Mutant reverse tetracycline transactivator for gene expression

Abstract

This article provides information on mutant reverse tetracycline transactivator (rtTA) protein and engineered nucleic acids (e.g., viral vectors, including lentiviral vectors, adenovirus vectors, AAV vectors, herpesvirus vectors, and retroviral vectors; and non-viral vectors, including RNA and plasmid DNA) encoding mutant rtTA, which can be used for, for example, regulating gene expression, inducing cell reprogramming, tissue repair, tissue regeneration, organ regeneration, reversing aging, treating diseases (e.g., acute injury, neurodegenerative diseases, chronic diseases, proliferative diseases, cardiovascular diseases, genetic diseases, inflammatory diseases, autoimmune diseases, neurological diseases, hematological diseases, pain conditions, mental disorders, metabolic disorders, cancer, aging, age-related diseases, and diseases affecting any tissue in a subject), or any combination thereof. This article also provides recombinant viruses comprising engineered nucleic acids (e.g., lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAVs), and methods for modulating (e.g., inhibiting or inducing) cell reprogramming, tissue repair, tissue regeneration, or any combination thereof by applying engineered nucleic acids or recombinant viruses comprising them to cells, tissues, or subjects (e.g., cells or tissues of subjects with a condition, said condition including any disease (e.g., eye disease), aging, neurodegenerative diseases, cancer, and age-related diseases), said application including the application of a mutant rtTA and an inducible nucleic acid encoding a transgene (e.g., engineered nucleic acid, including an expression vector).

Description

[0001] Related applications

[0002] This patent application claims the benefit of U.S. Provisional Application No. 62 / 738,894, filed September 28, 2018, pursuant to 35 U.S. SC §119(e), which is incorporated herein by reference in its entirety. Background Technology

[0003] Inducible gene expression holds immense promise for gene therapy and other biomedical applications. On-demand and tightly regulated gene expression can avoid toxicities associated with prolonged expression time or with supraphysiological expression of exogenous genes.

[0004] For example, the tetracycline-on (Tet-On) system typically utilizes reverse tetracycline transactivators (rtTA) to induce gene expression. Reverse tetracycline transactivators (rtTA) comprise a mutant tetracycline repressor DNA-binding protein (TetR) and a transactivation domain (e.g., Gossen et al., Science. 23 June 1995; 268(5218):1766-9). These transactivators can be activated in the presence of tetracycline (e.g., doxycycline) and subsequently bind to promoters containing tetracycline response elements (TREs) to induce gene expression (Gossen et al., Science. 23 June 1995; 268(5218):1766-9); Baron et al., Methods Enzymol. 2000; 327:401-21. The TRE contains at least one Tet operon (Tet-O) sequence (e.g., multiple repeats of the Tet-O sequence) and may be located upstream of a minimal promoter (e.g., a minimal promoter sequence derived from the immediate early promoter of human cytomegalovirus (hCMV)).

[0005] However, the tendency of Tet-On systems to initiate gene expression even in the absence of tetracycline (i.e., "leakage") hinders their use. Therefore, there is a need for improved Tet-On systems with little or no leakage and high tetracycline sensitivity. Invention Overview

[0007] This disclosure stems from the unexpected discovery that four mutations in residues corresponding to positions G72, G12, F67, and R171 in rtTA3 (SEQ ID NO:11) significantly increased the sensitivity of the Tet-On system in vivo and reduced leakage of the Tet-On system. In some embodiments, this document provides mutant rtTA (e.g., rtTA4), engineered nucleic acids encoding them (e.g., expression vectors, including viral and non-viral vectors), recombinant viruses (e.g., lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAVs) containing engineered nucleic acids (e.g., expression vectors), pharmaceutical compositions, and kits thereof. In some embodiments, the engineered nucleic acid encoding mutant rtTA4 also encodes a transgene (e.g., a protein-coding sequence, a gene-targeting nucleic acid, and / or a therapeutic sequence) operatively linked to a tetracycline response element (TRE) promoter. In some embodiments, the pharmaceutical composition and kit further comprise a second vector (e.g., multiple second vectors) or a second recombinant virus (e.g., multiple second recombinant viruses) containing a tetracycline response element (TRE) promoter operably linked to the transgene (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV). In some embodiments, the pharmaceutical composition and kit further comprise multiple vectors or multiple recombinant viruses (e.g., lentivirus, vaccinia virus, alphavirus, adenovirus, retrovirus, herpesvirus, or adeno-associated virus (AAV)) containing a tetracycline response element (TRE) promoter operably linked to the transgene. This document also provides methods for promoting gene expression comprising administering (1) any engineered nucleic acid (e.g., expression vector) encoding a mutant rtTA (e.g., rtTA4) as described herein, and (2) tetracycline, to cells, tissues, or a subject in need of such expression (e.g., a cell or tissue). In some embodiments, the method further comprises administering (3) a second nucleic acid (e.g., an engineered nucleic acid, including an expression vector) containing a TRE promoter operably linked to the transgene. In some embodiments, multiple second engineered nucleic acids (e.g., expression vectors) are administered.

[0008] The mutant rtTAs (e.g., rtTA4), engineered nucleic acids (e.g., expression vectors), recombinant viruses, pharmaceutical compositions, kits, and methods described herein can be used to regulate gene expression in vivo. Without being confined by any particular theory, the reduced leakage improves the toxicity profile of the rtTA4 Tet-On system compared to the rtTA3 Tet-On system and allows for transient transgene expression.

[0009] This disclosure provides a mutant rtTA (e.g., rtTA4). The mutant rtTA of this disclosure contains four mutations corresponding to positions G72, G12, F67 and R171 of rtTA3 (SEQ ID NO:11), and such a mutant rtTA containing the four mutations at these positions is referred to as rtTA4.

[0010] In some embodiments, the mutant rtTA further comprises at least one (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, or 240) mutation of residues corresponding to the position in rtTA3 (SEQ ID NO: 11), said mutation being not G72, G12, F67, or R171.

[0011] In some implementations, the mutation of the residue corresponding to the position in rtTA3 (SEQ ID NO:11) (i.e., not at position G72, G12, F67 or R171) is a point mutation, a truncated mutation, a deletion or an insertion.

[0012] In some implementations, G72 is mutated to G72V, G72I, G72L or G72P; G12 is mutated to G12S or G12T; F67 is mutated to F67S or F67T; and R171 is mutated to R171K or R171H.

[0013] In some embodiments, the four mutations are G72V or G72P, G12S, F67S, and R171K. The amino acid sequence encoding the mutant rtTA (e.g., rtTA4) may contain at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) of the sequence identical to SEQ ID NO:13, and contains mutations at residues corresponding to the following positions in rtTA3 (SEQ ID NO:11): G72, G12, F67, and R171. The nucleic acid encoding the mutant rtTA (e.g., rtTA4) may be codon-optimized and may contain at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) of the sequence identical to SEQ ID NO:12.

[0014] In another aspect of this disclosure, the nucleic acid (e.g., engineered nucleic acid, including expression vectors) (e.g., viral expression vectors, including lentiviruses, retroviruses, adenoviruses, herpesviruses, or adeno-associated viruses (AAVs)) comprises a nucleic acid sequence encoding a mutant rtTA (e.g., rtTA4) operatively linked to a promoter (e.g., a constitutive promoter or a tissue-specific promoter). In some embodiments, the nucleic acid (e.g., engineered nucleic acid, including expression vectors) encoding the mutant rtTA (e.g., rtTA4) 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:17. In some embodiments, the nucleic acid (e.g., engineered nucleic acid, including expression vectors) encoding the mutant rtTA (e.g., rtTA4) is composed of SEQ ID NO:17. In some embodiments, the nucleic acid encoding the mutant rtTA (e.g., rtTA4) (e.g., engineered nucleic acid, including an expression vector) comprises at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) the same sequence as the desmin-rtTA4 vector (SEQ ID NO:30). In some embodiments, the nucleic acid encoding the mutant rtTA (e.g., rtTA4) (e.g., engineered nucleic acid, including an expression vector) is composed of SEQ ID NO:30.

[0015] The promoter operatively linked to the nucleic acid encoding the mutant rtTA (e.g., rtTA4) can be a constitutive promoter (e.g., CP1, CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, Ac5, polyhedrin, TEF1, GDS, CaM3 5S, Ubi, H1, or U6 promoter). To allow for tissue-specific expression of rtTA, the promoter can be tissue-specific (e.g., eye-specific, bone-specific, lung-specific, breast-specific, pancreas-specific, muscle-specific, liver-specific, skin-specific, heart-specific, brain-specific, nerve tissue-specific, kidney-specific, testis-specific, ovary-specific, or intestinal-specific promoter).

[0016] In some embodiments, the nucleic acid encoding the mutant rtTA (e.g., rtTA4) (e.g., engineered nucleic acid, including expression vectors) also encodes a tetracycline repressor (e.g., tetR, tetRKRAB, TRSID), which can prevent rtTA from binding to the TRE promoter in the absence of tetracycline. In some embodiments, the nucleic acid sequence encoding the mutant rtTA (e.g., rtTA4) and the nucleic acid sequence encoding the tetracycline repressor (e.g., tetRKRAB) are operatively linked to the same promoter. In some embodiments, a spacer sequence (e.g., an internal ribosome entry site (IRES) or a 2A peptide) is present in the nucleic acid (e.g., engineered nucleic acid, including expression vectors) and separates at least two nucleic acid sequences, which can facilitate the generation of two separate amino acid sequences from a single expression vector.

[0017] In some embodiments, the nucleic acid encoding the mutant rtTA (e.g., rtTA4) (e.g., engineered nucleic acid, including expression vectors) also contains a marmot hepatitis virus (WHP) post-transcriptional regulatory element (WPRE), which can be used to enhance transgene expression (e.g., derived from a viral vector). In some 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.

[0018] In some embodiments, the nucleic acid encoding the mutant rtTA (e.g., rtTA4) (e.g., engineered nucleic acid, including expression vectors) is a viral vector (e.g., lentiviral vector, adenovirus vector, vaccinia virus, alphavirus, adeno-associated virus vector, adeno-associated virus (AAV) vector, or retroviral vector). In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 vector. The viral vector (e.g., the AAV vector) may also contain terminal inverted repeat sequences (ITRs). In some embodiments, the ITR contains a sequence that is at least 70% identical (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:22.

[0019] Another aspect of this disclosure provides recombinant viruses carrying engineered nucleic acids (e.g., expression vectors) that encode any mutant rtTA (e.g., rtTA4).

[0020] In another aspect of this disclosure, a pharmaceutical composition comprising any mutant rtTA (e.g., rtTA4) and a pharmaceutically acceptable excipient is provided. The pharmaceutical composition may also comprise a second nucleic acid encoding a transgene (e.g., an engineered nucleic acid, including an expression vector). The second nucleic acid (e.g., an engineered nucleic acid, including an expression vector) comprises a tetracycline response element (TRE) promoter operatively linked to the transgene (e.g., a TRE3G sequence identical to at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of SEQ ID NO:7). In some embodiments, the TRE promoter is a TRE2 or Ptight promoter. In some embodiments, the TRE promoter is a TRE2 promoter and comprises a sequence identical to at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of SEQ ID NO:23. In some implementations, the TRE promoter is the Ptight promoter and contains at least 70%, 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% of the same sequence as SEQ ID NO:24.

