] The expression vector containing one or more of the first, second, and third nucleic acids (e.g., engineered nucleic acids) can be any of the expression vectors described herein above. In some embodiments, the first nucleic acid, the second nucleic acid, the third nucleic acid, or any combination thereof, are present on separate expression vectors. In certain embodiments, two of the first nucleic acid, the second nucleic acid, the third nucleic acid, or any combination thereof are present on the same expression vector. In certain embodiments, all three nucleic acids (e.g., engineered nucleic acids) are present on the same expression vector. In certain embodiments, at least two of the first, second, or third nucleic acids (e.g., engineered nucleic acids) are operably linked to the same promoter. In certain embodiments, all three of the first, second, and third nucleic acids (e.g., engineered nucleic acids) are operably linked to the same promoter.

[0186] In some embodiments, the expression vector (e.g., a viral expression vector, including a lentiviral, retroviral, or adeno-associated viral vector) comprises an inducible promoter (e.g., a promoter comprising a tetracycline-responsive element (TRE) comprising a TRE3G sequence, a TRE2 sequence, or a P tight sequence), and the method further comprises administering an inducer (e.g., a chemical, a nucleic acid (e.g., an engineered nucleic acid) (e.g., a nucleic acid encoding an inducer (e.g., an engineered nucleic acid)), a protein, light, or temperature). In some embodiments, pH is used to induce expression of a nucleic acid operably linked to the promoter. In certain embodiments, the chemical capable of modulating the activity of the inducer is tetracycline (e.g., doxycycline). As a non-limiting example, a tetracycline-regulated transactivator (tTA) is an inducer whose activity is inhibited by tetracycline. As a non-limiting example, a reverse tetracycline-regulated transactivator (rtTA) is an inducer whose activity is activated by tetracycline. The inducer (e.g., rtTA or tTA) can be encoded by a fourth nucleic acid (e.g., an engineered nucleic acid) that is the administered nucleic acid. In certain embodiments, the inducer (e.g., a chemical, a nucleic acid (e.g., an engineered nucleic acid) (e.g., a nucleic acid comprising RNA and / or DNA encoding the inducer), a protein, light, a specific pH, or temperature) is introduced simultaneously with the nucleic acid (e.g., an engineered nucleic acid) encoding OCT4, SOX2, and KLF4. In certain embodiments, the inducer (e.g., a chemical, a nucleic acid (e.g., an engineered nucleic acid) (e.g., a nucleic acid comprising RNA and / or DNA encoding the inducer), a protein, light, a specific pH, or temperature) is introduced simultaneously with the nucleic acid (e.g., an engineered nucleic acid) encoding one or more (e.g., two or more, or three or more) transcription factors selected from OCT4; SOX2; KLF4; and any combination thereof. A promoter (e.g., a constitutive promoter including CAG and Ubc, or an inducible promoter) can be operably linked to the nucleic acid (e.g., an engineered nucleic acid) encoding the inducer.In certain embodiments, the promoter operably linked to the nucleic acid (eg, the engineered nucleic acid) encoding the inducer is a tissue-specific promoter.

[0187] In certain embodiments, the nucleic acid encoding the inducer (e.g., an engineered nucleic acid) is present on the same expression vector as at least one of the nucleic acids encoding OCT4, SOX2, KLF4, or a combination thereof (e.g., an engineered nucleic acid). In certain embodiments, the nucleic acid encoding the inducer (e.g., an engineered nucleic acid) is present on a separate expression vector from the nucleic acid encoding OCT4 (e.g., an engineered nucleic acid), the nucleic acid (e.g., an engineered nucleic acid), SOX2, and KLF4 (e.g., an engineered nucleic acid). In certain embodiments, the nucleic acids encoding OCT4, SOX2, and KLF4 (e.g., an engineered nucleic acid) are present on a first expression vector, and the fourth nucleic acid (e.g., an engineered nucleic acid) is present on a second expression vector. In some embodiments, the vector comprises a WPRE sequence and / or an hGH pA terminator sequence.

[0188] In some embodiments, the nucleic acid encoding OCT4, SOX2, KLF4, and / or inducer is not present on a viral vector. In some embodiments, the nucleic acid encoding one or more (for example, two or more or three or more) transcription factors selected from the group consisting of OCT4; SOX2; KLF4; and any combination thereof is not present on a viral vector. In some embodiments, the nucleic acid is delivered without a viral vector. In some embodiments, the delivery of nucleic acid that is not on a viral vector comprises administering naked nucleic acid, electroporation, using nanoparticles, and / or using liposomes.

[0189] The expression vector can be a viral vector (e.g., a lentiviral vector, adenoviral vector, retroviral vector, herpesvirus vector, alphavirus, vaccinia virus, or AAV vector). For example, a first expression vector encoding OCT4, SOX2, and KLF4 can include a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:121, SEQ ID NO:123, or SEQ ID NO:163. In certain embodiments, a fourth nucleic acid (e.g., an engineered nucleic acid) encoding an inducer further includes an SV-40-derived terminator sequence that includes a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:8 or 143.

[0190] In certain embodiments, the inducer is capable of inducing expression from an inducible promoter in the presence of tetracycline (e.g., doxycycline). In certain embodiments, the inducer is rtTA3 (e.g., rtTA3, including rtTA3 having a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 11; rtTA, including rtTA Advanced having a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 129). Advanced, rtTA4, including rtTA4 having a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 13, and rtTA2S-M2, including rtTA2S-M2 having a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 15. In certain embodiments, the method further includes administering tetracycline (e.g., doxycycline) to the cell, tissue, or subject. In certain embodiments, the method includes removing tetracycline (e.g., doxycycline) from the cell, tissue, or subject.

[0191] Recombinant viruses Aspects of the present disclosure provide recombinant viruses (e.g., lentiviruses, alphaviruses, vaccinia viruses, adenoviruses, herpes viruses, retroviruses, or AAVs) that can be useful for delivering transcription factors and / or inducers to cells, tissues, or organs of the central nervous system. The recombinant viruses (e.g., lentiviruses, alphaviruses, vaccinia viruses, adenoviruses, herpes viruses, retroviruses, or AAVs) can carry nucleic acids (e.g., engineered nucleic acids) (e.g., expression vectors) encoding transcription factors (e.g., OCT4, SOX2, KLF4, or any combination thereof) and / or encoding inducers. In some embodiments, the recombinant viruses carry nucleic acids encoding at least two transcription factors selected from OCT4, SOX2, and KLF4 (e.g., OCT4 and SOX2; KLF4 and SOX2; OCT4, KLF4, and SOX2; or OCT4 and KLF4). In some embodiments, the recombinant virus carries nucleic acids encoding at least three transcription factors selected from OCT4, SOX2, and KLF4 (e.g., OCT4, SOX2, and KLF4). In some cases, the recombinant virus of the present disclosure includes a nucleic acid encoding an inducer.

[0192] In certain embodiments, the recombinant virus is a recombinant AAV. In some embodiments, the recombinant AAV has tissue-specific targeting capability, such that the AAV transgene will be specifically delivered to one or more predetermined tissue(s). In some embodiments, the recombinant AAV can target the central nervous system. Generally, the AAV capsid is a relevant factor in determining the tissue-specific targeting capability of AAV. The AAV capsid may contain amino acid sequences derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV PHP.b, and variants thereof. In certain embodiments, the AAV serotype is a variant of AAV9 (e.g., AAV PHP.b). In certain embodiments, the AAV serotype is AAV PHP.eB. Non-limiting examples of tissue specificity of AAV serotypes are provided in Table 1. An "x" indicates that the indicated AAV serotype is capable of delivering the transgene to a particular tissue.

[0193] In some embodiments, the AAV serotype delivers engineered nucleic acids to the central nervous system. In some embodiments, the AAV serotype delivers engineered nucleic acids to the brain. Non-limiting examples of AAV serotypes that deliver engineered nucleic acids to the brain include AAV9, AAV10, AAV.PHP.b, AAV.PHP.eB, AAV.CAP-B10, and AAV.CAP-B22. In some embodiments, the AAV serotype used is not AAV2, not AAV9, or a combination thereof. Without being bound by any particular theory, AAV2 and / or AAV9 may have poor delivery to the brain. For example, see Goertsen et al., Nat Neurosci. 2022 Jan;25(1):106-115, which is incorporated herein by reference in its entirety. [Table 1]

[0194] Recombinant AAV containing specific capsid proteins can be produced using any suitable method. See, for example, U.S. Patent Application Publication No. 2003 / 0138772, incorporated herein by reference. AAV capsid protein sequences are also known in the art. See, for example, WO 2010 / 138263, incorporated herein by reference. Generally, recombinant AAV is produced in a host cell containing the following components: (1) a nucleic acid sequence (e.g., an engineered nucleic acid) encoding an AAV capsid protein or a fragment thereof, (2) a nucleic acid (e.g., an engineered nucleic acid) encoding a functional rep gene, (3) a recombinant AAV vector containing AAV terminal inverted repeats flanking a transgene (e.g., a nucleic acid (e.g., an engineered nucleic acid) encoding OCT4, KLF4, SOX2, or a combination thereof), and (4) helper functions that allow packaging of the recombinant AAV vector into an AAV capsid protein. In some cases, the recombinant AAV vector includes a nucleic acid encoding an inducer. In certain embodiments, helper functions are introduced via helper vectors known in the art (see, e.g., Chan et al. Nat Neurosci. 2017 Aug;20(8):1172-1179).

[0195] In some embodiments, the AAV capsid comprises a targeting moiety for one or more cells of the central nervous system. See, for example, WO2023004367 and WO2022232327. In some embodiments, the AAV capsid is AAV-BI30. See, for example, Krolak et al., Nat Cardiovasc Res. 2022 Apr;1(4):389-400.

[0196] In some cases, a suitable host cell line (e.g., HEK293T cells) can be used to produce the recombinant AAV disclosed herein according to conventional techniques. One or more expression vectors encoding one or more of the above-described components can be introduced into the host cell by exogenous nucleic acid (e.g., engineered nucleic acid), which can be cultured under suitable conditions that allow the production of AAV particles. When needed, a helper vector can be used to facilitate replication, facilitate assembly of AAV particles, or any combination thereof. In certain embodiments, the recombinant AAV vector is present on a nucleic acid (e.g., an engineered nucleic acid) that is separate from other components (e.g., a nucleic acid (e.g., an engineered nucleic acid) sequence encoding an AAV capsid protein or a fragment thereof, a nucleic acid (e.g., an engineered nucleic acid) encoding a functional rep gene, and helper functions that allow packaging of the recombinant AAV vector into an AAV capsid protein. In certain embodiments, the host cell may stably express one or more components required to produce AAV virus. In that case, the remaining components may be introduced into the host cell. The cell culture supernatant may be collected, and the viral particles contained therein may be collected by conventional methodologies.

[0197] In some embodiments, the methods described herein allow for the production of high viral titers (e.g., 2 x 10 per preparation). 12 Particles, 1 x 10 per mL 13 This involves the incorporation of one or more nucleic acids into a virus (e.g., an adeno-associated virus (AAV)) in a cellular or cellular environment (more than a single particle). This may be useful in reversing aging and treating neurological diseases. In some embodiments, the preparation involves the use of 10 T150 flasks of HEK293T cells.

[0198] Methods for activating OCT4, SOX2, and KLF4, alone or in combination, and their replacement Aspects of the present disclosure relate, in some embodiments, to activating OCT4, SOX2, and KLF4, alone or in combination, in cells, tissues, and / or organs of the central nervous system. In some embodiments, OCT4, SOX2, and KLF4, alone or in combination, are activated in the absence of c-Myc activation. The cells, tissues, and / or organs can be in vivo or ex vivo (e.g., in a subject). As used herein, activation encompasses any nucleic acid (e.g., nucleic acid including RNA, including DNA, or any combination thereof), protein, antibody, chemical, or any combination thereof, that can increase the biological activity of a protein of interest (e.g., OCT4, SOX2, and / or KLF4). Biological activity (e.g., gene expression, reprogramming ability, transcription factor activity, etc.) can be measured using any conventional method known in the art. In some embodiments, any nucleic acid (e.g., nucleic acid including RNA, including DNA, or any combination thereof), protein, antibody, chemical, or any combination thereof described herein replaces OCT4, SOX2, and / or KLF4. In some embodiments, any nucleic acid (e.g., nucleic acid including RNA, including DNA, or any combination thereof), protein, antibody, chemical, or any combination thereof described herein replaces OCT4, SOX2, KLF4, or any combination thereof. In some embodiments, any of the nucleic acids (e.g., engineered nucleic acids) encoding inducers, engineered proteins encoding inducers, chemicals that can regulate (e.g., activate or inhibit) the activity of inducers, and / or recombinant viruses encoding inducers described herein are used to activate inducers.

[0199] Activation of OCT4, SOX2, and KLF4 includes increasing the expression (eg, RNA and / or protein expression) of OCT4, SOX2, and KLF4, respectively, alone or in combination. In some embodiments, the expression of OCT4, SOX2, and KLF4 is increased by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% after administration to a cell, tissue, organ, and / or subject of a nucleic acid (e.g., a nucleic acid including RNA, including DNA, or any combination thereof) encoding OCT4, SOX2, and / or KLF4, a protein encoding OCT4, SOX2, and / or KLF4, an antibody capable of activating and encoding OCT4, SOX2, and / or KLF4, a chemical compound capable of activating and encoding OCT4, SOX2, and / or KLF4, or any combination thereof, each alone or in combination, compared to before administration. In some embodiments, the expression of OCT4, SOX2, and KLF4 is increased by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% after administration to a cell, tissue, organ, and / or subject of a nucleic acid (e.g., a nucleic acid including RNA, including DNA, or any combination thereof) encoding OCT4, SOX2, KLF4, or any combination thereof, a protein encoding OCT4, SOX2, KLF4, or any combination thereof, an antibody capable of activating and encoding OCT4, SOX2, KLF4, or any combination thereof, a chemical compound capable of activating and encoding OCT4, SOX2, KLF4, or any combination thereof, or any combination thereof, each alone or in combination, compared to before administration.

[0200] Activation of an inducer includes increasing the expression of the inducer (e.g., RNA and / or protein expression). In some embodiments, the expression of the inducer is increased by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% after administration of a nucleic acid encoding the inducer (e.g., a nucleic acid including RNA, including DNA, or any combination thereof), a protein encoding the inducer, a chemical compound capable of regulating the activity of the inducer, or any combination thereof, to a cell, tissue, organ, and / or subject compared to before administration.

[0201] Expression can be measured by any conventional method known in the art, including quantifying the levels of the protein of interest (e.g., using ELISA and / or Western blot analysis with an antibody that recognizes the protein of interest), or quantifying the levels of RNA (e.g., mRNA) for the gene of interest (e.g., using reverse transcription polymerase chain reaction).

[0202] In addition to the engineered nucleic acids discussed herein, OCT4, SOX2, KLF4 can be activated in cells, tissues, organs, and / or subjects by using engineered proteins, either alone or in combination.For example, the protein encoding OCT4, SOX2, and / or KLF4 can be produced (for example, recombinantly or synthetically) and administered to cells, tissues, organs, and / or subjects by any suitable route.For example, the protein encoding one or more transcription factors selected from the group consisting of OCT4; SOX2; KLF4; and any combination thereof can be produced (for example, recombinantly or synthetically) and administered to cells, tissues, organs, and / or subjects by any suitable route.