[0021] In some embodiments, the transgene is any protein-coding gene. In some embodiments, the transgene is a gene-targeting nucleic acid. In some embodiments, the transgene is a therapeutic sequence. In some embodiments, the therapeutic sequence can be used to treat acute injuries, neurodegenerative diseases, chronic diseases, proliferative diseases, cardiovascular diseases, genetic diseases, inflammatory diseases, autoimmune diseases, neurological diseases, hematological diseases, pain conditions, mental disorders, metabolic disorders, cancer, aging, age-related diseases, and diseases affecting any tissue in the subject.

[0022] In some implementations, the inducible nucleic acid (e.g., engineered nucleic acid, including expression vectors) (e.g., viral vectors) encodes OCT4, SOX2, and KLF4. See, for example, U.S. Provisional Application No. H0824.70296US00, filed September 28, 2018, entitled "CELLULAR REPROGRAMMING TO REVERSE AGING AND PROMOTE ORGAN AND TISSUEREGENERATION"; USSN 62 / 792,283, filed January 14, 2019, entitled "CELLULAR REPROGRAMMING TO REVERSE AGING AND PROMOTE ORGAN AND TISSUEREGENERATION"; USSN 62 / 865,877, filed June 24, 2019, entitled "CELLULAR REPROGRAMMING TO REVERSE AGING AND PROMOTE ORGAN AND TISSUE REGENERATION"; and USSN 62 / 880,488, filed June 24, 2019, entitled "CELLULAR REPROGRAMMING TO REVERSE AGING AND TISSUEREGENERATION". PROMOTE ORGAN AND TISSUE REGENERATION, filed July 30, 2019; and PCT application entitled CELLULAR REPROGRAMMING TO REVERSE AGING AND PROMOTE ORGAN AND TISSUEREGENERATION, Agent's Case No. H0824.70296WO00, filed on the same day as this application, are each incorporated herein by reference in their entirety.

[0023] In some embodiments, the nucleic acid encoding the mutant rtTA (e.g., rtTA4) (e.g., engineered nucleic acid, including an expression vector) and the nucleic acid encoding the transgene operatively linked to the TRE promoter (e.g., engineered nucleic acid, including an expression vector) are both viral vectors and are contained in a virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV). In some embodiments, the pharmaceutical composition comprises two viral vectors and / or two viruses.

[0024] In another aspect of this disclosure, cells and / or systems are provided that comprise any mutant rtTA (e.g., rtTA4), any engineered nucleic acid (e.g., expression vector) encoding the mutant rtTA and / or a transgene operatively linked to a TRE promoter, and / or any virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV).

[0025] In another aspect of this disclosure, a kit is provided comprising any mutant rtTA (e.g., rtTA4) described herein, any engineered nucleic acid (e.g., expression vector) encoding the mutant rtTA, any virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV), cells, systems, and / or any pharmaceutical composition.

[0026] Another aspect of this disclosure provides a method for promoting gene expression, comprising administering to cells, tissues, or a subject in need of it (1) any mutant rtTA (e.g., rtTA4), any engineered nucleic acid (e.g., expression vector) encoding a mutant rtTA (e.g., rtTA4), or a recombinant virus comprising any engineered nucleic acid (e.g., expression vector) encoding a mutant rtTA; (2) any engineered nucleic acid (e.g., expression vector) encoding a transgene operatively linked to a TRE promoter; and (3) a tetracycline (e.g., doxycycline). In some embodiments, the engineered nucleic acid (e.g., expression vector) encoding a mutant rtTA described herein, or the recombinant virus comprising any mutant rtTA expression vector, also encodes a tetracycline repressor (e.g., tetRKRAB). In some embodiments, the nucleic acid (e.g., engineered nucleic acid, including an expression vector) encoding a mutant rtTA of this disclosure, or the recombinant virus, also comprises a transgene operatively linked to a TRE promoter (e.g., TRE3G, TRE2, or P tight promoter). The method may also include withdrawal (i.e., cessation of tetracycline administration).

[0027] In some embodiments, the subject is a mammal (e.g., human or non-human). In some embodiments, the subject suffers from a disease (e.g., acute injury, neurodegenerative disease, chronic disease, proliferative disease, cardiovascular disease, genetic disease, inflammatory disease, autoimmune disease, neurological disease, hematological disease, pain condition, mental disorder, metabolic disorder, cancer, aging, age-related disease, and any disease affecting any tissue in the subject). In some embodiments, the method includes modulating cell reprogramming, tissue repair, treating a disease (e.g., acute injury, neurodegenerative disease, chronic disease, proliferative disease, cardiovascular disease, genetic disease, inflammatory disease, autoimmune disease, neurological disease, hematological disease, pain condition, mental disorder, metabolic disorder, cancer, aging, age-related disease, and any disease affecting any tissue in the subject), tissue regeneration, organ regeneration, reversing aging, or any combination thereof.

[0028] This document sets forth details of one or more embodiments of the present invention. Other features, objects, and advantages of the invention will become apparent from the detailed description, examples, drawings, and claims.

[0029] References cited in this application are incorporated herein by reference.

[0030] definition

[0031] “AAV” or “adeno-associated virus” is a non-enveloped virus capable of carrying and delivering nucleic acids (e.g., nucleic acids encoding transgenes, mutant rtTA4, or any combination thereof) and belongs to the genus Dependoparvovirus. Typically, AAVs do not integrate into the genome. The tissue-specific targeting ability of AAVs is generally determined by the AAV capsid serotype (see, for example, Table 1 below for examples of AAV serotypes and their utility in tissue-specific delivery). Non-restricted AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and their variants. In some embodiments, the AAV serotype is a variant of AAV9 (e.g., AAV PHP.b).

[0032] "Recombinant viruses" are viruses that have been isolated from their natural environment (e.g., from host cells, tissues, or subjects) or artificially generated (e.g., lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or adeno-associated viruses (AAVs)).

[0033] As used herein, the term "AAV vector" refers to a nucleic acid containing an AAV inverted terminal repeat (ITR) sequence side-attached to an expression cassette (e.g., an expression cassette containing a nucleic acid encoding a transgene alone or in combination, or an expression cassette encoding rtTA or tTA). An AAV vector may also contain a promoter sequence.

[0034] As used herein, the terms “application,” “administration,” or “giving” refer to the introduction of nucleic acids (e.g., engineered nucleic acids encoding transgenes and / or mutant rtTA), recombinant cells, recombinant viruses (e.g., lentiviruses, adenoviruses, alphaviruses, vacciniaviruses, retroviruses, herpesviruses, or AAVs), mutant rtTA (e.g., rtTA4), or any combination thereof or pharmaceutical compositions thereof. Engineered nucleic acids, recombinant cells, mutant rtTA4, viruses (e.g., lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAVs), or pharmaceutical compositions thereof, can be administered intravenously, intradermally, intra-arterially, intralesionally, intracranially, intra-articularly, intraprostatically, intrapleurally, intranasally, intravitreally, intravaginally, intrarectally, locally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesically, mucosally, intraperitoneally, intraumbilically, intraocularly, orally, topically, locally, systemically, by injection, infusion, continuous infusion, direct local perfusion of target cells, via catheter, in cream, in lipid composition (e.g., liposomes), or by other methods known to those skilled in the art or any combination thereof (see, for example, Remington's Pharmaceutical Sciences (1990), which is incorporated herein by reference). The nucleic acids disclosed herein (e.g., engineered nucleic acids, including expression vectors), recombinant cells, mutant rtTA proteins, or recombinant viruses (e.g., lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAVs) can be administered to any tissue, cell, organ, or subject.

[0035] The term "epigenome" or "epigenetics" refers to intracellular modifications and structural changes that control the expression of nucleic acids (such as engineered nucleic acids) or genomic information within cells. Epigenome alterations occur during embryonic development, disease progression, and aging, and drive these processes.

[0036] The term "cellular senescence" refers to cells that have exited the cell cycle and exhibit epigenetic markers consistent with senescence, or express senescence cellular markers such as senescence-associated β-galactosidases or inflammatory cytokines. Cellular senescence can be partial or complete.

[0037] The term "gene expression" refers to the degree to which a specific gene or all genes in a cell or tissue are transcribed into RNA. In some cases, RNA is translated into protein by the cell. The epigenome controls gene expression patterns.

[0038] The term "cell reprogramming" refers to the process of altering the epigenome of a cell using reprogramming factors (e.g., reversing or preventing epigenetic changes in cells that are the cause of dysfunction, deterioration, cell death, senescence, or aging). Cell reprogramming can be complete, reprogramming differentiated cells (e.g., somatic cells) into pluripotent stem cells. Cell reprogramming can be incomplete, allowing differentiated cells (e.g., somatic cells) to retain their cell species (e.g., lineage-specific stem cells). Cell reprogramming can be incomplete (e.g., failing to generate stem cells), allowing cells to regain vitality or exhibit more youthful properties (e.g., increased survival, reduced inflammation, or increased ability to divide). Cell reprogramming can provide additional cellular function or prevent cellular aging (e.g., transdifferentiation or transition to senescence). Cell reprogramming can induce transient or permanent changes in gene expression. In some embodiments, incomplete cell reprogramming is characterized by a lack of Nanog expression. In some embodiments, cell reprogramming prevents aging.

[0039] The terms “condition,” “disease,” and “disorder” are used interchangeably. Non-limiting examples of conditions, diseases, and disorders include acute injury, neurodegenerative diseases, chronic diseases, proliferative diseases, cardiovascular diseases, genetic diseases, inflammatory diseases, autoimmune diseases, neurological diseases, hematological diseases, pain conditions, mental disorders, metabolic disorders, cancer, aging, age-related diseases, and diseases affecting any tissue in the subject. For example, age-related conditions include heart failure, stroke, heart disease, atherosclerosis, neurodegenerative diseases (e.g., Parkinson's disease and Alzheimer's disease), cognitive decline, memory loss, diabetes, osteoporosis, arthritis, muscle loss, hearing loss (partial or complete), eye-related conditions (e.g., vision loss or retinal disease), glaucoma, and cancer. In some 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, aging of glial cells can be a cause of Alzheimer's disease. See, for example, Bussian et al., Nature. September 19, 2018.