[0203] In some embodiments, activating the expression of OCT4; SOX2; KLF4; their substitutions; or any combination thereof from the tetracycline-inducible expression vector comprises administering tetracycline (e.g., doxycycline) to a cell, organ, tissue, or subject. As one skilled in the art would understand, the route of tetracycline administration may depend on the type of cell, organ, tissue, and / or characteristic of the subject. In some embodiments, tetracycline is administered directly to a cell, organ, and / or tissue. By way of non-limiting example, tetracycline can be administered to the subject's eye by any suitable method, including eye drops containing tetracycline, sustained-release devices (e.g., micropumps, particles, and / or drug depots), and medicated contact lenses containing tetracycline. In some embodiments, tetracycline is administered systemically to a subject (e.g., via drinking water or intravenous injection). Tetracycline can be administered topically (e.g., via a cream) or via a subcutaneous pump (e.g., to deliver tetracycline to specific tissues). Tetracyclines may be administered intravenously, intradermally, intra-arterially, intralesionally, intratumorally, intracranially, intra-articularly, intraprostatically, intrapleurally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, intramucosally, pericardially, intraumbilically, intraocularly, orally, topically, locally, systemically, by injection, infusion, continuous infusion, by local perfusion bath directly onto target cells, via a catheter, in a cream, in particles (e.g., nanoparticles, microparticles), in lipid compositions (e.g., liposomes), or by other methods or combinations of any of the foregoing that would be known to one of skill in the art (see, e.g., Remington's Pharmaceutical Sciences (1990), incorporated herein by reference).

[0204] By way of non-limiting example, the engineered protein can be further modified or formulated for delivery to cells, tissues, organs, and / or subjects. For example, a protein transduction domain (i.e., a PTD or cell-penetrating peptide) can be attached to the engineered protein (e.g., OCT4, SOX2, and / or KLF4). By way of non-limiting example, a protein transduction domain (i.e., a PTD or cell-penetrating peptide) can be attached to the engineered protein encoding an inducer. Without being bound by theory, the protein transduction domain facilitates the delivery of cargo (e.g., proteins, nucleic acids, nanoparticles, viral particles, etc.) across the cell membrane. Protein transduction domains include cationic peptides, hydrophobic peptides, and / or cell-specific peptides. For example, see Zhou et al., Cell Stem Cell. 2009 May 8;4(5):381-4; Zahid et al., Curr Gene Ther. 2012 Oct;12(5):374-80.

[0205] In some embodiments, proteins encoding OCT4, SOX2, and / or KLF4, and / or proteins encoding inducers, are formulated into 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 into nanoparticles (e.g., for nuclear delivery). In certain embodiments, the nanoparticles further comprise proteins encoding inducers. For example, chitosan (poly(N-acetylglucosamine)) is a biodegradable polysaccharide that can be used to formulate nanoparticles by several methods. In some embodiments, chitosan polymer nanoparticles are loaded with proteins encoding OCT4, SOX2, and / or KLF4, and / or inducers, and delivered to the nucleus of cells. See, e.g., Tammam et al., Oncotarget. 2016 Jun 21;7(25):37728-37739.

[0206] In some embodiments, the chemicals, antibodies, and / or proteins replace OCT4, SOX2, and / or KLF4. In some embodiments, the chemicals, antibodies, proteins, or any combination thereof replace OCT4, SOX2, KLF4, or any combination thereof (e.g., OCT4 and SOX2; OCT4 and KLF4; KLF4 and SOX2; or KLF4, SOX2, and OCT4). For example, the chemicals, antibodies, and / or proteins can promote the expression of OCT4, SOX2, and / or KLF4. In certain cases, the chemicals, antibodies, and / or proteins can promote the expression of one or more transcription factors selected from OCT4; SOX2; KLF4; and any combination thereof. In some embodiments, the chemicals, antibodies, and / or proteins can activate downstream target genes of OCT4, SOX2, and / or KLF4. In some embodiments, a chemical substance, an antibody, a protein, or any combination thereof can activate downstream target genes of one or more transcription factors selected from the group consisting of OCT4; SOX2; KLF4; and any combination thereof. In some embodiments, when a chemical substance, an antibody, and / or a protein is used together with two other transcription factors to promote cell reprogramming, the chemical substance, the antibody, and / or a protein is said to replace OCT4, SOX2, and / or KLF4. In some embodiments, when a chemical substance, an antibody, a protein, or any combination thereof is used together with two other transcription factors to promote cell reprogramming, the chemical substance, the antibody, a protein, or any combination thereof is said to replace OCT4, SOX2, KLF4, or any combination thereof. For example, cell reprogramming can be determined by measuring gene expression (e.g., the expression of embryonic markers and / or pluripotency markers). In some embodiments, pluripotency markers include AP, SSEA1, and / or Nanog.

[0207] In some embodiments, the antibody is used to activate OCT4, SOX2, and / or KLF4. In some embodiments, the antibody is used to activate one or more transcription factors selected from OCT4, SOX2, KLF4, or any combination thereof. In some embodiments, the antibody does not target OCT4, SOX2, and / or KLF4. In some embodiments, the antibody does not target OCT4, SOX2, KLF4, or any combination thereof. In some embodiments, the antibody increases the expression of OCT4, SOX2, and / or KLF4. In some embodiments, the antibody increases the expression of OCT4, SOX2, KLF4, or any combination thereof. In some embodiments, the antibody does not increase the expression of OCT4, SOX2, and / or KLF4. In some embodiments, the antibody displaces OCT4, SOX2, and / or KLF4. In some embodiments, the antibody does not increase the expression of OCT4, SOX2, KLF4, or any combination thereof. In some embodiments, the antibody replaces OCT4, SOX2, KLF4, or any combination thereof. Any suitable method can be used to identify an antibody that can replace a transcription factor (e.g., OCT4, SOX2, and / or KLF4). Any suitable method can be used to identify an antibody that can replace a transcription factor (e.g., OCT4, SOX2, KLF4, or any combination thereof). For example, see Blanchard et al., Nat Biotechnol. 2017 Oct;35(10):960-968.

[0208] In some embodiments, another protein (e.g., a nucleic acid encoding a protein or a polypeptide encoding a protein) can be used to replace OCT4, SOX2, and / or KLF4. In some embodiments, another protein (e.g., a nucleic acid encoding a protein or a polypeptide encoding a protein) can be used to replace OCT4, SOX2, KLF4, or a combination thereof. For example, OCT4 can be replaced with Tet1, NR5A-2, Sall4, E-cadherin, NKX3-1, or any combination thereof. In some embodiments, OCT4, SOX2, and / or KLF4 can be replaced with NANOG and / or TET2. In some embodiments, OCT4, SOX2, KLF4, or any combination thereof can be replaced with NANOG and / or TET2. For example, see Nat Cell Biol. 2018 Aug;20(8):900-908; Gao et al., Cell Stem Cell. 2013 Apr 4;12(4):453-69. Nanog and Lin28 can replace Klf4. See, e.g., Yu et al., Science. 318, 1917-1920, 2007). In some embodiments, OCT4, SOX2, and / or KLF4 are replaced with Tet3 (tet methylcytosine dioxygenase 3). In some embodiments, OCT4, SOX2, KLF4, or any combination thereof are replaced with Tet3 (tet methylcytosine dioxygenase 3). In some embodiments, the nucleic acid encoding Tet1 DNA demethylase comprises a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to NM_030625.3 or NM_001253857.2. In some embodiments, the amino acid sequence encoding the Tet1 DNA demethylase comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to NP_085128.2 or NP_001240786.1.In some embodiments, the nucleic acid encoding the Tet2 DNA demethylase comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to NM_001127208.2, NM_001040400.2, NM_001346736.1, or NM_017628.4. In some embodiments, the amino acid encoding the Tet2 DNA demethylase comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to NP_060098.3, NP_001035490.2, NP_001333665.1, or NP_001120680.1. In some embodiments, the nucleic acid encoding the Tet3 DNA demethylase comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to NM_001287491.2, NM_001347313.1, NM_183138.2, or NM_001366022.1. In some embodiments, the amino acid encoding the Tet3 DNA demethylase comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to NP_001274420.1, NP_001334242.1, NP_898961.2, or NP_001352951.1. Tet1, Tet2, and / or Tet3 can be derived from any species. In some embodiments, Tet1, Tet2, and / or Tet3 are truncated versions of their wild-type counterparts. As a non-limiting example, Tet1, Tet2, and / or Tet3 are N-terminally truncated and catalytically active compared to their wild-type Tet1, Tet2, and / or Tet3 counterparts. In some embodiments, Tet1, Tet2, and / or Tet3 comprise only the catalytic domain of Tet1, Tet2, and / or Tet3. In some embodiments, Tet1, Tet2, and / or Tet3 comprise the catalytic domain of Tet1, Tet2, Tet3, or any combination thereof.Non-limiting examples of functional truncated Tet1 can be found in Hrit et al., Elife. 2018 Oct 16;7. Pii: e34870.

[0209] Additional methods for replacing OCT4, SOX2, and / or KLF4 to promote cellular reprogramming are known in the art. For example, Heng et al., Cell Stem Cell 6, 167-174 (2010); Eguchi et al., Proc. Natl Acad. Sci. USA 113, E8257-E8266 (2016); Gao et al., Cell Stem Cell 12, 453-469 (2013); Long et al., Cell Res. 25, 1171-1174 (2015); Hou et al., Science 341, 651-654 (2013); Redmer et al., EMBO Rep. 12, 720-726 (2011); Tan et al., J. Biol. Chem. 290, 4500-4511 (2014); Anokye-Danso et al. al., Cell Stem Cell 8, 376-388 (2011); Miyoshi et al., Cell Stem Cell 8, 633-638 (2011); Shu et al., Cell 153, 963-975 (2013); Yu, J. et al., Science 318, 1917-1920 (2007).

[0210] In some embodiments, the chemical substance replaces OCT4, SOX2, and / or KLF4 (for example, it can be used in place of OCT4, SOX2, and / or KLF4 in conjunction with two other transcription factors to promote cell reprogramming). In some embodiments, the chemical substance replaces OCT4, SOX2, KLF4, or any combination thereof (for example, it can be used in place of OCT4, SOX2, KLF4, or any combination thereof in conjunction with two other transcription factors to promote cell reprogramming). For example, SOX2 can be replaced by CHIR, FSK, or 616452. OCT4 can be replaced by DZNep. Since Sall4 can be used to replace OCT4 as mentioned above, any compound that replaces Sall4 can also be used to replace OCT4. For example, CHIR, FSK, and 616452 can be used to replace Sall4. Nanog can be replaced by 2i medium. See, e.g., Hou et al., Science. 2013 Aug 9;341(6146):651-4. See also, e.g., Zhao et al., Cell. 2015 Dec 17;163(7):1678-91.

[0211] In some embodiments, chemical reprogramming comprises using chemicals that reduce the toxicity of the chemicals that induce reprogramming.Non-limiting examples of chemicals that reduce the toxicity of chemical reprogramming include ROCK inhibitors (for example, Y27632 and Fasudil) and P38 MAPK inhibitors (for example, SB203580 and BIRB796).For example, see Li et al., Cell Stem Cell.2015 Aug 6;17(2):195-203.

[0212] OCT4, KLF4, SOX2, substitution, or any combination thereof can be activated (e.g., expression can be induced) in combination with activating a reprogramming enhancer and / or inhibiting a reprogramming barrier. Reprogramming enhancers can be activated using any suitable method known in the art. This includes overexpressing the enhancer, increasing the expression of the endogenous gene encoding the enhancer (e.g., using CRISPR technology), using chemicals and / or antibodies to increase the biological activity of the enhancer, and using chemicals and / or antibodies to promote the expression of the enhancer. Reprogramming barriers can be inhibited using any suitable method known in the art. This includes knocking down the expression of inhibitors (e.g., using siRNA, miRNA, shRNA), knocking out endogenous copies of inhibitors (e.g., using CRISPR technology, TALEN, zinc finger nucleases, etc.), using chemicals and / or antibodies to reduce the biological activity of inhibitors, and using chemicals and / or antibodies to reduce the expression of inhibitors.

[0213] Non-limiting examples of reprogramming enhancers and barriers are provided in Table 2. See also, for example, Ebrahimi, Cell Regen (Lond). 2015 Nov 11;4:10, which is incorporated herein by reference in its entirety. [Table 2]

[0214] Additional reprogramming enhancers that can be activated in combination with the activation of OCT4, KLF4, SOX2, their substitution, or any combination thereof include histone lysine demethylases (e.g., KDM2, KDM3, and KDM4).Histone lysine demethylases can be activated by being overexpressed in cells, tissues, organs, and / or subjects.Chemical activators of histone lysine demethylases are also encompassed by the present disclosure.For example, vitamin C can be used to activate KDM3 and / or KDM4.

[0215] In some embodiments, OCT4, SOX2, KLF4, substitutions thereof, or any combination thereof are activated in conjunction with the activation of C / EBPα and Tfcp2l 1. Without being bound by theory, C / EBPα and Tfcp2l 1, together with Klf4, may drive enhancer demethylation and activation mediated by Tet2 during reprogramming.

[0216] In some embodiments, OCT4, SOX2, KLF4, their substitutions, or any combination thereof are activated in cells, tissues, organs, and / or subjects in combination with cytokines that facilitate reprogramming.IL6 is a non-limiting example of cytokines.For example, see Mosteiro et al., Science. 2016 Nov 25;354(6315), which is incorporated herein by reference in its entirety.

[0217] In some embodiments, OCT4, SOX2, KLF4, their substitution, or any combination thereof is activated in cells, tissues, organs, and / or subjects in combination with miRNA activation (for example, miRNA administration and / or miRNA expression).For example, miRNA that promotes cell cycle progression can be introduced into cells, tissues, organs, and / or subjects.Non-limiting examples of miRNA that promote cell cycle progression include miR302-367, miR371-373, miR-200b, miR-200c, miR-205, miR290-295, miR-93, miR-106, and miR135b.

[0218] By way of non-limiting example, neural regeneration can be enhanced by combining activation of OCT4, SOX2, KLF4, their substitution, or any combination thereof with activation of enhancers, including, but not limited to, overexpression of a KLF family member (e.g., KLF7), overexpression of c-Myc, activation of STAT3, overexpression of SOX11, overexpression of Lin28, overexpression or delivery of soluble proteins encoding insulin-like growth factor 1 (IGF1) and osteopontin (OPN), and activation of B-RAF (e.g., introduction of a gain-of-function mutation). For example, Blackmore et al., Proc Natl Acad Sci US A. 2012 May 8;109(19):7517-22;Belin et al., Neuron. 2015 May 20;86(4):1000-1014;Bareyre et al., Proc Natl Acad Sci USA. 2011 Apr 12;108(15):6282-7;Norsworthy et al., Neuron. 2017 Jun 21;94(6):1112-1120.e4;Wang et al., Cell Rep. 2018 Sep 4;24(10):2540-2552.e6;Liu et al., Neuron. 2017 Aug 16;95(4):817-833;O'Donovan et al., J Exp Med, 2014. 211(5): pp. 801-14, which is incorporated herein by reference in its entirety.