[0040] As used herein, “eye disease” or “eye condition” refers to a disease or condition of the eye. Non-limiting examples of conditions affecting the eye include ectropion, lagophthalmos, eyelid laxity, ptosis, stye, xanthelasma palpebrarum, dermatitis, Demodex folliculorum, leishmaniasis, Loa filariasis, onchocerciasis, lice infestation (herpes simplex virus), leprosy, molluscum contagiosum, tuberculosis, yaws, herpes zoster, pustular dermatitis, dacryoadenitis, epiphora, exophthalmos, conjunctivitis, scleritis, keratitis, corneal ulcer / corneal abrasion, snow blindness / arc eye, Thygeson's superficial punctate keratopathy, corneal neovascularization, Fuchs' dystrophy, keratoconus, keratoconjunctivitis sicca, iritis, iris, uveitis, sympathetic ophthalmia, cataract, lens, choroidal retinitis, localized... Focal chorioretinitis, choroidal retinitis, choroiditis, retinitis, retinal choroidal retinitis, disseminated chorioretinitis, exudative retinopathy, posterior ciliary body inflammation, pars plana inflammation, choroidal retinitis, Harada's disease, choroidal retinitis, choroid, choroidal retinal scar, punctate scar, posterior pole (post-inflammatory) (post-traumatic), solar retinopathy, choroidal degeneration, atrophy, sclerosis, striae angiosporosis, choroidal trophic disorders, achoroidal syndrome, spiral atrophy of the choroidal areola (periapapillary), choroid, ornithineemia, choroidal hemorrhage, choroidal hemorrhage NOS (unless otherwise stated), choroid Retinal detachment, choroidal retina, choroidal retinal inflammation, infections and parasitic diseases, syphilitic choroidal retinitis, toxoplasmosis, tuberculosis, choroidal retina, retinal detachment, retina, choroid, visual distortion, retinal folds, hypertensive retinopathy, diabetic retinopathy, retinopathy, retinopathy of prematurity, age-related macular degeneration, macula, macular degeneration, bull's eye macular degeneration, epiretinal membrane, peripheral retinal degeneration, hereditary retinal dystrophy, retinitis pigmentosa, subretinal hemorrhage, retinal layer, central serous retinopathy, retinal detachment, macular edema, macular retinal disease, diabetic retinopathy, glaucoma Optic nerve diseases, ocular hypertension, open-angle glaucoma, angle-closure glaucoma, normal-tension glaucoma, floaters, Leber hereditary optic neuropathy, optic disc drusen, strabismus, oculomotor palsy, extraocular muscle, progressive extraocular muscle paralysis, esotropia, exotropia, refractive and accommodative disorders, hyperopia, myopia, astigmatism, anisometropia, presbyopia, extraocular muscle paralysis, amblyopia, Bernoulli's congenital cataract, scotoma, visual impairment, color blindness, achromatopsia / Maskun, cone cells, night blindness, blindness, river blindness, microphthalmia / iris cleft, optic nerve, brain, spinal cord, conjunctivitis, Arrow's pupil, pupil, corneal fungal disease, dry eye syndrome, and aniridia. In some implementations, the eye disease is an acute or chronic eye injury.

[0041] In some implementations, the eye disease is corneal abrasion.

[0042] In some implementations, eye disease is a corneal disease (e.g., a disease that affects the cornea or corneal cells).

[0043] In some implementations, the eye disease is Acanthamoeba keratitis, ectropion, amblyopia, unequal pupils, astigmatism, Bell's palsy, blepharitis, blurred vision, burning eyes, cataracts, macular degeneration, age-related macular degeneration, diabetic retinopathy, glaucoma, dry eye, poor vision (e.g., low vision), astigmatism, blepharitis, cataracts, meibomian gland cysts, conjunctivitis, diabetic retinopathy, dry eye, glaucoma, keratitis, keratoconus, macular degeneration, ocular hypertension, pinguecula, pterygium, retinitis pigmentosa, or ocular cancer (e.g., retinoblastoma, ocular melanoma, ocular lymphoma, medullary epithelioma, 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 implementations, the eye disease is keratoconus. In some implementations, the eye 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., type VII diseases of the eye and its adnexa).

[0044] Eye diseases can affect any part of the eye and / or its appendages. In some embodiments, an eye disease is a disorder of the eyelids, lacrimal system, and / or orbit. In some embodiments, an eye disease is a conjunctival disorder. In some embodiments, an eye disease is a disorder of the sclera, cornea, iris, and / or ciliary body. In some embodiments, an eye disease is a lens disorder. In some embodiments, an eye disease is a choroidal and / or retinal disorder. In some embodiments, an eye disease is glaucoma. In some embodiments, an eye disease is a vitreous and / or eyeball disorder. In some embodiments, an eye disease is a disorder of the optic nerve and / or visual pathway. In some embodiments, an eye disease is a disorder of the extraocular muscles, binocular movements, accommodation, and / or refractive power. In some embodiments, an eye disease is an extraocular muscle disorder, binocular movements, accommodation, and refractive power. In some embodiments, an eye disease is visual impairment and / or blindness.

[0045] The term "hereditary disease" refers to a disease caused by one or more abnormalities in a subject's genome, such as a disease present from birth. Hereditary diseases can be heritable and can be inherited from parents. Hereditary diseases can also be caused by mutations or alterations in a subject's DNA and / or RNA. In such cases, if the hereditary disease occurs phylogenetically, it will be heritable. Exemplary hereditary diseases include, but are not limited to, Aarskog-Scott syndrome, Arthur syndrome, achondroplasia, acrodysplasia, addiction, adrenoleukodystrophy, albinism, congenital macrostomia, Aragog syndrome, alkaptonuria, α-1-antitrypsin deficiency, Allport syndrome, Alzheimer's disease, asthma, autoimmune polyadenomas, androgen insensitivity syndrome, Happy Puppet syndrome, ataxia, ataxia-telangiectasia, atherosclerosis, attention deficit hyperactivity disorder (ADHD), autism, alopecia, Bedon syndrome, and Beckwith. Widman syndrome, Best's disease, bipolar disorder, brachydactyly, breast cancer, Burkitt lymphoma, chronic myeloid leukemia, peroneal muscular dystrophy, Crohn's disease, cleft lip, Coke's syndrome, Cofinlowy syndrome, colon cancer, congenital adrenal hyperplasia, Dylangie syndrome, Kestilo's elastin deficiency, Cowden syndrome, craniofrontal-nasal dysplasia syndrome, Krieger-Naja syndrome, Kuja disease, cystic fibrosis, deafness, depression, diabetes, malformations, Digeorg syndrome, Down syndrome, dyslexia, Duchenne muscular dystrophy, Doppowitz syndrome, ectodermal dysplasia, Elliva syndrome E.D. syndrome, epidermolysis bullosa, epilepsy, essential tremor, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Friedrich's ataxia, Gaucher disease, glaucoma, glucose-galactose malabsorption, glutaric aciduria, spiral atrophy, Sprinz-Goldberg syndrome (jaw-heartface syndrome), Goring syndrome, familial benign chronic pemphigus, hemifacial hypertrophy, hemochromatosis, hemophilia, hereditary motor and sensory neuropathy (HMSN), hereditary nonpolyposis colorectal cancer (HNPCC), Huntington's disease, high IgM immunodeficiency, juvenile diabetes mellitus, Cranfield's disease. Walter syndrome, Kabuki syndrome, Leigh disease, Long QT syndrome, lung cancer, malignant melanoma, bipolar disorder, Marfan syndrome, curly hair syndrome, miscarriage, mucopolysaccharidosis, multiple endocrine neoplasia, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, myotonic dystrophy, multiple neurofibromatosis, Niemann-Pick disease, Noonan syndrome, obesity, ovarian cancer, pancreatic cancer, Parkinson's disease, paroxysmal nocturnal hemoglobinuria, Pendleley syndrome, peroneal muscular dystrophy, phenylketonuria (PKU), polycystic kidney disease, Prader-Willi syndrome, primary biliary cirrhosis, prostate cancer, REAR syndrome.Reeves' disease, retinitis pigmentosa, retinoblastoma, Rett syndrome, Saffir-Lipo syndrome, schizophrenia, severe combined immunodeficiency, sickle cell anemia, spina bifida, spinal muscular atrophy, cerebellar atrophy, sudden adult death syndrome, Tangier disease, Dixack's disease, radius agenesis syndrome, anorectal-limb malformation syndrome, tuberous sclerosis, Turner syndrome, Ussell syndrome, retinocerebellar spinal cord hemangioma, Waardenburg syndrome, Weaver syndrome, Werner syndrome, Williams syndrome, Wilson's disease, xeroderma pigmentosum, and Zowieger's disease.

[0046] The term "musculoskeletal disorder" or "MSD" refers to injury and / or pain in the joints, ligaments, muscles, nerves, tendons, and structures supporting the limbs, neck, and back of a subject. In some embodiments, MSD is a degenerative disease. In some embodiments, MSD includes inflammatory conditions. Body parts of a subject that may be associated with MSD include the upper and lower back, neck, shoulders, and limbs (arms, legs, feet, and hands). In some implementations, MSD refers to bone diseases such as achondroplasia, acromegaly, callus, bone demineralization, fractures, bone marrow diseases, bone marrow tumors, congenital keratosis, leukemia (e.g., hairy cell leukemia, lymphocytic leukemia, myeloid leukemia, Philadelphia chromosome-positive leukemia, plasma cell leukemia, stem cell leukemia), systemic mastocytosis, myelodysplastic syndrome, paroxysmal nocturnal hemoglobinuria, myeloid sarcoma, myelofibrosis, multiple myeloma, polycythemia vera, Pearson's myelopancreatic syndrome, bone tumors, bone marrow tumors, Ewing sarcoma, osteochondroma, osteoclastoma, osteosarcoma, brachydactyly, progressive diaphyseal dysplasia syndrome, craniosynostosis, Krusson's craniofacial dysplasia, dwarfism, achondroplasia, Bloom syndrome, Coke's syndrome, Elleryway syndrome, and Seckel syndrome. Syndrome, vertebral epiphyseal dysplasia, congenital vertebral epiphyseal dysplasia, Werner syndrome, osteophyte formation, osteophytes, incomplete Kree-Weil syndrome, Marfan syndrome, multiple fibrous dysplasia, osteitis, osteoarthritis, osteochondritis, Molgio disease, Caspik disease, Leri-Weill achondroplasia, osteochondrosis, osteomalacia, osteogenesis imperfecta, osteolysis, Gorham-Stout syndrome Osteomalacia, osteomalacia, osteonecrosis, osteopenia, osteosclerosis, osteoporosis, bone petrification, vertebral epiphyseal dysplasia, periosteal pachydermosis, osseous Parkinson's disease, polydactyly, McMurphy syndrome, rickets, Rothmund-Thomson syndrome, Soto's syndrome, vertebral epiphyseal dysplasia, congenital vertebral epiphyseal dysplasia, syndactyly, Appel syndrome, type II syndactyly, or Werner syndrome. In some implementations, MSD refers to cartilage diseases such as chondroma, osteochondritis, Morki's disease, Caspik disease, or Leri-Weill chondrodysplasia. In some implementations, MSD refers to hernias such as lumbar disc herniation. In some implementations, MSD refers to joint diseases such as arthralgia, arthritis (e.g., gout (e.g., Kayser-Sydney syndrome, Lesner syndrome)), Lyme disease, osteoarthritis, psoriatic arthritis, reactive arthritis, rheumatic fever, rheumatoid arthritis, Felty syndrome, synovitis, Blau syndrome, nail-patella syndrome, spondyloarthritis, reactive arthritis, Strickler syndrome, synovial diseases, synovitis, or Blau syndrome. In some implementations, MSD refers to Rangi syndrome.In some implementations, MSD refers to muscle diseases such as Bass syndrome, mitochondrial encephalomyopathy, MELAS syndrome, MERRF syndrome, MNGIE syndrome, mitochondrial myopathy, Karns-Sell syndrome, myalgia, fibromyalgia, polymyalgia rheumatica, myoma, myositis, dermatomyositis, neuromuscular diseases, Karns-Sell syndrome, muscular atrophy, myasthenia gravis, congenital myasthenia gravis syndrome, Lambert-Eaton myasthenia gravis syndrome, myotonia, congenital myotonia, spinal muscular atrophy, tetany, extraocular muscle palsy, or rhabdomyolysis. In some implementations, MSD refers to Protis syndrome. In some implementations, MSD refers to rheumatic diseases such as arthritis (e.g., gout, such as Kayser-Sydney syndrome, Lesner syndrome, Lyme disease), osteoarthritis, psoriatic arthritis, reactive arthritis, rheumatic fever, rheumatoid arthritis, Felty syndrome, synovitis, Blau syndrome, polymyalgia rheumatica, rheumatic fever, rheumatic heart disease, or Sjögren's syndrome. In some implementations, MSD refers to Schjan's syndrome. In some implementations, MSD refers to skeletal diseases such as Leri-Weill dysplasia, skeletal malformations, Meniere's syndrome, pachydermosis, Rieger syndrome, spinal diseases, herniated discs, scoliosis, spina bifida, spondylitis, ankylosing spondylitis, spondyloarthritis, reactive arthritis, epiphyseal dysplasia, congenital vertebral epiphyseal dysplasia, or cervical spondylosis. In some implementations, the disease is a musculoskeletal disease.

[0047] "Proliferative disorders" are diseases caused by abnormal growth or expansion of cells (Walker, Cambridge Dictionary of Biology; Cambridge University Press: Cambridge, UK, 1990). Proliferative disorders can be associated with: 1) pathological proliferation of normal resting cells; 2) pathological migration of cells from their normal sites (e.g., metastasis of neoplastic cells); 3) pathological expression of proteolytic enzymes such as matrix metalloproteinases (e.g., collagenase, gelatinase, and elastase); or 4) pathological angiogenesis, such as in proliferative retinopathy and tumor metastasis. Exemplary proliferative disorders include cancer (i.e., "malignant tumors"), benign tumors, angiogenesis, inflammatory diseases, and autoimmune diseases.

[0048] The terms “tumor” and “nebula” are used interchangeably herein and refer to an abnormal mass of tissue in which the growth of the mass exceeds and is not coordinated with the growth of normal tissue. A tumor or neoplasm can be “benign” or “malignant”, depending on the following characteristics: degree of cell differentiation (including morphology and function), growth rate, local invasion, and metastasis. A “benign tumor” is typically fully differentiated, grows more slowly than a typical malignant tumor, and remains confined to its site of origin. Additionally, benign tumors do not have the ability to infiltrate, invade, or metastasize to distant sites. Exemplary benign tumors include, but are not limited to, lipomas, chondromas, adenomas, acanthomas, senile hemangiomas, seborrheic keratosis, freckles, and sebaceous hyperplasia. In some cases, certain “benign” tumors may subsequently develop into malignant tumors, which may be caused by additional genetic changes in a neoplastic subpopulation of the tumor, and these tumors are referred to as “precancerous tumors.” An exemplary precancerous tumor is a teratoma. Conversely, a “malignant tumor” is typically poorly differentiated (regressive) and has a typically rapid growth, accompanied by progressive invasion, invasion, and destruction of surrounding tissues. Furthermore, malignant tumors often have the ability to metastasize to distant sites. The terms "metastasis," "metastatic," or "tumor metastasis" refer to the spread or migration of cancer cells from a primary tumor or progenitor to another organ or tissue, and are usually identified by the presence of a "secondary tumor" or "secondary cell mass" of the same histological type as the primary tumor or progenitor (rather than the organ or tissue where the secondary (metastatic) tumor is located). For example, prostate cancer that has metastasized to the bone is called metastatic prostate cancer and includes cancerous prostate cancer cells growing in bone tissue.

[0049] The term "cancer" refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See, for example, Stedman's Medical Dictionary, 25th edition; edited by Hensyl; Williams & Wilkins: Philadelphia, 1990. Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal carcinoma; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendothelial sarcoma, angiosarcoma); appendix cancer; benign monoclonal gammaglobulinosis; cholangiocarcinoma (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., breast adenocarcinoma, breast papillary carcinoma, breast cancer, breast medullary carcinoma); brain cancer (e.g., meningioma, malignant glioma, glioma (e.g., astrocytoma, oligodendroglioma, medulloblastoma); bronchial cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; dorsal cord epithelioma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer). Colorectal adenocarcinoma; connective tissue carcinoma; epithelial carcinoma; ependymoma; endothelial sarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., esophageal adenocarcinoma, Barrett's adenocarcinoma); Ewing sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); common eosinophilia; gallbladder cancer; gastric cancer (e.g., gastric adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell carcinoma; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), pharyngeal cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic system cancer. Cancers including leukemia (such as acute lymphoblastic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myeloid leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myeloid leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphomas such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL, such as diffuse large cell lymphoma (DLC)). L) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, intranodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenström macroglobulinemia), hairy cell leukemia (HCL), immunogenic large cell lymphoma, B precursor lymphoblastic lymphoma, and primary central nervous system (CNS) lymphoma;And T-cell NHL such as T-precursor lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and degenerative large cell lymphoma); a mixture of one or more of the above-mentioned leukemias / lymphomas; and multiple myeloma (MM), heavy chain diseases (e.g., alpha chain disease, gamma chain disease, μ chain disease); hemangioma; hypopharyngeal carcinoma; inflammatory myofibroblastic tumor; immune cell amyloidosis; renal cell carcinoma (e.g., nephroblastoma, Also known as Wilms' tumor, renal cell carcinoma; liver cancer (e.g., hepatocellular carcinoma (HCC), malignant liver cancer); lung cancer (e.g., bronchial carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma); leiomyosarcoma (LMS); mast cell hyperplasia (e.g., generalized mast cell hyperplasia); muscle cancer; spinal dysplasia syndrome (MDS); mesothelioma; myeloproliferative disorders (MPD) (e.g., polycythemia vera (PV), essential thrombocythemia (ET), idiopathic myeloid metaplasia (AMM), also known as myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myeloid leukemia (CML), chronic neutrophilic leukemia). CNL), hypereosinophilic syndrome (HES); neuroblastoma; neurofibroma (e.g., neurofibromatosis type 1 or 2, schwannoma); neuroendocrine carcinoma (e.g., gastrointestinal pancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous tumor (IPMN), islet cell carcinoma); penile cancer (e.g., Paget's disease of the penis and scrotum); pineal tumor; primitive neuroectodermal tumor (PNT); plasmacytoma; paraneoplastic syndrome; intraepithelial tumor; prostate cancer (e.g., prostate cancer). Adenocarcinoma; rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland cancer; small bowel cancer; sweat gland cancer; synovial tumor; testicular cancer (e.g., seminoma, embryonal testicular carcinoma); thyroid cancer (e.g., papillary thyroid carcinoma, papillary thyroid carcinoma (PTC), medullary thyroid carcinoma); urethral cancer; vaginal cancer; and vulvar cancer (e.g., vulvar Paget's disease).

[0050] The term "inflammatory disease" refers to a disease caused, resulting from, or leading to inflammation. The term "inflammatory disease" can also refer to a dysregulated inflammatory response that causes an excessive response of macrophages, granulocytes, and / or T lymphocytes, resulting in abnormal tissue damage and / or cell death. Inflammatory diseases can be acute or chronic inflammatory conditions and can be caused by infectious or non-infectious factors. Inflammatory diseases include, but are not limited to, atherosclerosis, arteriosclerosis, autoimmune diseases, multiple sclerosis, systemic lupus erythematosus, polymyalgia rheumatica (PMR), gouty arthritis, degenerative arthritis, tendinitis, bursitis, psoriasis, cystic fibrosis, osteitis arthritis, rheumatoid arthritis, inflammatory arthritis, Sjögren's syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus vulgaris, pemphigusa, diabetes mellitus (e.g., type I), myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, and Goodpassuia disease. Mixed connective tissue disease, sclerosing cholangitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, pernicious anemia, inflammatory skin diseases, common interstitial pneumonia (UIP), asbestosis, silicosis, bronchiectasis, beryllium pneumoconiosis, talc lung, pneumoconiosis, sarcoidosis, desquamative interstitial pneumonia, lymphocytic interstitial pneumonia, giant cell interstitial pneumonia, cellular interstitial pneumonia, extrinsic allergic alveolitis, Wegener's granulomatosis and related forms of vasculitis (temporal arteritis and polyarteritis nodosa), inflammatory skin diseases, hepatitis, delayed-type hypersensitivity reactions (e.g., poison ivy dermatitis), pneumonia, respiratory tract diseases. Inflammation, adult respiratory distress syndrome (ARDS), encephalitis, immediate-type hypersensitivity reaction, asthma, hay fever, allergy, acute allergy, rheumatic fever, glomerulonephritis, pyelonephritis, cellulitis, cystitis, chronic cholecystitis, local ischemia (local ischemic injury), reperfusion injury, allogeneic transplant rejection, host-associated graft rejection, appendicitis, arteritis, blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis, chorioamnionitis, colitis, dacryoadenitis, dermatomyositis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrosis Myositis, gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, omphalitis, oophoritis, testis, osteitis, otitis media, pancreatitis, parotitis, pericarditis, lateral pharyngitis, pleurisy, phlebitis, localized pneumonia, proctitis, prostatitis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, parotitis, tonsillitis, urethritis, cystitis, uveitis, vaginitis, vasculitis, vulvitis, vulvovaginitis, vasculitis, chronic bronchitis, osteomyelitis, optic neuritis, temporal arteritis, transverse myelitis, necrotizing fasciitis, and necrotizing enterocolitis. Ocular inflammatory diseases include, but are not limited to, postoperative inflammation. In some implementations, inflammatory diseases are inflammatory aging (e.g., inflammation as a side effect of aging).

[0051] Autoimmune diseases are illnesses caused by an inappropriate immune response in a subject's body against substances and tissues normally present in the body. In other words, the immune system mistakes certain parts of the body for pathogens and attacks its own cells. This can be limited to certain organs (e.g., in autoimmune thyroiditis) or involve specific tissues in different locations (e.g., Goodpassuia disease, which can affect the basement membrane in both the lungs and kidneys). Treatment for autoimmune diseases typically involves immunosuppression, such as medications that reduce the immune response. Exemplary autoimmune diseases include, but are not limited to, glomerulonephritis, Goodpassu syndrome, necrotizing vasculitis, lymphadenitis, periarteritis nodosa, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis / polymyositis, antiphospholipid antibody syndrome, antiphospholipid scleroderma, pemphigus vulgaris, ANCA-associated vasculitis (e.g., Wegener's granulomatosis, microscopic polyangiitis), uveitis, Sjögren's syndrome, Crohn's disease, Reiter's syndrome, ankylosing spondylitis, Lyme disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, and cardiomyopathy.