[0219] In some embodiments, OCT4, SOX2, KLF4, their substitution, or any combination thereof is activated in cells, tissues, organs, and / or subjects in combination with the suppression or knockdown of reprogramming barriers.Non-limiting examples of reprogramming barriers include Chaf1a, Chaf1b, Ube2i, sumo2, and / or Nudt21.For example, see Brumbaugh et al., Cell.2018 Jan 11;172(1-2):106-120.e21;Cheloufi et al., Nature.2015 Dec 10;528(7581):218-24;and Borkent et al., Stem Cell Reports,2016.6(5):p.704-716, which are incorporated herein by reference in their entirety.

[0220] As a non-limiting example, the reprogramming barrier may be a DNA methyltransferase (DNMT), which can be inhibited to promote tissue, cell, and / or organ reprogramming. Most DNA methyltransferases use S-adenosyl-L-methionine as the methyl donor. DNMTs can be from any species. There are at least three different types of methyltransferases: M6A methyltransferase can methylate the amino group at the c-6 position of adenine on DNA (see, e.g., Enzyme Commission (EC) No. 2.1.1.72); m4C methyltransferase can produce N4-methylcytosine (see, e.g., Enzyme Commission (EC) No. 2.1.1.113); and M5C methyltransferase can produce C5-methylcytosine (see, e.g., Enzyme Commission (EC) No. 2.1.1.37).

[0221] Non-limiting examples of mammalian DNA methyltransferases (DNMTs) include DNMT1 and its isoforms DNMT1b and DNMT1o (oocyte-specific), DNMT3a, DNMT3b, and DNMT3L. GenBank Accession Nos. NM_001130823.3 (isoform a), NM_001318730.1 (isoform c), NM_001318731.1 (isoform d), and NM_001379.3 (isoform b) are non-limiting examples of nucleotide sequences encoding human DNMT1. Nucleic acids encoding DNMT1 can include sequences that are at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) to the sequences submitted under GenBank Accession Nos. NM_001130823.3 (isoform a), NM_001318730.1 (isoform c), NM_001318731.1 (isoform d), and / or NM_001379.3 (isoform b). GenBank Accession Nos. NP_001124295.1 (isoform a), NP_001305659.1 (isoform c), NP_001305660.1 (isoform d), and NP_001370.1 (isoform b) are non-limiting examples of amino acid sequences encoding human DNMT1. An amino acid sequence encoding DNMT1 can include a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to the sequence submitted under GenBank Accession Nos. NP_001124295.1 (isoform a), NP_001305659.1 (isoform c), NP_001305660.1 (isoform d), and / or NP_001370.1 (isoform b). Nucleic acids encoding human DNMT3A include GenBank Accession Nos. NM_001320892.1, NM_001320893.1, NM_022552.4, NM_153759.3, NM_175629.2, and NM_175630.1.Nucleic acids encoding DNMT3A can be at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to the sequences submitted under GenBank Accession Nos. NM_001320892.1, NM_001320893.1, NM_022552.4, NM_153759.3, NM_175629.2, and / or NM_175630.1. Amino acid sequences encoding human DNMT3A include GenBank Accession Nos. NP_001307821.1, NP_001307822.1, NP_072046.2, NP_715640.2, NP_783328.1, and NP_783329.1. The amino acid sequence encoding DNMT3A can be at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to the sequence submitted under GenBank Accession Nos. NP_001307821.1, NP_001307822.1, NP_072046.2, NP_715640.2, NP_783328.1, and / or NP_783329.1. Nucleic acids encoding human DNMT3B include GenBank Accession Nos. NM_001207055.1, NM_001207056.1, NM_006892.3, NM_175848.1, NM_175849.1, and NM_175850.2. Nucleic acids encoding DNMT3B can be at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to the sequences submitted under GenBank Accession Nos. NM_001207055.1, NM_001207056.1, NM_006892.3, NM_175848.1, NM_175849.1, and / or NM_175850.2. Amino acid sequences encoding human DNMT3B include GenBank Accession Nos. NP_001193984.1, NP_001193985.1, NP_008823.1, NP_787044.1, NP_787045.1, and NP_787046.1.The amino acid sequence encoding DNMT3B can be at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to the sequence submitted under GenBank Accession Nos. NP_001193984.1, NP_001193985.1, NP_008823.1, NP_787044.1, NP_787045.1, and / or NP_787046.1. Nucleic acids encoding human DNMT3L include those in GenBank Accession Nos. NM_013369.3 and NM_175867.2. Nucleic acids encoding DNMT3L can be at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to the sequence submitted under GenBank Accession Nos. NM_013369.3 and / or NM_175867.2. Amino acid sequences encoding human DNMT3L include those in GenBank Accession Nos. NP_037501.2 and NP_787063.1. Amino acid sequences encoding DNMT3L can be at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to the sequence submitted under GenBank Accession Nos. NP_037501.2 and / or NP_787063.1.

[0222] DNMTs can be inhibited using any suitable method known in the art. Suitable methods include knocking down DNMT mRNA, genetically knocking out DNMTs, and using DNMT inhibitors (e.g., chemical inhibitors). DNMT inhibitors are being investigated in clinical trials (e.g., phase III clinical trials) in the United States and elsewhere. Non-limiting examples of DNMT inhibitors include VIDAZA™ (azacitidine) (e.g., for the treatment of myelodysplastic syndrome and acute myeloid leukemia (AML)), DACOGEN™ (decitabine) (e.g., for the treatment of AML and chronic myeloid leukemia (CML)), and guadecitabine (SGI-110) (e.g., for the treatment of AML). In 2012, the European Union approved DACOGEN™ (decitabine) for use in patients with AML.

[0223] DNMTs can be inhibited by inhibiting DNMT stabilizers. Suitable methods for inhibiting DNMT stabilizers include knocking down mRNA encoding the stabilizer, gene knockout of the gene encoding the stabilizer, and using inhibitors (e.g., chemical inhibitors). As a non-limiting example, KDM1a, also known as Lsd1 or Aof2, is a stabilizer of DNMT1. For example, see Wang et al., Nat Genet. 2009 Jan;41(1):125-9. In some embodiments, KDM1a expression is knocked down using shRNA disclosed herein or known in the art. In some embodiments, KDM1a is inhibited to prevent damage induced by DNMT hypermethylation, which can be useful for promoting reprogramming.

[0224] In some embodiments, histone methyltransferase is a reprogramming barrier and is inhibited to facilitate cell, tissue, and / or organ reprogramming.Histone methyltransferase can be inhibited by any suitable method, including the use of chemical inhibitors.For example, 3-deazaneplanocin A (Dznep), epz004777, and BIX-01294 are examples of histone methyltransferase inhibitors.

[0225] In some embodiments, the reprogramming barrier is histone deacetylase (HDAC), and HDAC is inhibited to facilitate the reprogramming of cells, tissues, and / or organs.Non-limiting examples of HDAC inhibitors include valproic acid (VPA), trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), sodium butyrate (SB), belinostat (PXD101), panobinostat (LBH589), xinostat (JNJ-26481585), abexinostat (PCI-24781), divinostat (ITF2357), resminostat (4SC-201), phenylbutyrate (PBA), depsipeptide (romidepsin), entinostat (MS-275), mocetinostat (MGCD103), and tubastatin A (TBA).

[0226] In some embodiments, the reprogramming barrier is NF-κB, which is inhibited to facilitate cell, tissue, and / or organ reprogramming. Non-limiting examples of NF-κB inhibitors include BAY11-7082, TPCA1, and p65 siRNA. See, for example, the NF-κB Small Molecule Guide compiled by Abcam, which can be found on the Abcam website ( www.abcam.com / reagents / nf-kb-small-molecule-guide ) is available.

[0227] In some embodiments, the reprogramming barrier is a cytokine secreted by senescent cells, wherein the cytokine is inhibited to facilitate cell, tissue, and / or organ reprogramming. Non-limiting examples of cytokine inhibitors include anti-TNFα (Mahmoudi et al., Biorxiv, 2018) and drugs that kill senescent cells, including navitoclax.

[0228] In some embodiments, reprogramming barrier is microRNA (miRNA), and microRNA is inhibited to facilitate cell, tissue and / or organ reprogramming.Non-limiting examples of reprogramming barrier microRNA include miR Let-7 and miR-34.Without being bound by any particular theory, miR Let-7 inhibits cell cycle, so inhibiting miR Let-7 can increase the efficiency of reprogramming, and miR-34 inhibits the translation of p53, so inhibiting miR-34 can facilitate reprogramming.

[0229] In some embodiments, OCT4, SOX2, KLF4, their substitution, or any combination thereof is activated in cells, tissues, organs, and / or subjects in combination with the inhibition of PTEN, SOCS3, RhoA, and / or ROCK to enhance neural regeneration. In some embodiments, PTEN is deleted, SOCS3 is deleted, RhoA is knocked down, and / or ROCK is knocked down in cells, tissues, organs, and / or subjects. For a description of inhibition of PTEN, SOCS3, RhoA, and / or ROCK, see, e.g., Park et al., Science. 2008 Nov 7;322(5903):963-6; Smith et al., Neuron. 2009 Dec 10;64(5):617-23; Koch et al., Front Cell Neurosci. 2014 Sep 5;8:273; Koch et al., Cell Death Dis. 2014 May 15;5:e1225. Each reference is incorporated herein by reference in its entirety.

[0230] In some embodiments, OCT4, SOX2, KLF4, their substitutions, or any combination thereof are activated in cells, tissues, organs, and / or subjects in combination with neural electrical stimulation (e.g., high-contrast visual stimulation) to promote neural regeneration. For a description of high-contrast visual stimulation, see, for example, Lim et al., Nat Neurosci. 2016 Aug;19(8):1073-84. This reference is incorporated herein by reference in its entirety.

[0231] In some embodiments, OCT4, SOX2, KLF4, substitutions thereof, or any combination thereof are activated in cells, tissues, organs, and / or subjects in combination with gamma-band light stimulation to promote neural regeneration. For a description of gamma-band light stimulation, see, for example, McDermott et al., J Alzheimers Dis. 2018;65(2):363-392. This reference is incorporated herein by reference in its entirety.

[0232] In some embodiments, OCT4, SOX2, and / or KLF4 are activated in the central nervous system. In some embodiments, the central nervous system does not include the retina. In some embodiments, the central nervous system does not include the eye (e.g., the retina, uvea, pupil, lens, cornea, and / or sclera). In some embodiments, OCT4, SOX2, and / or KLF4 are activated in the brain. In some embodiments, OCT4, SOX2, and / or KLF4 are activated (e.g., expression of one or more of these transcription factors is induced) for one year or less (e.g., less than 11 months, less than 10 months, less than 9 months, less than 8 months, less than 7 months, less than 6 months, less than 5 months, less than 4 months, less than 3 months, less than 2 months, or less than 1 month). Non-limiting methods for controlling the induction of gene expression include the use of Tet-On or Tet-Off gene expression systems. For example, gene expression in the Tet-On system can be repressed by removing tetracycline, and gene expression in the Tet-Off system can be repressed by adding tetracycline.

[0233] Engineered cells, tissues, organs, and animals A further aspect of the present disclosure relates to engineered cells, tissues, or organs of the central nervous system, comprising any of the compositions disclosed herein, any of the expression vectors disclosed herein, or any of the recombinant viruses disclosed herein.Methods for producing engineered cells, tissues, and organs of the central nervous system are also encompassed by the present disclosure.Engineered cells can be useful, for example, in cell-based therapies (e.g., stem cell therapy). Stem cell therapies are currently undergoing clinical trials (see, e.g., David Cyranoski, Nature 557, 619-620 (2018)), but toxicity (e.g., off-target toxicity) is a concern. Without being bound by theory, the engineered cells of the present disclosure (e.g., cells engineered using AAV vectors encoding OCT4, KLF4, and / or SOX2, and / or inducers) may have lower toxicity because AAV does not integrate into the host cell genome and the use of the inducible system described herein to control expression of OCT4, KLF4, and / or SOX2 may allow for precise control of gene expression (e.g., amount and timing).

[0234] Any of the nucleic acids (e.g., engineered nucleic acids) (e.g., expression vectors) capable of inducing OCT4, KLF4, and / or SOX2 expression, the engineered proteins described herein, chemicals that activate (e.g., induce expression of) OCT4, KLF4, and / or SOX2, antibodies that activate (e.g., induce expression of) OCT4, KLF4, and / or SOX2, and / or recombinant viruses described herein (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV), alone or in combination, can be introduced into a host cell, host tissue, or organ to generate an engineered cell, engineered tissue, or engineered organ. Nucleic acids (e.g., engineered nucleic acids) capable of inducing expression of OCT4; KLF4; SOX2; or any combination thereof (e.g., expression vectors), engineered proteins described herein, chemicals that activate (e.g., induce expression of) OCT4; KLF4; SOX2; or any combination thereof, antibodies that activate (e.g., induce expression of) OCT4; KLF4; SOX2; or any combination thereof, and / or any of the recombinant viruses described herein (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV), alone or in combination, can be introduced into host cells, host tissues, or organs to generate engineered cells, engineered tissues, or engineered organs. In some embodiments, a nucleic acid encoding an inducer (e.g., an engineered nucleic acid), an engineered protein encoding an inducer, a chemical capable of modulating (e.g., activating or inhibiting) the activity of the inducer, and / or a recombinant virus encoding an inducer is also introduced into a host cell, host tissue, or organ to generate an engineered cell, engineered tissue, or engineered organ.

[0235] In some embodiments, a viral vector (e.g., an AAV vector comprising a vector having a TRE promoter operably linked to a nucleic acid encoding OCT4, KLF4, and SOX2) is packaged into a virus having an AAV-DJ capsid. In some embodiments, the AAV-DJ capsid increases the transduction efficiency of cultured cells compared to cells without the AAV-DJ capsid. In some embodiments, an AAV virus encoding OSK is administered to a cell. In some embodiments, an AAV virus encoding an inducer (e.g., an AAV-DJ virus) or a protein encoding an inducer is administered to the same cell. In some embodiments, the neural cell is a brain cell.

[0236] In some embodiments, the engineered cells are neurons. Neurons for use in the methods described herein can be any type of neuron. Neurons used in the methods can be from the central nervous system (CNS) (e.g., neurons from the brain or spinal cord) or the peripheral nervous system (PNS) (e.g., neurons from the somatic, autonomic, or enteric nervous systems). Neurons can be afferent neurons, efferent neurons, or interneurons. In some embodiments, the neurons used are of the type found in the brain, such as cortical neurons, cerebellar neurons, thalamic neurons, hippocampal neurons, hypothalamic neurons, corpus granulosus neurons, neurons from the basal ganglia (e.g., striatal neurons, neurons of the substantia nigra, or neurons of the globus pallidus), amygdala neurons, interneurons, or brainstem neurons. In some embodiments, the neurons are cortical neurons. In some embodiments, the neurons are not neurons from the eye (e.g., retinal ganglion cells). In some embodiments, the neuron is defined by its neurotransmitter (e.g., the neuron is a dopaminergic neuron, a cholinergic neuron, a GABAergic neuron, a glycinergic neuron, a glutamatergic neuron, or a serotonergic neuron). In some embodiments, the neuron is a motor neuron. In some embodiments, the neuron is a spinal motor neuron or a spinal interneuron. In some embodiments, the neuron is a cranial nerve or cranial ganglion. In some embodiments, the neuron is a sensory neuron from the PNS (e.g., a dorsal root ganglion (DRG) neuron, an autonomic ganglion neuron, e.g., a superior cervical ganglion (SCG) neuron), or an enteric neuron, e.g., a myenteric or submucosal neuron or ganglion). The neuron can be myelinated or unmyelinated.

[0237] Neurons for use in the methods described herein can be derived from any source. In some embodiments, neurons are primary neurons isolated from human subjects (for example, neurons isolated from excised brain tissue or tissue or organs removed during surgery). In some embodiments, neurons are primary neurons isolated from model organisms, such as mice, rats, guinea pigs, hamsters, cats, dogs, cows, horses, deer, elk, bison, bulls, camels, llamas, rabbits, sheep, goats, or non-human primates. For example, neurons can be cortical neurons, dorsal root ganglion neurons, or superior cervical ganglion neurons isolated from rats or mice. Neurons can be isolated from genetically modified organisms (for example, transgenic mice in which neurons are genetically modified to express fluorophores, knock out or overexpress genes of interest, or express constructs that allow the inducible control of neuronal activity or gene expression). In some embodiments, neurons are generated from stem cells, e.g., induced pluripotent stem cells (iPSCs, e.g., human iPSCs), embryonic stem cells (ESCs), neural stem cells (NSCs), adult stem cells (e.g., somatic stem cells or tissue-specific stem cells), hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, or germline stem cells. For example, the neurons can be human iPSC-derived motor neurons, human iPSC-derived cortical neurons, human iPSC-derived hippocampal neurons, or iPSC-derived dopaminergic neurons. In some embodiments, the neurons are from a neuronal cell line. In some embodiments, the cells are excitatory neurons. In some embodiments, the differentiated cells are used for transplantation purposes. In some embodiments, the engineered cells are cultured to generate engineered tissues. In some embodiments, the engineered cells are cultured to generate engineered organs.

[0238] Any of the engineered cells, tissues, or organs of the present disclosure can be present in an animal. For example, aspects of the present disclosure provide animals that contain, alone or in combination, any of the nucleic acids (e.g., engineered nucleic acids) (e.g., expression vectors) that can induce OCT4, KLF4, and / or SOX2 expression, the engineered proteins described herein, chemicals that activate (e.g., induce the expression of) OCT4, KLF4, and / or SOX2, antibodies that activate (e.g., induce the expression of) OCT4, KLF4, and / or SOX2, and / or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV) described herein. In some embodiments, the animal is a mouse, rat, guinea pig, hamster, cat, dog, cow, horse, deer, elk, bison, bull, camel, llama, rabbit, sheep, goat, or non-human primate. In some embodiments, the animal is a transgenic animal.

[0239] In some embodiments, the animal comprises a nucleic acid for use in the central nervous system, comprising a first nucleic acid (e.g., an engineered nucleic acid) encoding OCT4, a second nucleic acid (e.g., an engineered nucleic acid) encoding SOX2, and a third nucleic acid (e.g., an engineered nucleic acid) encoding KLF4, alone or in combination, in the absence of an exogenous nucleic acid (e.g., an engineered nucleic acid) capable of expressing c-Myc. In some embodiments, the nucleic acid encoding OCT4, SOX2, and / or KLF4 comprises an inducible promoter operably linked to any one of the first, second, or third nucleic acids or a combination thereof. In some embodiments, the inducible promoter is responsive to tetracycline. In some embodiments, the animal further comprises a nucleic acid encoding an inducer. In some embodiments, the nucleic acid encoding the inducer comprises a CAMKIIα promoter operably linked to a nucleic acid sequence encoding the inducer. In some embodiments, the inducer comprises a tetracycline transactivator (tTA) and / or a reverse tetracycline-regulated transactivator (rtTA). In some embodiments, the animal is a mouse, and further has overexpression of APP and PSEN1 with five familial AD mutations (APP KM670 / 671NL, APP I716V, APP V717I, PSEN1 M146L, PSEN1 L286V) under the control of Thy1 minigene.For example, see the description of the 5XFAD transgenic mouse in Oakley et al., J Neurosci. 2006;26:10129-10140 and Forner et al., Sci Data. 2021.

[0240] In some embodiments, the animal is a transgenic mouse comprising nucleic acids of the Col1a1 locus encoding OCT4, KLF4, and SOX2, wherein the nucleic acid sequences encoding OCT4, KLF4, and SOX2 are operably linked to a TetO promoter. In some embodiments, the animal further comprises a nucleic acid encoding rtTA-M2, wherein the nucleic acid encoding rtTA-M2 is operably linked to a CAMKIIα promoter. See, for example, Figure 4A.

[0241] composition Aspects of the present disclosure provide a composition for use in rejuvenating a cell, tissue, or organ, comprising: (a) a factor that induces OCT4 expression; (b) a factor that induces SOX2 expression; and (c) a factor that induces KLF4 expression, wherein the composition does not comprise a factor that induces c-MYC expression, and wherein the cell, tissue, or organ is a central nervous system cell, central nervous system tissue, or central nervous system organ, optionally, wherein the central nervous system does not include the eye, optionally, wherein the central nervous system does not include the retina, uvea, pupil, lens, cornea, and / or sclera, and optionally, wherein the cell, tissue, or organ is a brain cell, brain tissue, or brain.

[0242] In some embodiments, the brain cells are neurons.

[0243] In some embodiments, the neuron is an excitatory neuron.

[0244] In some embodiments, the brain tissue is neural tissue.

[0245] In some embodiments, the cell, tissue, or organ is in a subject, optionally wherein the composition is administered to a subject in need thereof, optionally wherein the subject has a neurological disorder.

[0246] In some embodiments, the composition does not induce OCT4, SOX2, or KLF4 expression in the retina.

[0247] In some embodiments, the composition does not induce OCT4, SOX2, or KLF4 expression in the eye (eg, the retina, uvea, pupil, lens, cornea, and / or sclera).

[0248] In some embodiments, each agent is independently a nucleic acid, a small molecule, or a polypeptide, optionally wherein the polypeptide is an antibody.

[0249] In some embodiments, the at least one agent comprises a nanoparticle.

[0250] In some embodiments, the at least one agent is encapsulated in at least one nanoparticle.

[0251] In some embodiments, the nucleic acid is DNA or RNA.

[0252] In some embodiments, the DNA is plasmid DNA.

[0253] In some embodiments, the RNA is mRNA.

[0254] In some embodiments, the factor that induces OCT4 expression is an engineered nucleic acid encoding OCT4.

[0255] In some embodiments, the factor that induces SOX2 expression is an engineered nucleic acid encoding SOX2.

[0256] In some embodiments, the factor that induces KLF4 expression is an engineered nucleic acid encoding KLF4.

[0257] In some embodiments, the factor that induces OCT4 expression is an engineered nucleic acid encoding OCT4, the factor that induces SOX2 expression is an engineered nucleic acid encoding SOX2, and the factor that induces KLF4 expression is an engineered nucleic acid encoding KLF4.

[0258] In some embodiments, the engineered nucleic acid is present on one or more expression vectors.

[0259] In some embodiments, the engineered nucleic acids are present on the same expression vector.

[0260] In some embodiments, one or more expression vectors include an inducible promoter operably linked to any one or combination of the engineered nucleic acids.

[0261] In some embodiments, the promoter is a TRE3G, TRE2 promoter, or a P tight promoter.

[0262] In some embodiments, the promoter comprises a tetracycline responsive element (TRE).

[0263] In some embodiments, the expression vector comprises an hGH pA terminator sequence, optionally wherein the hGH pA terminator sequence comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 139, 148, 153, 156, or 161.

[0264] In some embodiments, the expression vector comprises a WPRE sequence.

[0265] In some embodiments, the expression vector comprises a self-cleaving peptide.

[0266] In some embodiments, the self-cleaving peptide is a 2A peptide, optionally wherein the 2A peptide sequence comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 118 and / or is encoded by a nucleic acid that comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 144.

[0267] In some embodiments, the expression vector comprises inverted terminal repeats (ITRs) flanking the first nucleic acid, the second nucleic acid, the third nucleic acid, or a combination thereof, wherein the distance between the ITRs is 4.7 kb or less.

[0268] In some embodiments, the composition further comprises an inducer, or wherein the method further comprises administering an inducer to said subject.

[0269] In some embodiments, the inducer comprises tetracycline, a tetracycline transactivator (tTA), and / or a reverse tetracycline-regulated transactivator (rtTA), optionally wherein tTA comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 138 or 159, and optionally wherein tTA comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 137 or 158. %, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 11, 129, 13, or 15, and optionally, wherein rtTA is encoded by a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 10, 12, 14, or 128.

[0270] In some embodiments, the tetracycline is doxycycline.

[0271] In some embodiments, the composition comprises an expression vector having an engineered nucleic acid encoding tTA and / or rtTA, optionally wherein the engineered nucleic acid encoding tTA and / or rtTA comprises a WPRE sequence and / or an hGH pA sequence, optionally wherein the WPRE sequence is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 21, 135, 147, 152, 155, or 160, and / or the hGH pA sequence is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 139, 148, 153, 156, or 161.

[0272] In some embodiments, the expression vector encoding tTA and / or rtTA is the same expression vector as the engineered nucleic acid encoding OCT4, SOX2, and / or KLF4 or is a different expression vector.

[0273] In some embodiments, the rtTA is rtTA3, rtTA Advanced, rtTA2S-M2, or rtTA4, optionally wherein the rtTA comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to a sequence selected from the group consisting of SEQ ID NOs: 11, 13, 15, and 129.

[0274] In some embodiments, at least one expression vector is a viral vector, optionally wherein the at least one expression vector is packaged into a recombinant virus.

[0275] In some embodiments, the viral vector is a lentiviral, retroviral, adenoviral, alphavirus, vaccinia virus, human papillomavirus, or adeno-associated virus (AAV) vector.

[0276] In some embodiments, the AAV vector is packaged into an AAV-PHP.eB, AAV-PHP.b, AAV.CAP-B10, or AAV.CAP-B22 virus.

[0277] In some embodiments, the AAV vector is not AAV2 or AAV9.

[0278] In some embodiments, the subject is a human or non-human mammal.

[0279] In some embodiments, the expression vector having an engineered nucleic acid encoding a tTA and / or rtTA comprises a promoter operably linked to the nucleic acid encoding the tTA and / or rtTA.

[0280] In some embodiments, the promoter operably linked to the engineered nucleic acid encoding tTa or rtTA is a ubiquitous promoter.

[0281] In some embodiments, the ubiquitous promoter is UBC, CMV, PGK1, CAG, optionally wherein the ubiquitous promoter comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to 48, 132, 130, 136, or 162.

[0282] In some embodiments, the promoter operably linked to the engineered nucleic acid encoding tTa or rtTA is a neuron-specific promoter.

[0283] In some embodiments, the neuron-specific promoter is CaMKIIα, optionally wherein the CaMKIIα promoter comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 146, 149, or 154.

[0284] In some embodiments, the promoter operably linked to the engineered nucleic acid encoding tTA and / or rtTA is not a synapsin-I promoter, is not a CaMKII-gamma promoter, or a combination thereof, optionally wherein the synapsin-I promoter comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 157.

[0285] In some embodiments, the composition does not include a nucleic acid having a synapsin-I promoter, does not include a CaMKII gamma promoter, or a combination thereof, optionally wherein the synapsin-I promoter comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 157.

[0286] In some embodiments, the composition is a pharmaceutical composition.

[0287] In some embodiments, the composition comprises: (a) a viral vector comprising a nucleic acid encoding a tetracycline-regulated transactivator (tTA) or a reverse tetracycline-regulated transactivator (rtTA), wherein the nucleic acid encoding the tTA or rtTA is operably linked to a CaMKIIα promoter, optionally wherein the viral vector is an AAV vector, and optionally wherein the AAV vector is packaged into an AAV-PHP.eB virus; and (b) a viral vector comprising a first nucleic acid encoding OCT4, a second nucleic acid encoding SOX2, and a third nucleic acid encoding KLF4, wherein the first nucleic acid, the second nucleic acid, and the third nucleic acid are operably linked to a promoter comprising a tetracycline-responsive element (TRE), optionally wherein the viral vector is an AAV vector, and optionally wherein the AAV vector is packaged into an AAV-PHP.eB virus.

[0288] In some embodiments, the vector of (a) comprises a WPRE sequence and / or an hGH pA terminator sequence, optionally wherein the WPRE sequence comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 21, 135, 147, 152, 155, or 160, and / or wherein the hGH pA terminator sequence comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 139, 148, 153, 156, or 161.

[0289] In some embodiments, the viral vector in (a) is the same viral vector as (b).

[0290] In some embodiments, the composition comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:121, SEQ ID NO:123, or SEQ ID NO:163.

[0291] In some embodiments, the composition comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to any sequence disclosed herein.

[0292] In some embodiments, the composition comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO: 123.

[0293] In some embodiments, the composition comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:137, SEQ ID NO:158, SEQ ID NO:17, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:146, 149, or 154, SEQ ID NO:135, 147, 152, 155, or 160, SEQ ID NO:139, 148, 153, 156, or 161, or SEQ ID NO:126, optionally wherein the composition comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO:127.

[0294] In some embodiments, the viral vector of part (a) comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:137, SEQ ID NO:158, SEQ ID NO:17, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:146, 149, or 154, SEQ ID NO:135, 147, 152, 155, or 160, SEQ ID NO:139, 148, 153, 156, or 161, or SEQ ID NO:126.

[0295] In some embodiments, the viral vector of part (b) comprises a sequence that is at least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:121, SEQ ID NO:123, or SEQ ID NO:163.

[0296] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0297] The compositions of the present disclosure may include, alone or in combination, at least one of any of the nucleic acids (e.g., engineered nucleic acids) (e.g., expression vectors) capable of inducing OCT4, KLF4, and / or SOX2 expression described herein and / or nucleic acids encoding inducers, engineered proteins, engineered cells, chemicals that activate (e.g., induce expression of) OCT4, KLF4, and / or SOX2, antibodies that activate (e.g., induce expression of) OCT4, KLF4, and / or SOX2, and / or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV). In some embodiments, the composition includes an inducer (e.g., a nucleic acid encoding an inducer). In certain embodiments, the compositions of the present disclosure comprise at least one of any of the nucleic acids (e.g., engineered nucleic acids) (e.g., expression vectors) described herein that can induce expression of OCT4, KLF4, SOX2, or any combination thereof, engineered proteins, engineered cells, chemicals that activate (e.g., induce expression of) OCT4, KLF4, SOX2, or any combination thereof, antibodies that activate (e.g., induce expression of) OCT4, KLF4, SOX2, or any combination thereof, and / or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV), alone or in combination. In some embodiments, the compositions comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acids (e.g., engineered nucleic acids) (e.g., expression vectors encoding OCT4, KLF4, and / or SOX2) that can induce OCT4, KLF4, and / or SOX2 expression.In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acids (e.g., engineered nucleic acids) (e.g., expression vectors encoding OCT4, KLF4, SOX2, or any combination thereof) that can induce the expression of OCT4, KLF4, SOX2, or any combination thereof. In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different viruses (e.g., lentivirus, alphavirus, vaccinia virus, adenovirus, retrovirus, herpesvirus, human papillomavirus, or AAV), each carrying one or more different transgenes. In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different chemicals that activate (e.g., induce the expression of) OCT4, KLF4, and / or SOX2. In some embodiments, the composition comprises one, two, three, four, five, six, seven, eight, nine, ten, or more different chemicals that activate (e.g., induce expression of) OCT4; KLF4; SOX2; or any combination thereof. In some embodiments, the composition further comprises one or more nucleic acids (e.g., engineered nucleic acids) encoding an inducer, one or more engineered proteins encoding the inducer, one or more chemicals that can modulate (e.g., activate or inhibit) the activity of the inducer, and / or one or more recombinant viruses encoding the inducer. In some embodiments, the composition comprises engineered cells (e.g., differentiated cells). In some embodiments, the composition comprises engineered proteins encoding OCT4, SOX2, and / or KLF4. In some embodiments, the composition comprises engineered proteins encoding OCT4, SOX2, KLF4, or any combination thereof. In some embodiments, the composition further comprises an engineered protein encoding an inducer.