[0052] The term "liver disease" or "liver disorders" refers to damage or disease of the liver. Non-limiting examples of liver diseases include intrahepatic cholestasis (e.g., Aragog syndrome, biliary cirrhosis), fatty liver (e.g., alcoholic fatty liver, Reye's syndrome), hepatic vein thrombosis, Wilson's disease (i.e., Wilson's disease), hepatomegaly, liver abscess (e.g., amoebic liver abscess), cirrhosis (e.g., alcoholic, biliary, and experimental cirrhosis), alcoholic liver disease (e.g., fatty liver, hepatitis, cirrhosis), and parasitic liver diseases (e.g., echinococcosis, liver fluke disease, amoebic liver abscess). Jaundice (e.g., hemolytic, hepatocellular, cholestatic jaundice), cholestasis, portal hypertension, hepatomegaly, ascites, hepatitis (e.g., alcoholic hepatitis, animal hepatitis), chronic hepatitis (e.g., autoimmune hepatitis B, hepatitis C, hepatitis D, drug-induced chronic hepatitis), toxic hepatitis, viral hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E), granulomatous hepatitis, secondary biliary cirrhosis, hepatic encephalopathy, varicose veins. Primary biliary cirrhosis, primary sclerosing cholangitis, hepatocellular adenoma, hemangioma, gallstones, liver failure (e.g., hepatic encephalopathy, acute liver failure), angiomyolipoma, calcified liver metastases, cystic liver metastases, fibrolamellar hepatocellular carcinoma, hepatic adenoma, hepatocellular carcinoma, liver cysts (e.g., simple cysts, polycystic liver disease, hepatobiliary cystadenoma, choledochal cyst), mesenchymal tumors (mesenchymal hamartoma, infantile angioendothelioma, hemangioma, hepatic purpura, lipoma, inflammatory pseudotumor), epithelial tumors. (e.g., hamartoma, cholangioadenoma), focal nodular hyperplasia, nodular regenerative hyperplasia, hepatoblastoma, hepatocellular carcinoma, cholangioadenocarcinoma, cystadenocarcinoma, vascular tumor, adenoangiosarcoma, Kaposi's sarcoma, hemangioendothelioma, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous cell carcinoma, primary lymphoma, hepatic purpura, erythrocyte hepatic porphyria, hepatic porphyria (e.g., acute intermittent porphyria, tarda cutaneous porphyria), and Selweger syndrome.

[0053] The term "splenic disease" refers to diseases of the spleen. Examples of spleen diseases include, but are not limited to, splenomegaly, splenic cancer, asplenia, splenic trauma, idiopathic purpura, Felty syndrome, Hodgkin's disease, and immune-mediated spleen injury.

[0054] The term "lung disease" or "pulmonary disease" refers to diseases of the lungs. Examples of lung diseases include, but are not limited to, bronchiectasis, bronchitis, bronchopulmonary dysplasia, interstitial lung disease, occupational lung disease, emphysema, cystic fibrosis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), asthma (e.g., intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma), chronic bronchitis, chronic obstructive pulmonary disease (COPD), emphysema, interstitial lung disease, sarcoidosis, asbestosis, aspergillosis, pneumonia (e.g., lobar pneumonia, multilobar pneumonia, bronchopneumonia, interstitial pneumonia), pulmonary fibrosis, tuberculosis, rheumatoid lung disease, pulmonary embolism, and lung cancer (e.g., non-small cell lung cancer (e.g., adenocarcinoma, squamous cell lung cancer, large cell lung cancer), small cell lung cancer).

[0055] "Hematologic disorders" include diseases that affect hematopoietic cells or tissues. Hematologic disorders include those associated with abnormal hematologic contents and / or function. Examples of hematologic disorders include those caused by bone marrow irradiation or chemotherapy for cancer, such as pernicious anemia, hemorrhagic anemia, hemolytic anemia, aplastic anemia, sickle cell anemia, sideroblastic anemia, anemia associated with chronic infections such as malaria, trypanosomiasis, HTV, hepatitis virus, or other viruses, myelopathic anemia due to bone marrow deficiency, renal failure due to anemia, anemia, polycythemia vera, infectious mononucleosis (EVI), acute non-lymphocytic leukemia (ANLL), acute myeloid leukemia (AML), and acute promyelocytic leukemia (APL). Acute myelomonocytic leukemia (AMMoL), polycythemia vera, lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, Wilms' tumor, Ewing's sarcoma, retinoblastoma, hemophilia, disorders associated with increased risk of thrombosis, herpes, thalassemia, antibody-mediated conditions such as transfusion reactions and myelocytosis, mechanical trauma to red blood cells such as microangiopathic hemolytic anemia, thrombotic thrombocytopenic purpura and disseminated intravascular coagulation, infections caused by parasites such as Plasmodium, and chemical damage caused by, for example, lead poisoning and hypersplenism.

[0056] The term "neurological disease" refers to any disease of the nervous system, including diseases involving the central nervous system (brain, brainstem, and cerebellum), the peripheral nervous system (including cranial nerves), and the autonomic nervous system (parts of which are located in both the central and peripheral nervous systems). Neurodegenerative diseases are types of neurological disorders characterized by the loss of nerve cells, including but not limited to Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), tauopathy (including frontotemporal dementia), and Huntington's disease. Examples of neurological diseases include, but are not limited to, headaches, stupor and coma, dementia, seizures, sleep disorders, trauma, infections, tumors, neuro-ophthalmological disorders, movement disorders, demyelinating diseases, spinal cord disorders, and disorders of the peripheral nerves, muscles, and neuromuscular junctions. Addictions and mental illnesses, including but not limited to bipolar disorder and schizophrenia, are also included in the definition of neurological diseases. Other examples of neurological disorders include acquired epileptic aphasia; acute disseminated encephalomyelitis; leukoadrenal atrophy; agenesis of the corpus callosum; cognitive impairment; Eckaldi syndrome; Alexander disease; Alpert disease; alternating hemiplegia; Alzheimer's disease; amyotrophic lateral sclerosis; anencephaly; happy puppet syndrome; hemangioma; hypoxia; aphasia; apraxia; arachnoid cysts; arachnoiditis; Archibal malformations; arteriovenous malformations; Asperger's syndrome; ataxia-telangiectasia; attention deficit hyperactivity disorder; autism; autonomic dysfunction; back pain; Betton's disease; Bechtel's disease; Behr's palsy; benign spontaneous blepharospasm; benign focal blepharospasm; muscular atrophy; benign intracranial hypertension; Binswanger's disease; blepharospasm; pigmentary disorders; brachial plexus injury; brain abscess; brain injury; brain tumors (including glioblastoma multiforme). Tumors; spinal tumors; lateral spinal cord compression-spinal cord hemisection syndrome; Carnafan disease; carpal tunnel syndrome (CTS); burning pain; central pain syndrome; central pontine myelinolysis; head disorders; cerebral aneurysm; cerebral arteriosclerosis; cerebral atrophy; cerebral gigantism; cerebral palsy; progressive peroneal muscular atrophy; 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 paralysis; cortical basal degeneration; cranial arteritis; craniosynostosis; Cujjah disease; cumulative trauma disorder; Cushing's syndrome; giant cell inclusion body disease (CIBD); cytomegalovirus infection; oculofoot chorea syndrome; fourth ventricle atresia syndrome; Dawson's disease; De Morsier syndrome; brachial plexus palsy; dementia; cutaneous myositis; diabetic neuropathy; diffuse sclerosis; familial autonomic dysfunction; dyslexia; dyslexia; dystonia; early infantile epileptic encephalopathy; empty sella syndrome; encephalitis; encephalocele; trigeminal hemangioma; epilepsy; Elber's palsy; essential tremor; diffuse somatic angiokeratoma;Fal's syndrome; syncope; familial spastic paralysis; febrile seizures; Fischer syndrome; Friedreich ataxia; frontotemporal dementia and other "Tau protein diseases"; cerebroside disorders; Göstadmann syndrome; giant cell arteritis; giant cell inclusion body disease; spherocytic leukodystrophy; Guillain-Barré syndrome; HTLV-1 related myelopathy; protein globulin depigmentation syndrome; traumatic brain injury; headache; hemifacial spasm; hereditary spastic paraplegia; Reifsomnia; herpes zoster otitis media; herpes zoster; Hirayama syndrome; HIV-related dementia and neuropathy (see also neurological manifestations of AIDS); holohemorrhagic malformation; Huntington's disease and other polyglutamic-acylamine repeat sequence diseases; hydrocephalus; hydrocephalus; hypercortisolism; Hypoxia; Immune-mediated encephalomyelitis; Inclusion body myositis; Pigmentary disorders; Convulsions and thirst; Phytanate storage disease; Infantile Reiferson disease; Infantile spasms; Inflammatory myopathy; Intracranial cysts; Increased intracranial pressure; Jubert syndrome; Karns-Sell syndrome; Kennedy disease; Kingsburn syndrome; Congenital short neck syndrome; Clapham's disease; Kugelberg-Weyland disease; Kuru disease; Lafra disease; Lambert-Eaton myasthenia gravis syndrome; Acquired epileptic aphasia syndrome; Lateral medullary (Wallenberg) syndrome; Learning disability; Leigh disease; Lennears syndrome; Lesnain syndrome; Leukodystrophy; Lewy body dementia; Asylum; Locked-in syndrome; Gray's disease (also known as motor neuron disease or amyotrophic lateral sclerosis); Lumbar disc herniation Symptoms; Lyme disease - neurological sequelae; Machado-Joseph disease; macrocephaly; macrocephaly; Ménière syndrome; Meniere's disease; meningitis; hair knot syndrome; metachromatic leukodystrophy; microcephaly; migraine; Miller Fisher syndrome; minor stroke; mitochondrial myopathy; Möbius syndrome; unilateral muscular atrophy; motor neuron disease; abnormal vascular network disease of the basal brain; mucopolysaccharidosis; multi-infarct dementia; multifocal motor neuropathy; multiple sclerosis and other demyelinating diseases; multiple system atrophy with orthostatic hypotension; muscular atrophy; myasthenia gravis; diffuse demyelinating sclerosis; infantile myoclonic encephalopathy; myoclonus; myopathy; congenital myotonia; narcolepsy; neurofibromatosis; neuroleptic malignant syndrome; neurological disorders of AIDS Manifestations; neurological sequelae of lupus; neurogenic myotonia; neuronal ceroid lipofuscin deposition; neuronal migration disorder; Niemann-Pick disease; O'Sullivan-McLeod syndrome; occipital neuralgia; occult spinal canal closure sequence; Otahara syndrome; olivopontocerebellar atrophy; oculoclonus myoclonus; optic neuritis; orthostatic hypotension; overuse syndrome; sensory disturbances; Parkinson's disease; congenital myotonic dysplasia; epithelial neoplasms; paroxysmal seizures; Burrow syndrome; Peyronie's disease; periodic paralysis; peripheral neuropathy; painful neuropathy and neuropathic pain; persistent vegetative state; pervasive developmental disorder; phototropic sneezing reflex; phytate storage disease; Picker's disease; nerve compression;Pituitary tumor; polymyositis; pore-shaped brain; sequelae of poliomyelitis; postherpetic neuralgia (PHN); post-infectious encephalomyelitis; orthostatic hypotension; Powell syndrome; primary lateral sclerosis; prion disease; progressive; unilateral facial atrophy; progressive multifocal leukoencephalopathy; progressive sclerotic gray matter atrophy; progressive supranuclear palsy; pseudotumor cerebri; geniculate ganglion syndrome (type I and II); Rasmussen encephalitis; reflex sympathetic dystrophy syndrome; Rif-Sym disease; repetitive motion injury; repetitive pressure injury; restless legs syndrome; retrovirus-associated myelopathy; Rett syndrome; Reye's syndrome; St. Vitus chorea; Sandhof's disease; Sheld's disease; schizophrenia; septal dysplasia; shaken baby syndrome; herpes zoster; Hedgren's syndrome; Sjögren's syndrome; sleep apnea; cerebral macrosomia; spasticity Spina bifida; spinal cord injury; spinal cord tumor; spinal muscular atrophy; stiff-person syndrome; stroke; Steg-Weber syndrome; subacute sclerosing panencephalitis; subarachnoid hemorrhage; subcortical arteriosclerotic encephalopathy; Sydenham's chorea; syncope; syringomyelia; delayed motor disorder; amaurotic familial dementia; temporal arteritis; tethered cord syndrome; myotonic cataract; thoracic outlet syndrome; painful tetany; Todd's paralysis; Tourette syndrome; transient ischemic attack; infectious cavernous encephalopathy; transverse myelitis; traumatic brain injury; tremor; trigeminal neuralgia; tropical spastic paraplegia; tuberous sclerosis; vascular dementia (multi-infarct dementia); vasculitis, including temporal arteritis; Hippel-Lindau disease (VHL); Wallenberg syndrome; Wednisch-Hoffmann disease; West syndrome; neck injury; Williams syndrome; Wilson's disease; and Zelveger syndrome.