[0298] In some embodiments, the composition comprises: a vector comprising a nucleic acid encoding a tetracycline-regulated transactivator (tTA) or a reverse tetracycline-regulated transactivator (rtTA), wherein the nucleic acid encoding tTA or rtTA is operably linked to a CaMKIIα promoter; and / or a vector comprising a first nucleic acid encoding OCT4, a second nucleic acid encoding SOX2, and a third nucleic acid encoding KLF4, wherein the first, second, and third nucleic acids are operably linked to a promoter comprising a tetracycline-responsive element (TRE). In some embodiments, one or more vectors are viral vectors. In some embodiments, one or more viral vectors are packaged into an AAV-PHP.eB virus.

[0299] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.Suitable carriers can be easily selected by those skilled in the art, taking into account the indications for example, nucleic acids (for example, engineered nucleic acids) that can activate (for example, induce the expression of) OCT4, KLF4, and / or SOX2, chemical substances, antibodies that activate (for example, induce the expression of) OCT4, KLF4, and / or SOX2, engineered proteins, engineered cells, and / or recombinant viruses (for example, lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV) that are intended. Suitable carriers can be easily selected by those skilled in the art, taking into account, for example, the indications for which the nucleic acid (e.g., an engineered nucleic acid) (e.g., an expression vector) capable of inducing the expression of OCT4; KLF4; SOX2; or any combination thereof, a chemical that activates (e.g., induces the expression of) OCT4; KLF4; SOX2; or any combination thereof, an antibody that activates (e.g., induces the expression of) OCT4; KLF4; SOX2; or any combination thereof, an engineered protein, an engineered cell, and / or a recombinant virus (e.g., a lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV) is intended. Suitable carriers can be easily selected by those skilled in the art, taking into account, for example, the indications for which the nucleic acid encoding the inducer (e.g., an engineered nucleic acid), the engineered protein encoding the inducer, the chemical substance capable of regulating (e.g., activating or inhibiting) the activity of the inducer, and / or the recombinant virus containing the inducer (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV) is intended. For example, one suitable carrier includes saline, which can be formulated with various buffer solutions (e.g., phosphate-buffered saline).Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The choice of carrier is not a limitation of the disclosure.

[0300] Optionally, the compositions of the present disclosure may include, in addition to, for example, any of the nucleic acids (e.g., engineered nucleic acids) disclosed herein, engineered cells containing OCT4, KLF4, and / or SOX2 and / or inducers, engineered proteins, chemicals that activate (e.g., induce expression of) OCT4, KLF4, and / or SOX2, antibodies that activate (e.g., induce expression of) OCT4, KLF4, and / or SOX2, and / or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV), and carrier(s), other pharmaceutical ingredients, for example, preservatives or chemical stabilizers. Optionally, the compositions of the present disclosure may contain, in addition to, for example, a nucleic acid (e.g., an engineered nucleic acid) (e.g., an expression vector) disclosed herein, OCT4; KLF4; SOX2; or any combination thereof; and / or engineered cells containing an inducer, engineered protein, a chemical that activates (e.g., induces the expression of) OCT4; KLF4; SOX2; or any combination thereof, an antibody that activates (e.g., induces the expression of) OCT4; KLF4; SOX2; or any combination thereof, and / or any of the recombinant viruses described herein (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV), and any of the carrier(s), other pharmaceutical ingredients, for example, preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.The compositions of the present disclosure may further include a nucleic acid (e.g., an engineered nucleic acid) encoding the inducer, an engineered protein encoding the inducer, a chemical that can modulate (e.g., activate or inhibit) the activity of the inducer, and / or a recombinant virus encoding the inducer.

[0301] Nucleic acids (e.g., engineered nucleic acids) (e.g., expression vectors) capable of inducing the expression of OCT4; KLF4; SOX2; or any combination thereof described herein, engineered cells, chemicals that activate (e.g., induce the expression of) OCT4; KLF4; SOX2; or any combination thereof, engineered proteins encoding OCT4; KLF4; SOX2; or any combination thereof, antibodies that activate (e.g., induce the expression of) OCT4; KLF4; SOX2; or any combination thereof, and / or recombinant viruses encoding the same (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV) are administered in an amount sufficient to transfect cells of the desired tissue (e.g., from the central nervous system, including the brain) and to provide sufficient levels of gene transfer and expression without undue adverse effects. Either a nucleic acid (e.g., an engineered nucleic acid) encoding the inducer, an engineered protein encoding the inducer, a chemical capable of modulating (e.g., activating or inhibiting) the activity of the inducer, and / or a recombinant virus encoding the inducer is administered in an amount sufficient to transfect cells of the desired tissue (e.g., tissue from the central nervous system, including, e.g., the brain) and to provide sufficient levels of gene transfer and expression without undue adverse effects. Examples of pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to a selected organ (e.g., direct delivery to the central nervous system, including, e.g., the brain).Nucleic acids (e.g., engineered nucleic acids) (e.g., expression vectors) capable of inducing OCT4, KLF4, and / or SOX2 expression, engineered cells, chemicals that activate OCT4, KLF4, and / or SOX2 (e.g., induce their expression), engineered proteins, antibodies that activate OCT4, KLF4, and / or SOX2 (e.g., induce their expression), and / or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus), as described herein, can be used to induce OCT4, KLF4, and / or SOX2 expression. , herpesvirus, human papillomavirus, or AAV) can be delivered intravenously, intradermally, intra-arterially, intralesionally, intratumorally, intracranially, intra-articularly, intraprostatically, intrapleurally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, intramucosally, pericardially, intraumbilically, intraocularly, orally, topically, locally, systemically, by injection, infusion, continuous infusion, by local perfusion bath directly onto target cells, via a catheter, in a cream, in a lipid composition (e.g., liposomes), or by other methods or combinations of any of the foregoing that would be known to one of skill in the art.The present invention relates to nucleic acids (e.g., engineered nucleic acids) (e.g., expression vectors) capable of inducing the expression of OCT4; KLF4; SOX2; or any combination thereof, engineered cells, chemicals that activate (e.g., induce the expression of) OCT4; KLF4; SOX2; or any combination thereof, engineered proteins, antibodies that activate (e.g., induce the expression of) OCT4; KLF4; SOX2; or any combination thereof, and / or recombinant viruses (e.g., lentiviruses, adenoviruses, alphaviruses, Any of the viruses (vaccinia virus, retrovirus, herpes virus, human papilloma virus, or AAV) can be delivered intravenously, intradermally, intra-arterially, intralesionally, intratumorally, intracranially, intra-articularly, intraprostatically, intrapleurally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, intramucosally, pericardially, intraumbilically, intraocularly, orally, topically, locally, systemically, by injection, infusion, continuous infusion, by local perfusion bath directly onto target cells, via a catheter, in a cream, in a lipid composition (e.g., liposomes), or by other methods or combinations of any of the foregoing that would be known to one of skill in the art. Any of the nucleic acids encoding the inducers, chemicals capable of modulating the activity of the inducers, engineered proteins encoding the inducers, and / or recombinant viruses encoding the inducers can be delivered intravenously, intradermally, intraarterially, intralesionally, intratumorally, intracranially, intraarticularly, intraprostatically, intrapleurally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, intramucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, systemically, by injection, infusion, continuous infusion, by direct local perfusion of 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 by any combination of the foregoing. In some embodiments, any of the compositions described herein is administered to the central nervous system. In some embodiments, any of the compositions described herein is not administered to the eye (e.g., is not administered to the retina). In some embodiments, any of the compositions described herein is administered to the brain. If desired, routes of administration can be combined.

[0302] In some embodiments, the nucleic acid is delivered non-virally (e.g., not on a viral vector and / or not by a virus). In some embodiments, the nucleic acid (e.g., RNA or DNA) encoding OCT4, SOX2, and / or KLF4 and / or an inducer is administered by a liposome. In some embodiments, the nucleic acid (e.g., RNA or DNA) encoding OCT4, SOX2, KLF4, or any combination thereof and / or an inducer is administered by a liposome. In some embodiments, the nucleic acid (e.g., RNA or DNA) encoding OCT4, SOX2, and / or KLF4 and / or an inducer is administered by a particle. In some embodiments, the nucleic acid (e.g., RNA or DNA) encoding OCT4, SOX2, KLF4, or any combination thereof and / or an inducer is administered by a particle. In some embodiments, the nucleic acid is RNA (e.g., mRNA).

[0303] In some embodiments, a pharmaceutical composition comprising an expression vector encoding OCT4, KLF4, and / or SOX2 and / or an inducer, or a pharmaceutical composition comprising a virus carrying an expression vector, is administered to a cell, tissue, organ, or subject. In some embodiments, a pharmaceutical composition comprising an expression vector encoding an inducer, or a pharmaceutical composition comprising a virus carrying an expression vector, is administered to a cell, tissue, organ, or subject. For example, the cell, tissue, or organ may be the central nervous system. The subject may have a neurological disorder. In some embodiments, the virus and / or expression vector encoding OCT4, KLF4, and / or SOX2 is administered systemically. In some embodiments, the virus and / or expression vector encoding the inducer is administered systemically. In some embodiments, the virus and / or expression vector encoding OCT4, KLF4, and / or SOX2 and / or the inducer is administered locally (e.g., directly to the tissue or organ of interest, including, for example, tissue or organ from the central nervous system). In some embodiments, the tissue or organ is brain tissue or the brain. In some embodiments, viruses and / or expression vectors encoding OCT4, KLF4, and / or SOX2 and / or encoding inducers are administered via retro-orbital vein injection. In some embodiments, viruses and / or expression vectors encoding OCT4, KLF4, and / or SOX2 and / or encoding inducers are administered via intrathecal administration. In some embodiments, inducers (e.g., nucleic acids encoding inducers, proteins encoding inducers, or viruses encoding inducers) and / or chemicals capable of regulating (e.g., activating or inhibiting) the activity of inducers are administered using the same administration route as OCT4, KLF4, and / or SOX2 (e.g., nucleic acids encoding OCT4, KLF4, and / or SOX2).In some embodiments, the inducer (e.g., a nucleic acid encoding the inducer, a protein encoding the inducer, or a virus encoding the inducer) and / or a chemical substance capable of modulating (e.g., activating or inhibiting) the activity of the inducer is administered via a different route of administration than OCT4, KLF4, and / or SOX2 (e.g., a nucleic acid encoding OCT4, KLF4, and / or SOX2).

[0304] In some embodiments, a pharmaceutical composition comprising an expression vector encoding OCT4; KLF4; SOX2; or any combination thereof, or a pharmaceutical composition comprising a virus carrying an expression vector, is administered to a cell, tissue, organ, or subject. In some embodiments, a pharmaceutical composition comprising an expression vector encoding an inducer, or a pharmaceutical composition comprising a virus carrying an expression vector, is administered to a cell, tissue, organ, or subject. In some embodiments, a virus and / or expression vector encoding OCT4; KLF4; SOX2; or any combination thereof is administered systemically. In some embodiments, a virus and / or expression vector encoding an inducer is administered systemically. In some embodiments, a virus and / or expression vector encoding OCT4; KLF4; SOX2; or any combination thereof is administered locally (e.g., directly to a tissue or organ of interest, including the central nervous system). In some embodiments, a virus and / or expression vector encoding an inducer is administered locally (e.g., directly to a tissue or organ of interest, including the central nervous system). In some embodiments, the inducer (e.g., a nucleic acid encoding the inducer, a protein encoding the inducer, or a virus encoding the inducer) and / or a chemical substance capable of modulating (e.g., activating or inhibiting) the activity of the inducer is administered using the same route of administration as OCT4; KLF4; SOX2; or any combination thereof (e.g., OCT4; KLF4; SOX2; OCT4 and SOX2; OCT4 and KLF4; KLF4 and SOX2; or a nucleic acid encoding KLF4, OCT4, and SOX2).In some embodiments, the inducer (e.g., a nucleic acid encoding the inducer, a protein encoding the inducer, or a virus encoding the inducer) and / or a chemical substance capable of modulating (e.g., activating or inhibiting) the activity of the inducer is administered via a route of administration different from OCT4; KLF4; SOX2; or any combination thereof (e.g., OCT4; KLF4; SOX2; OCT4 and SOX2; OCT4 and KLF4; KLF4 and SOX2; or a nucleic acid encoding KLF4, OCT4, and SOX2).

[0305] In some embodiments, the expression vector is an inducible vector, in which a nucleic acid encoding OCT4, KLF4, and / or SOX2 and / or an inducer is operably linked to an inducible TRE promoter (e.g., TRE3G, TRE2, or P tight). In some embodiments, the expression vector is an inducible vector, in which a nucleic acid encoding OCT4; KLF4; SOX2; or any combination thereof, and / or an inducer is operably linked to an inducible TRE promoter (e.g., TRE3G, TRE2, or P tight). In some embodiments, the virus and / or inducible vector is administered together with tetracycline (e.g., doxycycline). In some embodiments, the virus and / or expression vector comprising a TRE promoter is administered separately from tetracycline (e.g., doxycycline). For example, any of the viruses and / or expression vectors comprising a TRE promoter described herein can be administered systemically, and tetracycline can be administered locally (e.g., to the organ or tissue of interest). In some embodiments, any of the viruses and / or expression vectors comprising a TRE promoter described herein can be administered locally (e.g., directly to a tissue or organ of interest, including the central nervous system), and tetracycline can be administered systemically. As a non-limiting example, a virus and / or expression vector comprising a TRE promoter can be administered directly to a subject's eye (e.g., by injection), and tetracycline (e.g., doxycycline) can be administered systemically (e.g., orally as a pill).

[0306] In some embodiments, tetracycline is administered intravenously, intradermally, intraarterially, intralesionally, intratumorally, intracranially, intraarticularly, intraprostatically, intrapleurally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, intramucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, systemically, by injection, infusion, continuous infusion, by direct local perfusion of target cells, via a catheter, in a cream, or in a lipid composition. In some embodiments, tetracycline is administered directly to cells, organs, and / or tissues. Non-limiting examples include tetracycline-containing eye drops, sustained-release devices (e.g., micropumps, particles, and / or drug depots), and medicated contact lenses containing tetracycline. In some embodiments, tetracycline is administered systemically to a subject (e.g., via drinking water or intravenous injection). Tetracyclines can be administered topically (eg, by cream) or by subcutaneous pump (eg, to deliver tetracycline to specific tissues).