[0057] "Pain conditions" include, but are not limited to, neuropathic pain (e.g., peripheral neuropathic pain), central pain, afferent pain, chronic pain (e.g., chronic nociceptive pain and other forms of chronic pain such as postoperative pain, such as pain after hip, knee, or other replacement surgery), preoperative pain, stimulation of pain receptors (nociceptive pain), acute pain (e.g., hallucinations and transient acute pain), non-inflammatory pain, inflammatory pain, cancer-related pain, wound pain, burn pain, postoperative pain, pain associated with medical procedures, pain caused by pruritus, bladder pain syndrome, pain associated with premenstrual anxiety disorder and / or premenstrual syndrome, pain associated with chronic fatigue syndrome, pain associated with preterm birth, pain associated with withdrawal symptoms of drug addiction, joint pain, arthritis pain (e.g., pain associated with crystallizing 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, toothache, toothache / maxillofacial pain, visceral pain, etc. The pain conditions considered herein may include a mixture of the various types of pain described above and herein (e.g., nociceptive pain, inflammatory pain, neuropathic pain, etc.). In some embodiments, a particular type of pain may be dominant. In other embodiments, the pain condition includes two or more types of pain, with none being dominant. A skilled clinician can determine the dosage to achieve a therapeutically effective amount for a particular subject based on the pain condition.

[0058] The term "mental disorder" refers to mental illness and includes the disorders and impairments 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, but are not limited to, 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 phobias), childhood disorders (e.g., attention deficit / hyperfunction disorder, conduct disorder, and confrontational defiance disorder), eating disorders (e.g., anorexia nervosa and bulimia nervosa), mood disorders (e.g., depression, bipolar disorder, cyclothymic mood disorder, depressive disorder, and major depressive disorder), and personality disorders (e.g., antisocial personality disorder, avoidant personality disorder, borderline personality disorder, dependent personality disorder, histrionic personality disorder, narcissistic personality disorder, etc.). Obsessive-compulsive personality disorder, paranoid personality disorder, schizophrenia-like personality disorder, and schizotypal personality disorder; mental disorders (e.g., short-term psychotic disorder, delusional disorder, schizophrenic affective disorder, schizophrenia-like disorder, syndrome, and sharing disorder); drug-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; psychosis; dementia; multi-infarct dementia; learning and memory disorders (e.g., amnesia and age-related memory loss); and Tourette syndrome.

[0059] The term "metabolic disorder" refers to any impairment involving changes in the normal metabolism of carbohydrates, lipids, proteins, nucleic acids, or combinations thereof. Metabolic disorders are associated with deficiencies or excesses in metabolic pathways that lead to imbalances in the metabolism of nucleic acids, proteins, lipids, and / or carbohydrates. Factors affecting metabolism include, but are not limited to, endocrine (hormonal) control systems (e.g., the insulin pathway, enteroendocrine hormones, including GLP-1, PYY, etc.) and neural control systems (e.g., GLP-1 in the brain). Examples of metabolic disorders include, but are not limited to, diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes), hyperglycemia, hyperinsulinemia, insulin resistance, and obesity.

[0060] In some implementations, the disease is characterized by cellular dysfunction. For example, the disease may be a mitochondrial disease. Non-restrictive mitochondrial diseases include Friedrich's ataxia, Alphers' disease, Bass syndrome, β-oxidation deficiency, carnitine deficiency, CPTI deficiency, and mitochondrial DNA deletion. Cellular dysfunction may include mitochondrial dysfunction, RNA replication dysfunction, DNA replication dysfunction, translational dysfunction, and / or protein folding dysfunction.

[0061] In some implementations, the disease or condition is caused by a mental disorder (wood), bleeding, injury (such as a fracture, gunshot wound, cut, or scar left during surgery, such as a cesarean section).

[0062] In some implementations, the disease is an infectious disease (e.g., a disease caused by a pathogen and / or virus). Non-limiting examples of infectious diseases include tuberculosis, HIV / AIDS, rabies, plague, cholera, dengue fever, measles, malaria, meningitis, pertussis, Lyme disease, influenza, hepatitis C, typhoid fever, and poliomyelitis.

[0063] As used herein, the terms "effective amount" and "therapeuticly effective amount" refer to the amount or concentration of the compound of the invention that, when administered to a subject, effectively treats at least partially the condition suffered by the subject.

[0064] As used herein, a “functional” or “active” protein is a protein that retains its biological activity (e.g., the ability to act as a transcription factor or inducer). Conversely, a nonfunctional or inactive protein is a protein that cannot perform one or more of its wild-type functions.

[0065] A “eukaryotic cell” is a cell that includes a nucleus surrounded by a membrane. Non-limiting examples of eukaryotic cells include animal cells, plant cells, fungal or protist cells, wherein the animal cell is optionally a mammalian cell.

[0066] The term "gene" refers to a nucleic acid segment that expresses a protein, including regulatory sequences preceding (5' non-coding sequence) and following (3' non-coding sequence). A "natural gene" is a gene found naturally and carrying its own regulatory sequences. A "chimeric gene" or "chimeric construct" is any gene or construct that is not a natural gene and contains regulatory and coding sequences that are not simultaneously present in nature. Therefore, a chimeric gene or chimeric construct can contain regulatory and coding sequences derived from different sources, or regulatory and coding sequences derived from the same source but arranged in a non-natural manner. An "endogenous gene" is a natural gene located in its natural position within the genome of an organism. An "exogenous" gene is a gene that is not normally present in the host organism but is introduced into the host organism through gene transfer. Exogenous genes can include natural genes inserted into non-natural organisms, or chimeric genes. A "transgenic" gene is a gene introduced into the genome through a transformation procedure.

[0067] "Homologous" or "homogeneous" refers to a sequence (e.g., a nucleic acid or amino acid sequence) that shares a specific percentage of 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% percentage identity). Homologous sequences include, but are not limited to, parallel homologous or orthologous sequences. Parallel homologous sequences originate from gene duplication within the species' genome, while orthologous homologous sequences diverge after speciation events. Functional homologs retain one or more biological activities of the wild-type protein. In some embodiments, functional homologs of the transgenic protein retain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the biological activity (e.g., transcription factor activity) of the wild-type counterpart.

[0068] "Inverted terminal repeats" or "ITRs" are nucleic acid sequences that are inversely complementary to each other. Generally, in AAV vectors, ITRs are present on either side of the cassette (e.g., an expression cassette containing nucleic acids encoding transgenes, mutant rtTAs, or any combination thereof). AAV ITRs include ITRs from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and their AAV variants.

[0069] The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” “nucleic acid molecule,” “nucleic acid sequence,” and “oligonucleotide” refer to a series of nucleotide bases (also called “nucleotides”) in DNA and RNA, and mean any chain of two or more nucleotides. The terms “nucleic acid” or “nucleic acid sequence,” “nucleic acid molecule,” “nucleic acid fragment,” or “polynucleotide” can be used interchangeably with “gene,” “gene-encoded mRNA,” and “cDNA.”

[0070] Nucleic acids can be single-stranded or double-stranded chimeric mixtures or derivatives or modified variants thereof. For example, oligonucleotides can be modified on the base moiety, sugar moiety, or phosphate backbone, for example, to improve the stability of the molecule, its hybridization parameters, etc. Nucleotide sequences typically carry genetic information, including information used by cellular structures to produce proteins and enzymes. These terms include double-stranded or single-stranded genomes and cDNA, RNA, any polynucleotides that are synthesized and genetically manipulated, and both sense and antisense polynucleotides. This includes single-stranded and double-stranded molecules, i.e., DNA-DNA, DNA-RNA, and RNA-RNA hybrids, as well as “protein nucleic acids” (PNAs) formed by coupling bases to an amino acid backbone. This also includes nucleic acids containing carbohydrates or lipids. Exemplary DNAs include 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), kinematic DNA (kDNA), proviruses, lysogenic bacteria, repetitive DNA, satellite DNA, and viral DNA. Exemplary RNAs include single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), small interfering RNA (siRNA), messenger RNA (mRNA), pre-mRNA, small 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, flexizyme, nucleolar small RNA (snoRNA), splice leader RNA, viral RNA, and viral satellite RNA.

[0071] The nucleic acids described herein can be synthesized using standard methods known in the art, such as using automated DNA synthesizers (commercially available from Biosearch, Applied Biosystems, etc.). For example, phosphate thioester oligonucleotides can be synthesized using the method described in Stein et al., Nucl. Acids Res., 16, 3209, (1988), and methyl phosphonate oligonucleotides can be prepared using glass polymer carriers with controllable pore sizes (Sarin et al., Proc. Natl. Acad. Sci. USA 85, 7448-7451, (1988)). Various methods have been developed for delivering antisense DNA or RNA into cells, such as direct injection of antisense molecules into tissue sites or systemic administration of modified antisense molecules (antisense molecules linked to peptides or antibodies that specifically bind to receptors or antigens expressed on the surface of target cells) designed to target desired cells. Alternatively, RNA molecules can be produced by transcribing DNA sequences encoding antisense RNA molecules in vitro and in vivo. Such DNA sequences can be introduced into various vectors containing appropriate RNA polymerase promoters, such as T7 or SP6 polymerase promoters. Alternatively, depending on the promoter used, antisense cDNA constructs that constitutively or inducibly synthesize antisense RNA can be stably introduced into cell lines. However, it is often difficult to achieve intracellular antisense concentrations sufficient to inhibit the translation of endogenous mRNA. Therefore, a preferred method utilizes recombinant DNA constructs in which antisense oligonucleotides are under the control of strong promoters. Transfection of target cells in a patient using such constructs results in sufficient transcription of single-stranded RNA, which forms complementary base pairs with the endogenous target gene transcript and thereby prevents the translation of the target gene mRNA. For example, a vector can be introduced into the body to be absorbed by cells and direct the transcription of antisense RNA. As long as such a vector can transcribe to produce the desired antisense RNA, it can remain in a free state or be integrated into the chromosome. Such vectors can be constructed using standard recombinant DNA techniques in the art. The vector can be a plasmid, virus, or other vector known in the art for replication and expression in mammalian cells. Expression of sequences encoding antisense RNA can be carried out in mammalian (preferably human) cells using any promoter known in the art. Such promoters can be inducible or constitutive. Any type of plasmid, granule, yeast artificial chromosome, or viral vector can be used to prepare recombinant DNA constructs that can be directly introduced into tissue sites.