[0307] For example, the dose of recombinant virus (e.g., lentivirus, alphavirus, vaccinia virus, adenovirus, retrovirus, herpesvirus, human papillomavirus, or AAV) virion required to achieve a particular therapeutic effect, e.g., a dose in units of genome copies per kilogram of body weight (GC / kg), will vary based on several factors, including, but not limited to, the route of recombinant virus (e.g., lentivirus, alphavirus, vaccinia virus, adenovirus, retrovirus, herpesvirus, human papillomavirus, or AAV) virion administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. Those skilled in the art can readily determine the dose range of recombinant virus (e.g., lentivirus, alphavirus, vaccinia virus, adenovirus, retrovirus, herpesvirus, human papillomavirus, or AAV virion) for treating a patient with a specific disease or disorder based on the aforementioned and other factors.

[0308] An effective amount of a recombinant virus (e.g., lentivirus, alphavirus, vaccinia virus, adenovirus, retrovirus, herpesvirus, human papillomavirus, or AAV) is an amount sufficient to target and infect an animal and target the desired tissue. In some embodiments, an effective amount of a recombinant virus (e.g., lentivirus, alphavirus, vaccinia virus, adenovirus, retrovirus, herpesvirus, human papillomavirus, or AAV) is an amount sufficient to generate a stable somatic transgenic animal model. The effective amount will depend primarily on factors such as the subject's species, age, weight, health, and tissue to be targeted. Therefore, it may vary between animals and tissues. For example, an effective amount of a recombinant virus (e.g., lentivirus, alphavirus, vaccinia virus, adenovirus, retrovirus, herpesvirus, human papillomavirus, or AAV) is generally about 10 9 ~1016 The volume ranges from about 1 ml to about 100 ml of solution containing genome copies. In some cases, about 10 11 ~10 13 A dosage of between 10 and 100 recombinant virus (e.g., lentivirus, adenovirus, retrovirus, alphavirus, vaccinia virus, herpesvirus, human papillomavirus, or AAV) genome copies is appropriate. In certain embodiments, a dosage of between 10 and 100 recombinant virus (e.g., lentivirus, adenovirus, retrovirus, alphavirus, vaccinia virus, herpesvirus, human papillomavirus, or AAV) genome copies is appropriate. 10 or 10 11 Recombinant viral (e.g., lentivirus, adenovirus, retrovirus, alphavirus, vaccinia virus, herpesvirus, human papillomavirus, or AAV) genome copies are effective for targeting visual tissue (e.g., retinal tissue). In some cases, stable transgenic animals are generated by multiple doses of recombinant viral (e.g., lentivirus, adenovirus, retrovirus, herpesvirus, human papillomavirus, alphavirus, vaccinia virus, or AAV) genes.

[0309] In some embodiments, a dose of a recombinant virus (e.g., a lentivirus, adenovirus, retrovirus, herpesvirus, human papillomavirus, alphavirus, vaccinia virus, or AAV) is administered to a subject no more than once per calendar day (e.g., a 24-hour period). In some embodiments, a dose of a recombinant virus (e.g., a lentivirus, alphavirus, vaccinia virus, adenovirus, retrovirus, herpesvirus, human papillomavirus, or AAV) is administered to a subject no more than once per 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, a dose of a recombinant virus (e.g., a lentivirus, alphavirus, vaccinia virus, adenovirus, retrovirus, herpesvirus, human papillomavirus, or AAV) is administered to a subject no more than once per calendar week (e.g., 7 calendar days). In some embodiments, a dose of a recombinant virus (e.g., a lentivirus, an alphavirus, a vaccinia virus, an adenovirus, a retrovirus, a herpes virus, a human papilloma virus, or an AAV) is administered to a subject no more than every other week (e.g., once every two calendar weeks). In some embodiments, a dose of a recombinant virus (e.g., a lentivirus, an alphavirus, a vaccinia virus, an adenovirus, a retrovirus, a herpes virus, a human papilloma virus, or an AAV) is administered to a subject no more than once per calendar month (e.g., once every 30 calendar days). In some embodiments, a dose of a recombinant virus (e.g., a lentivirus, an alphavirus, a vaccinia virus, an adenovirus, a retrovirus, a herpes virus, a human papilloma virus, or an AAV) is administered to a subject no more than once per six calendar months. In some embodiments, a dose of recombinant virus (e.g., lentivirus, alphavirus, vaccinia virus, adenovirus, retrovirus, herpesvirus, human papillomavirus, or AAV) is administered to a subject no more than once per calendar year (e.g., 365 days, or 366 days in leap years).

[0310] In some embodiments, particularly where high recombinant virus (e.g., lentivirus, alphavirus, vaccinia virus, adenovirus, retrovirus, herpesvirus, human papillomavirus, or AAV) concentrations are present (e.g., approximately 10 13 Compositions of recombinant viruses (e.g., lentiviruses, alphaviruses, vaccinia viruses, adenoviruses, retroviruses, herpesviruses, human papillomaviruses, or AAV) are formulated to reduce aggregation of AAV particles in the composition. Suitable methods for reducing aggregation can be used, including, for example, adding detergents, adjusting pH, adjusting salt concentrations, etc. (see, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference).

[0311] By way of non-limiting example, AAV-mediated transgene delivery has been shown to be feasible and non-toxic in humans. For example, AAV can be delivered to the eye. See, e.g., Smalley, Nat. Biotechnol. 2017 Nov 9;35(11):998-999.

[0312] The formulation of pharmaceutically acceptable excipients and carrier solutions is known to those skilled in the art, as is the development of suitable dosage and treatment regimens for using the specific compositions described herein in various treatment regimens.Typically, these preparations can contain at least about 0.1% or more of active compound, but the percentage of active ingredient(s) can of course vary, and can conveniently be between about 1 or 2% and about 70% or 80% or more of the total weight or volume of the preparation.Of course, the amount of active compound in each therapeutically useful composition can be prepared in such a way that a suitable dosage is obtained in any given unit dose of compound.Factors such as solubility, bioavailability, biological half-life, administration route, product shelf life and other pharmacological considerations are considered by those who prepare such pharmaceutical preparations, and therefore different dosages and treatment regimens may be desirable.

[0313] In some embodiments, nucleic acids (e.g., engineered nucleic acids) capable of inducing OCT4, KLF4, and / or SOX2 expression (e.g., expression vectors), engineered cells containing OCT4, KLF4, and / or SOX2, engineered proteins encoding Oct4, KLF4, and / or SOX2, chemicals that activate OCT4, KLF4, and / or SOX2 (e.g., induce their expression), antibodies that activate OCT4, KLF4, and / or SOX2 (e.g., induce their expression), and / or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV) are delivered directly to a target tissue, e.g., to a tissue of interest (e.g., neural tissue), by a suitably formulated pharmaceutical composition, e.g., as disclosed herein.

[0314] In some embodiments, nucleic acids (e.g., engineered nucleic acids) capable of inducing the expression of OCT4; KLF4; SOX2; or any combination thereof (e.g., expression vectors), engineered cells containing OCT4; KLF4; SOX2; or any combination thereof, engineered proteins encoding Oct4, KLF4, SOX2, or a combination thereof, chemicals that activate (e.g., induce the expression of) OCT4, KLF4, SOX2, or a combination thereof, antibodies that activate (e.g., induce the expression of) OCT4, KLF4, SOX2, or a combination thereof, and / or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV) are delivered directly to the target tissue, e.g., to the tissue of interest (e.g., neural tissue), e.g., by a suitably formulated pharmaceutical composition disclosed herein.

[0315] In some embodiments, a nucleic acid (e.g., an engineered nucleic acid) (e.g., an expression vector) encoding an inducer, an engineered cell containing the inducer, an engineered protein encoding the inducer, a chemical capable of modulating the activity of the inducer, and / or a recombinant virus (e.g., a lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) encoding the inducer is delivered directly to the target tissue, e.g., directly to the tissue of interest (e.g., neural tissue), e.g., by a suitably formulated pharmaceutical composition disclosed herein.

[0316] However, in certain circumstances, separately or in addition, nucleic acids (e.g., engineered nucleic acids) capable of inducing OCT4, KLF4, and / or SOX2 expression (e.g., expression vectors) and / or nucleic acids encoding inducers, nucleic acids (e.g., engineered nucleic acids) capable of inducing expression of a combination of transcription factors selected from OCT4, KLF4 and / or nucleic acids encoding inducers, engineered cells, engineered proteins, chemicals that activate (e.g., induce expression of) OCT4, KLF4, and / or SOX2, chemicals that activate (e.g., induce expression of) a combination of transcription factors selected from OCT4, KLF4, and SOX2, It may be desirable to deliver either a chemical compound capable of modulating (e.g., activating or inhibiting) the activity of an inducer, an antibody that activates (e.g., induces expression of) OCT4, KLF4, and / or SOX2, an antibody that activates (e.g., induces expression of) OCT4; KLF4; SOX2; or any combination thereof, and / or a recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV) via another route, e.g., subcutaneously, intrapancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally, or orally, intraperitoneally, or by inhalation. In some embodiments, the administration modalities described in U.S. Pat. Nos. 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety) can be used to deliver recombinant viruses (e.g., lentivirus, alphavirus, vaccinia virus, adenovirus, retrovirus, herpesvirus, human papillomavirus, or AAV). In some embodiments, the administration mode is by intrainterstitial injection. In some embodiments, the administration mode is by retro-orbital vein injection. In some embodiments, the administration mode is by intrathecal administration.

[0317] In some embodiments, the mode of administration does not include administration to the subject's eye (e.g., the retina, uvea, pupil, lens, cornea, and / or sclera). In some embodiments, the mode of administration does not include administration to the subject's retina. In some embodiments, OCT4, KLF4, SOX2, and / or inducers are not expressed in the retina. In some embodiments, OCT4, KLF4, SOX2, and / or inducers are not activated in the retina. In some embodiments, OCT4, KLF4, SOX2, and / or inducers are not expressed in the eye. In some embodiments, OCT4, KLF4, SOX2, and / or inducers are not activated in the eye.

[0318] In some embodiments, nucleic acids (e.g., mRNA) encoding OCT4, SOX2, KLF4, or any combination thereof are nanoformulated into polyplexes, which can be useful, for example, for non-invasive aerosol inhalation and delivery of nucleic acids to the lung (e.g., the lung epithelium). For example, see Patel et al., Adv Mater. 2019 Jan 4:e1805116. doi:10.1002 / adma.201805116 for a description of nanoformulated mRNA polyplexes, which is incorporated herein by reference in its entirety.

[0319] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In most cases, the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0320] For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent may first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, any suitable sterile aqueous medium may be employed. For example, a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of subcutaneous infusion fluid or injected at the proposed infusion site (see, e.g., "Remingto's Pharmaceutical Science," 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person administering will, in any event, determine the appropriate dose for the individual host.

[0321] Sterile injectable solutions are prepared by incorporating nucleic acids (e.g., engineered nucleic acids) (e.g., expression vectors) capable of inducing OCT4, KLF4, and / or SOX2 expression, engineered cells, engineered proteins, chemicals that activate OCT4, KLF4, and / or SOX2 (e.g., that induce their expression), antibodies that activate OCT4, KLF4, and / or SOX2 (e.g., that induce their expression), and / or active recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, human papillomavirus, or AAV) in the required amounts, for example, into an appropriate solvent with various other ingredients listed herein, as required, followed by filtered sterilization. In certain embodiments, sterile injectable solutions are prepared by incorporating the nucleic acid encoding the inducer (e.g., an engineered nucleic acid), the engineered protein encoding the inducer, a chemical capable of regulating the activity of the inducer, and / or an active recombinant virus encoding the inducer (e.g., a lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV) in the required amounts, for example, into a suitable solvent with various other ingredients listed herein, followed by filtration sterilization. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the required other ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying techniques. These produce a powder of the active ingredient plus any additional desired ingredients from the previously sterile-filtered solution.

[0322] Compositions comprising nucleic acids (e.g., engineered nucleic acids) (e.g., expression vectors) encoding OCT4, KLF4, and / or SOX2 disclosed herein, engineered cells, engineered proteins, chemicals that activate (e.g., induce expression of) OCT4, KLF4, and / or SOX2, antibodies that activate (e.g., induce expression of) OCT4, KLF4, and / or SOX2, and / or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, human papillomavirus, or AAV) can also be formulated in neutral or salt forms. The compositions disclosed herein include nucleic acids (e.g., engineered nucleic acids) (e.g., expression vectors) encoding OCT4, KLF4, SOX2, or any combination thereof, engineered cells, engineered proteins, chemicals that activate (e.g., induce the expression of) OCT4, KLF4, SOX2, or any combination thereof, antibodies that activate (e.g., induce the expression of) OCT4, KLF4, SOX2, or any combination thereof, and / or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV) can also be formulated in neutral or salt form.The compositions can include inducers (e.g., nucleic acids encoding inducers or proteins encoding inducers and / or recombinant viruses encoding inducers) and / or chemicals that can regulate the activity of inducers. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, etc. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, ferric hydroxide, and the like, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like.Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as, for example, injectable solutions, drug release capsules, and the like.

[0323] Carriers include any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutical active substances is known in the art. Supplementary active ingredients can also be incorporated into the compositions.

[0324] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used to introduce the compositions of the present disclosure into suitable host cells. In particular, any of nucleic acids (e.g., engineered nucleic acids) (e.g., expression vectors) that can induce OCT4, KLF4, and / or SOX2 expression, any of engineered proteins, any of chemicals that activate (e.g., induce the expression of) OCT4, KLF4, and / or SOX2, any of antibodies that activate (e.g., induce the expression of) OCT4, KLF4, and / or SOX2, engineered cells, and / or any of recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV) can be encapsulated in lipid particles, liposomes, vesicles, nanospheres, nanoparticles, etc. In some embodiments, any of nucleic acids (e.g., engineered nucleic acids) (e.g., expression vectors) capable of inducing the expression of OCT4, KLF4, SOX2, or any combination thereof, any of engineered proteins, any chemicals that activate (e.g., induce the expression of) OCT4, KLF4, SOX2, or any combination thereof, any of antibodies that activate (e.g., induce the expression of) OCT4, KLF4, SOX2, or any combination thereof, engineered cells, and / or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV) can be encapsulated in lipid particles, liposomes, vesicles, nanospheres, nanoparticles, etc. Inducers (e.g., nucleic acids encoding inducers or proteins encoding inducers and / or recombinant viruses encoding inducers) and / or chemicals that can regulate the activity of inducers can be encapsulated in lipid particles, liposomes, vesicles, nanospheres, nanoparticles, etc.

[0325] In some embodiments, the delivery vehicle targets the cargo. For example, any of the nucleic acids, engineered proteins, chemicals, antibodies, and / or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV) described herein can be delivered via nanoparticles that deliver cargo to certain tissues or cell types. For example, nanoparticles coated with galactose polymers are known to release their cargo in senescent cells as a result of their endogenous beta-galactosidase activity. For example, see Lozano-Torres et al., J Am Chem Soc. 2017 Jul 5; 139(26):8808-8811.

[0326] In some embodiments, nucleic acids, engineered proteins, chemicals, antibodies, and / or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV) are formulated into poly(glycoamidoamine) brush nanoparticles. See, e.g., Dong et al., Nano Lett. 2016 Feb 10;16(2):842-8.