[0072] Nucleic acids may be side-linked with natural regulatory (expression control) sequences or heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome-binding site sequences, enhancers, response elements, repressors, signal sequences, polyadenylated sequences, introns, 5′- and 3′-uncoding regions, etc. The nucleic acid can also be modified using many methods known in the art. Non-limiting examples of such modifications include methylation, “capping,” substitution of one or more naturally occurring nucleotides with analogs, and internucleotide modifications, such as those having uncharged bonds (e.g., methylphosphonates, triphosphates, phosphamidates, carbamates, etc.) and charged bonds (e.g., thiophosphates, dithiophosphates, etc.). Polynucleotides may contain one or more additional covalently linked portions, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelating agents (e.g., metals, radioactive metals, iron, metal oxides, etc.), and alkylating agents. Polynucleotides can be derivatized by forming trimethyl phosphate, triethyl phosphate, or alkyl aminophosphate linkages. Furthermore, the polynucleotides described herein can be modified using labels that can directly or indirectly provide a detectable signal. Exemplary labels include radioisotopes, fluorescent molecules, isotopes (e.g., radioisotopes), biotin, etc.

[0073] "Recombinant nucleic acid molecules" or "engineered nucleic acids" are nucleic acid molecules that have undergone molecular biological manipulation; that is, nucleic acid molecules that are not naturally occurring or genetically engineered. Furthermore, the terms "recombinant DNA molecules" or "engineered nucleic acids" refer to nucleic acid sequences that are not naturally occurring, or nucleic acid sequences that can be prepared by artificially combining two originally separate nucleic acid sequence fragments (i.e., by joining normally discontinuous DNA fragments together). The term “recombination generation” refers to an artificial combination of nucleic acid fragments, typically achieved through chemical synthesis or artificial manipulation, such as genetic engineering techniques using restriction enzymes, ligases, and similar recombination techniques described, for example, by Sambrook et al., Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory, Plainview, NY; (1989), or Ausubel et al., Current Protocols in Molecular Biology, Current Protocols (1989), and DNA Cloning: A Practical Approach, Volumes I and II (edited by DNGlover), IREL Press, Oxford, (1985); each of which is incorporated herein by reference.

[0074] Such manipulations can be performed to replace one codon with a redundant codon encoding the same or conserved amino acid, often while introducing or removing sequence recognition sites. Alternatively, nucleic acid fragments with desired functions can be linked together to produce a single genetic entity containing the desired combination of functions not naturally present. Restriction enzyme sites are often targets for such manipulations, but other site-specific targets can also be introduced by design, such as promoters, DNA replication sites, regulatory sequences, control sequences, open reading frames, or other useful features.

[0075] As used herein, “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein to which the substitution occurs. Variants can be prepared according to methods known to those skilled in the art for altering polypeptide sequences, such as those compiled in references such as *Molecular Cloning: A Laboratory Manual*, edited by J. Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or *Current Protocols in Molecular Biology*, edited by F. Mausubel et al., John Wiley & Sons, Inc., New York. Conservative substitution of amino acids includes substitutions between amino acids within the following group: (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.

[0076] "Recombinant cells" or "engineered cells" are cells that contain recombinant nucleic acids.

[0077] "The residue in sequence X corresponding to position a in sequence Y" refers to the amino acid sequence alignment tool known in the art (e.g., Clustal Omega or...). When comparing sequences X and Y, the residue at the corresponding position of a in sequence X.

[0078] The term "leaky" when used in relation to inducible systems (e.g., Tet-On or Tet-Off systems) refers to transgene expression by an inducible promoter in the absence of gene induction. For example, in a Tet-On system, transgene expression in the absence of tetracycline (e.g., doxycycline) is considered a "leaky" system. Similarly, in a Tet-Off system, transgene expression in the presence of tetracycline (e.g., doxycycline) is considered a "leaky" system. The level of "leaking" in an inducible system can be determined by measuring the level of gene expression (e.g., by Western blotting, RNA analysis, or ELISA) in the absence of gene induction (e.g., in the absence of tetracycline in a Tet-On system or in the presence of tetracycline in a Tet-Off system).

[0079] The term "promoter" refers to the control region of a nucleic acid sequence in which the initiation and transcription rate of the remainder of the sequence are controlled. Promoters may also contain subregions to which regulatory proteins and molecules, such as RNA polymerases and other transcription factors, can bind. Promoters can be constitutive, inducible, activatable, repressive, tissue-specific, or any combination thereof. A promoter drives the expression of the nucleic acid sequence it regulates or drives the transcription of the nucleic acid sequence it regulates. In this context, a promoter is considered "operationally linked" when it is in the correct functional position and orientation relative to the nucleic acid sequence it regulates to control ("drive") the initiation of transcription of that sequence, the expression of that sequence, or a combination thereof.

[0080] Promoters can facilitate the ubiquitous or tissue-specific expression of operable, linked nucleic acid sequences 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, EF1a, SV40, PGK1 (human or mouse), Ubc, human β-actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIIa, GAL1, GAL10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, and U6, as known to those skilled in the art (see, for example, Addgene website:blog.addgene.org / plasmids-101-the-promoter-region).

[0081] Non-limiting examples of ubiquitous promoters include tetracycline-responsive promoters (under relevant conditions), CMV, EF1α, SV40 promoters, PGK1, Ubc, CAG, human β-actin gene promoters, and promoters containing an upstream activating sequence (UAS). In some embodiments, the promoter is a mammalian promoter. Non-limiting examples of tissue-specific promoters include brain-specific, liver-specific, muscle-specific, nerve cell-specific, lung-specific, heart-specific, bone-specific, intestinal-specific, skin-specific promoters, and eye-specific promoters. For example, a muscle-specific promoter is a desmin promoter (e.g., 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:29).

[0082] Non-limiting examples of constitutive promoters include CP1, CMV, EF1α, SV40, PGK1, Ubc, human β-actin, β-tubulin, CAG, Ac5, polyhedroin, TEF1, GDS, CaM3 5S, Ubi, H1, and U6. The Ubc promoter may contain at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) the same sequence as SEQ ID NO:18.

[0083] An "inducible promoter" is a promoter characterized by initiating or enhancing transcriptional activity in the presence, under the influence of, or in contact with an inducer. The inducer can be an endogenous or typically exogenous condition, compound, reagent, or protein that contacts the engineered nucleic acid in a manner that is active in inducing transcriptional activity by the inducible promoter. In some embodiments, the inducer is a tetracycline-sensitive protein (e.g., rtTA).

[0084] Inducible promoters as used in this disclosure include any inducible promoters described herein or known to those skilled in the art. Examples of inducible promoters include, but are not limited to, promoters regulated by chemical / biochemical and physical means, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., ahydrotetracycline (aTc)-responsive promoters), and other tetracycline-responsive promoter systems, including tetracycline repressor proteins (tetRTetR, e.g., SEQ ID NO:26; or TetRKRAB, e.g., SEQ ID NO:26). NO:27), tetracycline operon sequence (tetO) and tetracycline transactivator fusion protein (tTA), and tetracycline operon sequence (tetO) and reverse tetracycline transactivator fusion protein (rtTA)), steroid-regulated promoters (e.g., promoters based on 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 yeast, mouse and human metallothionein (proteins that bind and multivalently chelate metal ions) genes), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g. heat shock promoters), and light-regulated promoters. Non-limiting examples of inducible systems using light-regulated promoters are provided in Wang et al., Nat. Methods. 2012 Feb 12; 9(3):266-9.

[0085] As used herein, a “TRE promoter” is a promoter containing a tetracycline response element (TRE). As used herein, a TRE contains 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 one 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 also contains a minimal promoter located downstream of the tet-O sequence. A minimal promoter is a promoter containing the minimal elements of the 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 containing Tet-O). For example, the minimum promoter may be a minimum CMV promoter containing at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) of the same sequence as SEQ ID NO:20. For example, the TRE promoter may be a TRE3G promoter (e.g., a TRE3G promoter containing at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) of the same sequence as SEQ ID NO:7). In some embodiments, the TRE promoter is a TRE2 promoter containing at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) of the same sequence as SEQ ID NO:23. In some implementations, the TRE promoter is a P tight promoter containing at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) of the same sequence as SEQ ID NO:24.

[0086] As used herein, “reverse tetracycline transactivator” (“rtTA”) is an inducer that binds to a TRE promoter (e.g., TRE3G, P tight, or TRE2 promoter) in the presence of a tetracycline (e.g., doxycycline) and is capable of driving the expression of a transgene operatively linked to a TRE promoter. rtTA typically comprises a mutant tetracycline repressor DNA-binding protein (TetR) and a transactivation domain (see, for example, Gossen et al., Science. June 23, 1995; 268(5218):1766-9). Any suitable transactivation domain may be used. Non-limiting examples include VP64, P65, RTA, and MPH MS2-P65-HSF1. In some embodiments, the rtTA of this disclosure comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 transactivation domains. When bound to tetracycline, the mutant TetR domain is able to bind to the TRE promoter.

[0087] rtTA can be rtTA3, rtTA4, or variants thereof. As used herein, the rtTA3 amino acid sequence comprises the following amino acids at positions corresponding to residues in SEQ ID NO:11: glycine at residue 72, glycine at residue 12, phenylalanine at residue 67, and arginine at residue 171. In some embodiments, the nucleic acid encoding rtTA3 comprises at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%) the same sequence as SEQ ID NO:10, and / or the nucleic acid sequence encodes an rtTA3 protein comprising the following amino acids at positions corresponding to residues in SEQ ID NO:11: glycine at residue 72, glycine at residue 12, phenylalanine at residue 67, and arginine at residue 171. The rtTA3 nucleotide sequence may be composed of SEQ ID NO:10. In some embodiments, the amino acid sequence encoding rtTA3 comprises 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%) the same sequence as (SEQ ID NO:11), and contains the following amino acids corresponding to the specified positions in SEQ ID NO:11: glycine at residue 72, glycine at residue 12, phenylalanine at residue 67, and arginine at residue 171. The amino acid sequence of rtTA3 may consist of SEQ ID NO:11.

[0088] As used herein, the rtTA4 amino acid sequence contains mutations at positions corresponding to the following residues in SEQ ID NO:11: G72; G12; F67; and R171. In some embodiments, G72; G12; F67; and R171 may be mutated to any residue. In some embodiments, the nucleic acid encoding rtTA4 contains 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%) the same sequence as SEQ ID NO:12 and encodes a protein having mutations at the following residues corresponding to the following positions in rtTA3 (SEQ ID NO:11): G72; G12; F67; and R171. The rtTA4 nucleic acid sequence may consist of SEQ ID NO:12.

[0089] In some embodiments, the amino acid sequence encoding rtTA4 comprises 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%) the same sequence as SEQ ID NO:13, and / or encodes a protein having mutations at the following residues corresponding to the following positions in rtTA3 (SEQ ID NO:11): G72; G12; F67; and R171. In some embodiments, relative to rtTA3, the amino acid sequence of rtTA4 comprises the following mutations: a valine (V) or proline (P) mutation at the residue corresponding to position G72 in SEQ ID NO:11, a serine (S) mutation at the residue corresponding to position G12 in SEQ ID NO:11, a serine (S) mutation at the residue corresponding to position F67 in SEQ ID NO:11, and a lysine (K) mutation at the residue corresponding to position R171 in SEQ ID NO:11. The amino acid sequence of rtTA4 can be composed of SEQ ID NO:13.