[0327] In some embodiments, nucleic acids, engineered proteins, chemicals, antibodies, and / or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, human papillomavirus, or AAV) are formulated into lipid nanoparticles. For an example, see Cullis and Hope Mol Ther. 2017 Jul 5;25(7):1467-1475. In some embodiments, the lipid nanoparticles contain one or more membrane fusion proteins, which deliver plasmids directly to the cytoplasm. Alternatively, factors such as OCT4; KLF4; SOX2; or any combination thereof can be fused directly to a targeting protein, with or without nanoparticle encapsulation. In some embodiments, the lipid nanoparticles are Fusogenix lipid nanoparticles. In some embodiments, the lipid nanoparticles are "wrapped liposomes" (WL). For an example, see Yamauchi et al., Biochim Biophys Acta. 2006 Jan;1758(1):90-7. In some embodiments, the lipid nanoparticles are PEGylated liposomes (e.g., DOXIL™) (e.g., Allen & Hansen, Biochim Biophys Acta. 1991 Jul 1;1066(1):29-36.), 1,2-dioleoyl-sn-glycerol-3 phosphatidylethanolamine (DOPE), the neutral helper lipid phosphatidylethanolamine (PE), or combinations thereof (e.g., Farhood et al., Biochim Biophys Acta. 1995 May 4;1235(2):289-95; Zhou & Huang, Biochim Biophys Acta. 1994 Jan 19;1189(2):195-203.). In some embodiments, the lipid nanoparticles or fusion proteins contain or employ molecules or proteins to mimic methods employed by viruses for intracellular delivery of macromolecules (see, e.g., Kobayashi et al., Bioconjug Chem. 2009 May 20;20(5):953-9).For example, various pH-sensitive peptides, such as vesicular stomatitis virus protein (VSV G), phage coat proteins and / or shGALA, and / or fusion-associated small transmembrane (FAST) proteins, such as avian reovirus (ARV), Nelson Bay reovirus (NBV), and baboon reovirus (BBV), aquareovirus reovirus (AQV), and reptilian reovirus (RRV), and / or bombesin targeting peptides are used. See, for example, Peisajovich et al., Eur J Biochem. 2002 Sep;269(17):4342-50; Sakurai et al., 2011. See also Nesbitt, Targeted Intracellular Therapeutic Delivery Using Liposomes Formulated with Multifunctional FAST proteins, Western University Thesis, 2012.

[0328] In some embodiments, nucleic acids (e.g., RNA or DNA, including plasmids) encoding OCT4, KLF4, SOX2, or a combination thereof, and / or nucleic acids encoding inducers are encapsulated in Fusogenix lipid nanoparticles. In some embodiments, nucleic acids encoding inducers (e.g., rtTA or tTA) are encapsulated in Fusogenix lipid nanoparticles. In some embodiments, the lipid nanoparticles contain viral membrane proteins. Without being bound by theory, the lipid nanoparticles may be non-toxic because they contain membrane fusion proteins that are not viral membrane fusion proteins. Non-limiting examples of membrane fusion proteins include those disclosed in U.S. Pat. No. 7,851,595, U.S. Pat. No. 8,252,901, WO 2012 / 040825, and WO 2002 / 044206.

[0329] In some embodiments, the compositions of the present disclosure (e.g., including nucleic acids encoding OCT4, KLF4, SOX2, or combinations thereof and / or nucleic acids encoding inducers) are delivered non-virally. Methods for non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic aci...

Claims

1. 1. A composition for use in rejuvenating a cell, tissue, or organ, comprising: a) Factors that induce OCT4 expression; b) a factor that induces SOX2 expression; and c) factors that induce KLF4 expression; Including, the composition does not comprise a factor that induces c-MYC expression, and wherein the cell, tissue, or organ is a central nervous system cell, central nervous system tissue, or central nervous system organ, optionally, wherein the central nervous system does not include the retina, and optionally, wherein the cell, tissue, or organ is a brain cell, brain tissue, or brain; The composition.

2. 1. A method of rejuvenating a cell, tissue, and / or organ comprising administering to the cell, tissue, or organ a composition, the composition comprising: a) Factors that induce OCT4 expression; b) a factor that induces SOX2 expression; and c) factors that induce KLF4 expression; Including, the composition does not comprise a factor that induces c-MYC expression, and wherein the cell, tissue, or organ is a central nervous system cell, central nervous system tissue, or central nervous system organ, optionally, wherein the central nervous system does not include the retina, and optionally, wherein the cell, tissue, or organ is a brain cell, brain tissue, or brain; The method.

3. 3. The composition or method of claim 1 or 2, wherein the brain cells are neurons or glial cells.

4. The composition or method of claim 3, wherein the neuron is an excitatory neuron.

5. The composition or method of any one of claims 1 to 4, wherein the brain tissue is neural tissue.

6. 6. The composition or method of any one of claims 1 to 5, wherein the cell, tissue or organ is in a subject, optionally wherein the composition is administered to a subject in need thereof, optionally wherein the subject has a neurological disorder.

7. The composition or method of any one of claims 1 to 6, wherein the composition does not induce OCT4, SOX2, or KLF4 expression in the retina.

8. 8. The composition or method of any one of claims 1 to 7, wherein the composition induces expression of OCT4, SOX2, and KLF4 for a period of time sufficient to rejuvenate cells, tissues, and / or organs.

9. 9. The composition or method of any one of claims 1 to 8, wherein the period of time sufficient to rejuvenate a cell, tissue or organ is approximately one month.

10. The composition or method of any one of claims 1 to 9, wherein expression of OCT4, SOX2 and KLF4 is induced for less than 2 months.

11. The composition or method of any one of claims 1 to 10, wherein expression of OCT4, SOX2 and KLF4 is induced for at most one month.

12. 12. The composition or method of any one of claims 1 to 11, wherein rejuvenating a cell, tissue or organ comprises restoring epigenetic information in the cell, tissue and / or organ.

13. 13. The composition or method of any one of claims 1 to 12, wherein rejuvenating a cell, tissue, or organ comprises restoring epigenetic information lost in the cell, tissue, or organ due to aging, injury, disease, or any combination thereof.

14. 14. The composition or method of any one of claims 1 to 13, wherein rejuvenating a cell, tissue or organ comprises re-establishing an epigenetic state of the cell, tissue or organ that is similar to the epigenetic state formed shortly after fertilization or terminal differentiation.

15. The composition or method of any one of claims 1 to 14, wherein each agent is independently a nucleic acid, a small molecule, or a polypeptide, optionally wherein the polypeptide is an antibody.

16. The composition or method of any one of claims 1 to 15, wherein at least one agent comprises a nanoparticle.

17. The composition or method of any one of claims 15 to 16, wherein the at least one agent is encapsulated in the at least one nanoparticle.

18. The composition or method of any one of claims 15 to 17, wherein the nucleic acid is DNA or RNA.

19. 20. The composition or method of claim 18, wherein the DNA is plasmid DNA.

20. 20. The composition or method of claim 18, wherein the RNA is mRNA.

21. 21. The composition or method of any one of claims 15 to 20, wherein the factor that induces OCT4 expression is an engineered nucleic acid encoding OCT4.

22. The composition or method of any one of claims 15 to 21, wherein the factor that induces SOX2 expression is an engineered nucleic acid encoding SOX2.

23. 23. The composition or method of any one of claims 15 to 22, wherein the factor that induces KLF4 expression is an engineered nucleic acid encoding KLF4.

24. 21. The composition or method of any one of claims 15 to 20, wherein the factor that induces OCT4 expression is an engineered nucleic acid encoding OCT4, the factor that induces SOX2 expression is an engineered nucleic acid encoding SOX2, and the factor that induces KLF4 expression is an engineered nucleic acid encoding KLF4.

25. 24. The composition or method of claim 21, 22, or 23, wherein the engineered nucleic acid is present on one or more expression vectors.

26. 26. The composition or method of claim 25, wherein the engineered nucleic acids are present on the same expression vector.

27. 27. The composition or method of any one of claims 25 or 26, wherein the one or more expression vectors include an inducible promoter operably linked to any one or combination of the engineered nucleic acids.

28. 28. The composition or method of claim 27, wherein the promoter is a TRE3G, TRE2 promoter, or a P tight promoter.

29. 29. The composition or method of claim 27 or claim 28, wherein the promoter comprises a tetracycline responsive element (TRE).

30. The composition or method of any one of claims 25 to 29, wherein the expression vector comprises an hGH pA terminator sequence, optionally wherein the hGH pA terminator sequence comprises a sequence that is at least 70% identical to SEQ ID NO: 139, 148, 153, 156, or 161.

31. The composition or method of any one of claims 25 to 30, wherein the expression vector comprises a WPRE sequence.

32. 32. The composition or method of any one of claims 25 to 31, wherein the expression vector comprises a self-cleaving peptide.

33. The composition or method of claim 32, wherein the self-cleaving peptide is a 2A peptide, and optionally, wherein the 2A peptide sequence comprises a sequence that is at least 70% identical to SEQ ID NO: 118 and / or is encoded by a nucleic acid that comprises a sequence that is at least 70% identical to SEQ ID NO:

144.

34. 34. The composition or method of any one of claims 25 to 33, wherein the expression vector comprises inverted terminal repeats (ITRs) flanking the first nucleic acid, the second nucleic acid, the third nucleic acid, or a combination thereof, wherein the distance between the ITRs is 4.7 kb or less.

35. 35. The composition or method of any one of claims 1 to 34, wherein the composition further comprises an inducer or the method further comprises administering an inducer to said subject.

36. The composition or method of claim 35, wherein the inducer comprises tetracycline, a tetracycline transactivator (tTA), and / or a reverse tetracycline-regulated transactivator (rtTA), optionally wherein tTA comprises a sequence at least 70% identical to 138 or 159, optionally wherein tTA is encoded by a sequence at least 70% identical to 137 or 158, optionally wherein rtTA comprises a sequence at least 70% identical to SEQ ID NO: 11, 129, 13, or 15, optionally wherein rtTA is encoded by a sequence at least 70% identical to SEQ ID NO: 10, 12, 14, or 128.

37. 37. The composition or method of claim 36, wherein the tetracycline is doxycycline.

38. The composition or method of claim 36 or claim 37, wherein the composition comprises an expression vector having an engineered nucleic acid encoding tTA and / or rtTA, optionally wherein the engineered nucleic acid encoding tTA and / or rtTA comprises a WPRE sequence and / or an hGH pA sequence, optionally wherein the WPRE sequence is at least 70% identical to SEQ ID NO: 21, 135, 147, 152, 155, or 160, and / or the hGH pA sequence is at least 70% identical to SEQ ID NO: 139, 148, 153, 156, or 161.

39. 39. The composition or method of claim 38, wherein the expression vector encoding tTA and / or rtTA is the same expression vector as the engineered nucleic acid encoding OCT4, SOX2, and / or KLF4 or is a different expression vector.

40. 40. The composition or method of any one of claims 36-39, wherein the rtTA is rtTA3, rtTA Advanced, rtTA2S-M2, or rtTA4, optionally wherein the rtTA comprises a sequence that is at least 70% identical to a sequence selected from the group consisting of SEQ ID NOs: 11, 13, 15, and 129.

41. 41. The composition or method of any one of claims 25 to 40, wherein at least one expression vector is a viral vector, optionally wherein at least one expression vector is packaged into a recombinant virus.

42. 42. The composition or method of claim 41, wherein the viral vector is a lentivirus, retrovirus, adenovirus, alphavirus, vaccinia virus, human papilloma virus, or adeno-associated virus (AAV) vector.

43. The composition or method of claim 41 or claim 42, wherein the AAV vector is packaged into an AAV-PHP.eB, AAV-PHP.b, AAV.CAP-B10, or AAV.CAP-B22 virus.

44. The composition or method of any one of claims 1 to 43, wherein the AAV vector is not AAV2 or AAV9.

45. 45. The composition or method of any one of claims 1 to 44, wherein the subject is a human or non-human mammal.

46. 46. ​​The composition or method of any one of claims 38 to 45, wherein the expression vector having an engineered nucleic acid encoding tTA and / or rtTA comprises a promoter operably linked to the nucleic acid encoding tTA and / or rtTA.

47. 47. The composition or method of claim 46, wherein the promoter operably linked to the engineered nucleic acid encoding tTA or rtTA is a ubiquitous promoter.

48. 48. The composition or method of claim 47, wherein the ubiquitous promoter is UBC, CMV, PGK1, CAG, and optionally, wherein the ubiquitous promoter comprises a sequence that is at least 70% identical to 48, 132, 130, 136, or 162.

49. 47. The composition or method of claim 46, wherein the promoter operably linked to the engineered nucleic acid encoding tTA or rtTA is a neuron-specific promoter.

50. The composition or method of claim 49, wherein the neuron-specific promoter is CaMKIIα, optionally wherein the CaMKIIα promoter comprises a sequence that is at least 70% identical to SEQ ID NO: 146, 149, or 154.

51. 51. The composition or method of any one of claims 46-50, wherein the promoter operably linked to the engineered nucleic acid encoding tTA and / or rtTA is not a synapsin-I promoter, is not a CaMKII-gamma promoter, or is a combination thereof, optionally wherein the synapsin-I promoter comprises a sequence that is at least 70% identical to SEQ ID NO:

157.

52. 52. The composition or method of any one of claims 1 to 51, wherein the composition is administered via retro-orbital vein injection.

53. 52. The composition or method of any one of claims 1 to 51, wherein the composition is administered via intrathecal administration.

54. 52. The composition or method of any one of claims 1 to 51, wherein the composition is administered systemically, optionally wherein the systemic injection is an intravenous injection.

55. 55. The composition or method of any one of claims 1 to 54, wherein the composition is not administered to the retina of the subject.

56. 56. The composition or method of any one of claims 1 to 55, wherein the composition is used to improve cognitive function in a subject.

57. 57. The composition or method of any one of claims 1 to 56, wherein the composition is used to improve memory in a subject.

58. The composition or method of any one of claims 1 to 57, wherein the composition does not comprise a nucleic acid having a synapsin-I promoter, does not comprise a CaMKII-gamma promoter, or a combination thereof, optionally wherein the synapsin-I promoter comprises a sequence that is at least 70% identical to SEQ ID NO:

157.

59. 59. The composition or method of any one of claims 1 to 58, wherein the composition is administered to a subject having or suspected of having a neurological disorder.

60. 60. The composition or method of claim 59, wherein the neurological disorder is a neurodegenerative disorder.

61. 61. The composition or method of claim 60, wherein the neurodegenerative disorder is Alzheimer's disease, Parkinson's disease, dementia, Friedreich's ataxia, amyotrophic lateral sclerosis, or vascular dementia.

62. 62. The composition or method of any one of claims 1 to 61, wherein the composition is a pharmaceutical composition.

63. The composition comprises: (a) a viral vector comprising a nucleic acid encoding a tetracycline-regulated transactivator (tTA) or a reverse tetracycline-regulated transactivator (rtTA), wherein the nucleic acid encoding the tTA or the rtTA is operably linked to a CaMKIIα promoter, optionally, wherein the viral vector is an AAV vector, and optionally, wherein the AAV vector is packaged into an AAV-PHP.eB virus; and (b) a viral vector comprising a first nucleic acid encoding OCT4, a second nucleic acid encoding SOX2, and a third nucleic acid encoding KLF4, wherein the first, second, and third nucleic acids are operably linked to a promoter comprising a tetracycline responsive element (TRE), optionally wherein the viral vector is an AAV vector, and optionally wherein the AAV vector is packaged into an AAV-PHP.eB virus; 63. The composition or method of any of claims 1 to 62, comprising:

64. The composition or method of claim 63, wherein the vector (a) comprises a WPRE sequence and / or an hGH pA terminator sequence, optionally wherein the WPRE sequence comprises a sequence that is at least 70% identical to SEQ ID NO: 21, 135, 147, 152, 155, or 160, and / or wherein the hGH pA terminator sequence comprises a sequence that is at least 70% identical to SEQ ID NO: 139, 148, 153, 156, or 161.

65. 64. The composition or method of claim 63, wherein the viral vector in (a) is the same viral vector as in (b).

66. 66. The composition or method of any of claims 1-65, wherein the composition comprises a sequence that is at least 70% identical to SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:121, SEQ ID NO:123, or SEQ ID NO:

163.

67. 66. The composition or method of any one of claims 1-29, 32-33, 35-42, 45-48, 51-62, and 65, wherein the composition comprises a sequence that is at least 70% identical to SEQ ID NO:

123.

68. 67. The composition or method of any one of claims 1 to 66, wherein the composition comprises SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:137, SEQ ID NO:158, SEQ ID NO:17, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:146, 149, or 154, SEQ ID NO:135, 147, 152, 155, or 160, SEQ ID NO:139, 148, 153, 156, or 161, or a sequence which is at least 70% identical to SEQ ID NO:126, optionally wherein the composition does not comprise a sequence which is at least 70% identical to SEQ ID NO:

127.

69. 66. The composition or method of any one of claims 63 to 65, wherein the viral vector of part (a) comprises SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:137, SEQ ID NO:158, SEQ ID NO:17, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:146, 149, or 154, SEQ ID NO:135, 147, 152, 155, or 160, SEQ ID NO:139, 148, 153, 156, or 161, or a sequence that is at least 70% identical to SEQ ID NO:

126.

70. 70. The composition or method of any one of claims 63 to 65 and 69, wherein the viral vector of part (b) comprises a sequence that is at least 70% identical to SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:121, SEQ ID NO:123, or SEQ ID NO:

163.

71. The composition or method of any of claims 1 to 70, wherein the composition further comprises a pharma- ceutically acceptable carrier.

72. An expression vector comprising a sequence which is at least 70% identical to a sequence selected from SEQ ID NOs: 123-127.

73. 73. A recombinant virus comprising the expression vector of claim 72.

74. 74. An engineered cell, tissue or organ of the central nervous system comprising a composition according to any one of claims 1, 3-51, 58 and 62-71, an expression vector according to claim 72, or a recombinant virus according to claim 73.

75. A kit comprising: (a) a container containing the composition according to any one of claims 1, 3 to 51, 58, and 62 to 68, the expression vector according to claim 72, or the recombinant virus according to claim 73, and (b) instructions for rejuvenating a cell, tissue, or organ of the central nervous system, optionally in a subject in need thereof, optionally where the cell, tissue, or organ from the central nervous system is a brain cell, brain tissue, or brain. Including the kit.

76. A kit comprising: (a) a first container housing a viral vector comprising a nucleic acid encoding a tetracycline-regulated transactivator (tTA) or a reverse tetracycline-regulated transactivator (rtTA), wherein the nucleic acid encoding the tTA or rtTA is operably linked to a CaMKIIα promoter; (b) a second container housing a viral vector comprising a first nucleic acid encoding OCT4, a second nucleic acid encoding SOX2, and a third nucleic acid encoding KLF4, wherein the first, second, and third nucleic acids are operably linked to a promoter comprising a tetracycline responsive element (TRE); and (c) instructions for rejuvenating a cell, tissue, or organ, optionally in a subject in need thereof, optionally wherein the viral vector of (a) and / or (b) is an AAV vector, optionally wherein the AAV vector is packaged into an AAV-PHP.b virus, optionally wherein the AAV-PHP.b virus is an AAV.PHP.eB virus, and optionally wherein the cell, tissue, or organ from the central nervous system is a brain cell, brain tissue, or brain. Including the kit.

77. 77. The kit of claim 76, wherein the viral vector of part (a) comprises SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:137, SEQ ID NO:158, SEQ ID NO:17, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:146, 149, or 154, SEQ ID NO:135, 147, 152, 155, or 160, SEQ ID NO:139, 148, 153, 156, or 161, or a sequence that is at least 70% identical to SEQ ID NO:

126.

78. 78. The kit of claim 76 or 77, wherein the viral vector of part (b) comprises a sequence that is at least 70% identical to SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:121, SEQ ID NO:123, or SEQ ID NO:

163.

79. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of any one of claims 1 to 78, wherein KLF4 comprises a sequence that is at least 70% identical to SEQ ID NO:6 or SEQ ID NO:

45.

80. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of any one of claims 1 to 79, wherein SOX2 comprises a sequence that is at least 70% identical to SEQ ID NO:4 or SEQ ID NO:

43.

81. 61. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of any one of claims 1 to 60, wherein OCT4 comprises a sequence that is at least 70% identical to SEQ ID NO:2 or SEQ ID NO:

41.

82. The composition, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit comprises: (a) a nucleic acid sequence encoding OCT4, KLF4, and SOX2 operably linked to a TRE promoter; And (b) a nucleic acid sequence encoding rtTA operably linked to a UbC promoter; 82. The composition, method, expression vector, recombinant virus, engineered cell, tissue or organ, or kit of any one of claims 1 to 81, comprising a nucleic acid having the formula:

83. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of claim 82, wherein the nucleic acid sequences encoding OCT4, KLF4, and SOX2 further encode a 2A peptide.

84. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of claim 82 or 83, wherein the nucleic acid having (a) and (b) further encodes a neomycin resistance gene and / or comprises a WPRE sequence.

85. 85. The composition, method, expression vector, recombinant virus, engineered cell, tissue or organ, or kit of claim 84, wherein the neomycin resistance gene is operably linked to the PGK promoter.

86. A nucleic acid having (a) and (b), (a) rtTA Advanced (SEQ ID NO: 129); (b) human OCT4 (SEQ ID NO:41); (c) P2A (SEQ ID NO: 118); (d) human SOX2 (SEQ ID NO: 43); (e) T2A (SEQ ID NO: 9); (f) human KLF4 (SEQ ID NO: 45); and (g) neomycin resistance gene (SEQ ID NO: 134); The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of any one of claims 81 to 85, encoding at least one protein sequence that is at least 70% identical to a sequence selected from:

87. A nucleic acid having (a) and (b), (a) reverse complement of rtTA Advanced (SEQ ID NO: 128); (b) the reverse complement of the UbC promoter (SEQ ID NO: 130); (c) P tight TRE promoter (SEQ ID NO:24); (d) human OCT4 (SEQ ID NO: 40); (e) P2A (SEQ ID NO: 119); (f) human SOX2 (SEQ ID NO: 42); (g) T2A (SEQ ID NO: 120); (h) human KLF4 (SEQ ID NO: 131); (i) PGK promoter (SEQ ID NO: 132); (j) a neomycin resistance gene (SEQ ID NO: 133); and (k) WPRE (SEQ ID NO: 135), The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of any one of claims 81 to 86, comprising at least one sequence that is at least 70% identical to a sequence selected from:

88. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of any one of claims 81-86, wherein the nucleic acid having (a) and (b) comprises a sequence that is at least 70% identical to pLVX-rtTA-hOSK-all-in-one(human) (SEQ ID NO: 123).

89. 82. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of any one of claims 1 to 81, wherein the composition, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit comprises a nucleic acid encoding tTA, wherein tTA comprises a sequence that is at least 70% identical to SEQ ID NO:

138.

90. 90. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of claim 89, wherein the nucleic acid encoding tTA is at least 70% identical to SEQ ID NO:

137.

91. 90. The composition, method, expression vector, recombinant virus, engineered cell, tissue or organ, or kit of claim 88 or 89, wherein the nucleic acid encoding the tTA comprises an hGH pA sequence and / or a CMV promoter.

92. 92. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of claim 91, wherein the hGH pA sequence is at least 70% identical to SEQ ID NO:

139.

93. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of claim 91 or 92, wherein the CMV promoter is at least 70% identical to SEQ ID NO:

136.

94. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of any one of claims 89-93, wherein the nucleic acid encoding tTA comprises a sequence that is at least 70% identical to pAAV-CMV-tTA(Advanced) (SEQ ID NO:32).

95. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of any one of claims 1 to 81 and 89 to 94, wherein the composition, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit comprises nucleic acids encoding OCT4, SOX2, and KLF4 operably linked to a TRE promoter.

96. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of claim 95, wherein the nucleic acid sequence encoding OCT4, SOX2, and KLF4 operably linked to the TRE promoter further encodes a 2A peptide and / or SV40 pA.

97. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of claim 96, wherein the 2A peptide comprises a sequence that is at least 70% identical to T2A (sequence number 9) or P2A (sequence number 118).

98. Nucleic acid sequences encoding OCT4, SOX2, and KLF4 are (a) mouse OCT4 (SEQ ID NO:2); (b) human OCT4 (SEQ ID NO: 40); (c) mouse SOX2 (SEQ ID NO: 4); (d) human SOX2 (SEQ ID NO: 42); (e) human KLF4 (SEQ ID NO: 131); and (f) mouse KLF4 (SEQ ID NO: 6); 98. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of claim 97, encoding at least one sequence that is 70% identical to a sequence selected from:

99. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of any one of claims 95 to 98, wherein the TRE promoter operably linked to a nucleic acid sequence encoding OCT4, SOX2, and KLF4 is at least 70% identical to SEQ ID NO:

7.

100. A nucleic acid sequence encoding OCT4, SOX2, and KLF4 operably linked to a TRE promoter, (a) TRE3G (SEQ ID NO: 7): (b) Mouse Oct4 (SEQ ID NO: 1): (c) P2A (SEQ ID NO: 144); (d) mouse Klf4 (SEQ ID NO: 145); (e) SV40 pA (SEQ ID NO: 143); (f) mouse Sox2 (SEQ ID NO:3); and (g) T2A (SEQ ID NO: 120), 100. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of any one of claims 95 to 99, comprising at least one sequence that is 70% identical to a sequence selected from:

101. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of any one of claims 1 to 81 and 89 to 100, wherein the composition, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit comprises a nucleic acid encoding an inducer operably linked to the CaMKIIα promoter.

102. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of claim 101, wherein the inducer is tTA or rtTA.

103. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of claim 102, wherein the inducer comprises a sequence that is at least 70% identical to tTA Advanced (SEQ ID NO: 138), rtTA2S-M2 (SEQ ID NO: 15), or rtTA3 (SEQ ID NO: 11).

104. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of any one of claims 101 to 103, wherein the CaMKIIα promoter comprises a sequence that is at least 70% identical to SEQ ID NO:146, SEQ ID NO:149, or SEQ ID NO:

154.

105. The composition, method, expression vector, recombinant virus, engineered cell, tissue or organ, or kit of any one of claims 101 to 104, wherein the nucleic acid encoding the inducer further comprises a WPRE and / or an hGH pA sequence.

106. The composition, method, expression vector, recombinant virus, engineered cell, tissue or organ, or kit of any one of claims 105, wherein the WPRE sequence is at least 70% identical to SEQ ID NO: 147, 152, or 155.

107. 107. The composition, method, expression vector, recombinant virus, engineered cell, tissue or organ, or kit of claim 105 or 106, wherein the hGH pA sequence is at least 70% identical to SEQ ID NO: 148, 153, or 156.

108. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of any one of claims 101 to 107, wherein the nucleic acid encoding the inducer comprises a sequence that is at least 70% identical to a sequence selected from tTA-Advanced (SEQ ID NO: 137), rtTA2S-M2 (SEQ ID NO: 14), or rtTA3 (SEQ ID NO: 10).

109. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of any one of claims 101-108, wherein the nucleic acid encoding the inducer comprises a sequence that is at least 70% identical to a sequence selected from pAAV-CaMKIIα-tTA2 (SEQ ID NO: 124), pAAV-CaMKIIα-rtTA2S-M2 (SEQ ID NO: 125), or pAAV-CaMKIIα-rtTA3 (SEQ ID NO: 126).

110. 110. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of any one of claims 1 to 109, wherein the composition, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit does not comprise a synapsin-I promoter operably linked to a nucleic acid sequence encoding an inducer.

111. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of claim 110, wherein the composition, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit does not contain a sequence that is at least 70% identical to SEQ ID NO:

157.

112. The composition, method, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit of claim 111, wherein the composition, expression vector, recombinant virus, engineered cell, tissue, or organ, or kit does not contain a sequence that is at least 70% identical to pAAV-ihSyn1-tTA (sequence number 127).

113. Nervous system cells, central nervous system tissues, or central nervous system organs, a) Factors that induce OCT4 expression; b) a factor that induces SOX2 expression; and c) factors that induce KLF4 expression; Including, wherein the nervous system cells, central nervous system tissues, or central nervous system organs do not contain factors that induce c-MYC expression; An animal comprising a central nervous system cell, tissue, or organ.

114. 1. A method of rejuvenating a cell, tissue, and / or organ comprising administering to the cell, tissue, or organ a composition, the composition comprising: (a) Factors that induce OCT4 expression; (b) a factor that induces SOX2 expression; and (c) Factors that induce KLF4 expression; Including, wherein the composition does not comprise a factor that induces c-MYC expression, wherein the cell, tissue, or organ is a central nervous system cell, central nervous system tissue, or central nervous system organ, optionally, wherein the central nervous system does not include a retina, and optionally, wherein the cell, tissue, or organ is a brain cell, brain tissue, or brain; wherein the method increases expression of one or more of the following sets of genes: associative learning, excitatory synapse assembly, central nervous system neuron axonogenesis, central nervous system neurogenesis, memory, control of GABAergic synaptic transmission, control of postsynaptic organization, learning, control of neurogenesis, central nervous system neuron differentiation, and / or synapse maturation. method.

115. 1. A method of rejuvenating a cell, tissue, and / or organ comprising administering to the cell, tissue, or organ a composition, the composition comprising: (a) Factors that induce OCT4 expression; (b) a factor that induces SOX2 expression; and (c) Factors that induce KLF4 expression; Including, wherein the composition does not contain a factor that induces c-MYC expression, wherein the cell, tissue, or organ is a central nervous system cell, central nervous system tissue, or central nervous system organ, optionally wherein the central nervous system does not include the retina, and optionally wherein the cell, tissue, or organ is a brain cell, brain tissue, or brain; wherein the method increases hypomethylated CpG sites in gene loci involved in positive regulation of epidermal development, midgut development, negative regulation of chromatin organization, negative regulation of histone methylation, camera eye lens development, negative regulation of adipocyte differentiation, negative regulation of histone modifications, lung morphogenesis, DNA methylation of cytosines, and / or development associated with neural progenitor cell proliferation; and / or wherein the method increases hypermethylated CpG sites in developmentally relevant loci associated with skeletal muscle cell differentiation, cardiac looping, cardiac left-right asymmetry determination, uterine morphogenesis, regulation of the Notch signaling pathway, embryonic cranial skeletal morphogenesis, animal organogenesis, tricuspid valve morphogenesis, lens fiber cell differentiation, and / or positive regulation of the BMP signaling pathway; method.

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