[0090] A polycistronic vector is a vector that encodes more than one amino acid sequence (e.g., a vector encoding at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100 proteins). Polycistronic vectors allow the expression of multiple amino acid sequences from a nucleic acid sequence. The nucleic acid sequences encoding individual proteins can be joined or separated, resulting in unjoined proteins. For example, an internal ribosome entry site (IRES) or a peptide cleavage signal can be placed between the nucleic acid sequences encoding each transcription factor in the vector. Exemplary peptide cleavage signals include 2A peptides (e.g., T2A, P2A, E2A, and F2A). The 2A peptide may contain at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) of the same sequence as SEQ ID NO:9. In some embodiments, the nucleic acid of this disclosure (e.g., engineered nucleic acid, including expression vectors) is a polycistronic expression vector.

[0091] "Protein," "peptide," or "polypeptide" includes polymers of amino acid residues linked together by peptide bonds. The term refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, a protein will be at least three amino acids long. A protein can refer to a single protein or a collection of proteins. The proteins of the present invention preferably contain only native amino acids, but alternatively may use non-native amino acids (i.e., compounds not found in nature but which can be introduced into the polypeptide chain) and / or amino acid analogs as known in the art. Furthermore, one or more amino acids in the protein can be modified, for example by adding chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation or functionalization, or other modifications. Proteins can also be single molecules or multi-molecular complexes. Proteins can be fragments of naturally occurring proteins or peptides. Proteins can be naturally occurring, recombinant, synthetic, or any combination thereof.

[0092] "Prokaryotic cells" are cells that lack membrane-bound organelles. Non-limiting examples of prokaryotes include archaea and bacteria.

[0093] As used in this article, “reversing aging” refers to altering physical characteristics associated with aging. All animals typically experience a period of growth and maturation, followed by a progressive and irreversible physiological decline, ending in death. The length of time from birth to death is called the lifespan of an organism, and each organism has a characteristic average lifespan. Aging is the physical manifestation of changes over time, as measured as a percentage of average lifespan.

[0094] The intended "subjects" include, but are not limited to, humans (i.e., males or females of any age group, such as child subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults)) and / or other non-human animals, such as mammals (e.g., primates (e.g., macaques, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and / or turkeys). In some embodiments, the animal is a mammal. The animal can be male or female and at any developmental stage. The non-human animal can be a transgenic animal.

[0095] As used herein, a “terminator” or “termination sequence” is a nucleic acid sequence that causes transcription to terminate. A terminator can be unidirectional or bidirectional. It consists of a DNA sequence involved in specifically terminating the synthesis of RNA transcripts by RNA polymerase. The termination sequence prevents downstream nucleic acid sequences from being transcribed and activated by an upstream promoter. Therefore, in some embodiments, a terminator that terminates RNA transcript production is considered.

[0096] The most commonly used type of terminator is the forward terminator. When placed downstream of the nucleic acid sequence that is normally transcribed, a forward transcription terminator will cause transcription to stop. In some implementations, a bidirectional transcription terminator can be used, which typically causes transcription to terminate on both the forward and reverse strands. In some implementations, a reverse transcription terminator can be used, which typically terminates transcription only on the reverse strand.

[0097] Non-limiting examples of mammalian termination 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 some embodiments, the termination sequence is SV40 and contains at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) the same sequence as SEQ ID NO:8.

[0098] As used herein, the “Tet-Off” system is an inducible system that can inhibit the expression of a specific transgene in the presence of a tetracycline (e.g., doxycycline (DOX)). Conversely, the Tet-Off system can induce the expression of a specific transgene in the absence of a tetracycline (e.g., doxycycline DOX). In some embodiments, the Tet-Off system comprises a tetracycline-responsive promoter and a tetracycline-controlled transactivator (tTA) operatively linked to the transgene (e.g., encoding a protein, a gene-targeting nucleic acid, and / or a therapeutic gene). The transgene with a tetracycline-responsive promoter (e.g., TRE3G, TRE2, or Ptight promoter) and the tetracycline-controlled transactivator can be encoded in the same vector or in separate vectors.

[0099] As used herein, the “Tet-On” system is an inducible system that can induce the expression of a specific transgene in the presence of a tetracycline (e.g., doxycycline (DOX)). In some embodiments, the Tet-On system comprises a tetracycline-responsive promoter operatively linked to the transgene (e.g., a therapeutic sequence, a gene-targeting nucleic acid, and / or a nucleic acid encoding a protein) and a reverse tetracycline-controlled transactivator (rtTA).

[0100] Expression cassettes encoding tetracycline-responsive promoters (e.g., promoters containing TRE, including TRE3G, P-tight, and TRE2) and trans-activators controlled by inverse tetracycline can be encoded on the same vector or on separate vectors.

[0101] The term "tetracycline repressor" or "TetR" refers to a protein capable of binding to a Tet-O sequence (e.g., the Tet-O sequence in a TRE) in the absence of tetracycline (e.g., doxycycline) and preventing the binding of rtTA (e.g., rtTA3, rtTA4, or variants thereof) in the absence of tetracycline (e.g., doxycycline). TetR prevents gene expression by a promoter containing a TRE in the absence of tetracycline (e.g., doxycycline). In the presence of tetracycline, TetR cannot bind to a promoter containing 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, TetR is a TetR fusion (e.g., TRSID, which can be generated by fusing TetR to the mSIN30 interacting domain (SID) of Mad1). See, for example, Zhang et al., J Biol Chem. 30 November 2001; 276(48):45168-74.

[0102] As used herein, the term "therapeutic sequence" is any transgenic sequence encoding a therapeutic nucleic acid and / or protein (including prophylactic nucleic acids and / or proteins and diagnostic nucleic acids and / or proteins). For example, a non-limiting list of transgenic sequences as therapeutic sequences is described in O'Connor et al., Nat Rev Genet. 2006 Apr; 7(4):261-76. Non-limiting examples of therapeutic proteins include antibodies, enzymes, kinases, hormones, growth factors, cytokines, plasma proteins, fusion proteins, membrane cleavage proteins, and coagulation factors. In some embodiments, the therapeutic protein is an inflammatory factor. In some embodiments, the therapeutic protein is an anti-inflammatory agent. In some embodiments, the therapeutic protein is an immunomodulator. In some embodiments, the therapeutic protein is an anticancer agent. In some embodiments, the therapeutic protein is a metabolite. In some embodiments, the therapeutic protein is an antiviral agent / viricidal agent. In some embodiments, the therapeutic protein is an antibacterial agent / bactericidal agent.

[0103] The term "tissue" refers to any biological tissue (including cell populations, body parts, or organs) or a portion thereof, including blood vessels and / or lymphatic vessels, of a subject, which is the object to be delivered by the compounds, particles, and / or compositions of the present invention. Tissue can be abnormal or unhealthy tissue requiring treatment. Tissue can also be normal or healthy tissue that has a higher risk of becoming abnormal or unhealthy than normal, which may require preventative measures. In some embodiments, tissue is considered healthy but, under current or future conditions, its performance or survival is not optimal. For example, in agricultural practice, environmental conditions, including climate and growth conditions (e.g., nutrition), can benefit from any of the methods described herein. In some embodiments, tissue is the central nervous system. In some embodiments, tissue refers to tissue derived from certain embodiments, with cells or tissue originating from the eye, ear, nose, mouth (including gums and tooth roots), bone, lung, mammary gland, breast, pancreas, stomach, esophagus, muscle (including cardiac muscle), liver, blood vessels, skin (including hair), heart, brain, nerve tissue, kidney, testis, prostate, penis, cloaca, fins, ovary, or intestine. In some implementations, the tissue is damaged (e.g., due to congenital defects, injury, accident, or iatrogenic damage) and / or is aging tissue. In some implementations, the tissue is deep tissue accessible by a fiber optic probe.

[0104] In the context of damaged tissue, the term "tissue repair" refers to the restoration of tissue structure, function following tissue damage, or a combination thereof. Tissue repair includes tissue regeneration, cell growth, tissue replacement, and / or the rearrangement (reprogramming) of existing tissue.

[0105] The term "tissue regeneration" refers to the generation of new tissue or cells within a tissue of the same type as the tissue of interest (e.g., the same type of damaged tissue or cells). In some embodiments, the methods provided herein facilitate organ regeneration.

[0106] The term "tissue replacement" refers to the generation of tissues that are different from the tissues of interest (e.g., tissues with impaired connective tissue replacement).

[0107] As used herein, the terms “treatment,” “management,” and “curing” refer to reversing, alleviating, delaying the onset of a disease or disorder or one or more symptoms thereof, or inhibiting the progression of a disease or disorder or one or more symptoms thereof, as described herein. In some embodiments, treatment may be given after one or more symptoms have already appeared. In other embodiments, treatment may be given in the absence of symptoms. For example, treatment may be given to a susceptible individual before the onset of symptoms, or treatment may be administered with another damaging agent (e.g., based on a history of symptoms, based on genetic or other susceptibility factors, disease therapy, or any combination thereof). Treatment may also continue after symptoms have subsided, for example, to prevent or delay their recurrence.

[0108] The term "variant" or "mutant" refers to a sequence that contains modifications relative to the wild-type sequence (e.g., the rtTA3 sequence). Non-restrictive modifications to amino acid sequences include insertions, deletions, truncation mutations, and point mutations. Non-restrictive modifications to nucleic acid sequences include frameshift mutations, nucleotide insertions, and nucleotide deletions.

[0109] The term "WPRE" refers to the post-transcriptional regulatory element (WPRE) of marmot hepatitis virus (WHP). WPREs generate tertiary structures in nucleic acids (e.g., expression vectors) and are capable of enhancing transgene expression (e.g., from viral vectors). In some 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.

[0110] These and other exemplary substitutions are described in more detail in the detailed description of the invention, embodiments, and claims. The invention is not intended to be limited in any way to the foregoing list of exemplary substitutions.

[0111] Brief description of the attached figures

[0112] Figure 1This is a vector map illustrating the characteristics of an adeno-associated virus (AAV) vector encoding inverse tetracycline transactivator 4 (rtTA4). Ubc is a constitutive promoter operatively linked to the nucleic acid encoding rtTA4. SV40pA is an SV40-derived termination sequence. The sequence of this vector is provided in SEQ ID NO:17.

[0113] Figure 2A-2M Including Figure 1 The diagram shows a series of schematic representations of the features mapped onto the nucleic acid sequence of the vector encoding rtTA4.

[0114] Figure 3 It shows Figure 2A-2M The location and size of each feature depicted.

[0115] Figures 4A-4B This includes data showing in vivo leakage of the tetracycline-on (Tet-On) system using rtTA3 (SEQ ID NO:11) as an inducer. Figure 4A This is a non-limiting example of a Tet-On system comprising two nucleic acids. The first nucleic acid comprises a UBC promoter operatively linked to a sequence encoding rtTA3, a self-cleaving peptide (2A peptide), and mKate (far-red fluorescent protein). Figure 4A The second nucleic acid encodes an inducible promoter having a tetracycline response element (TRE) operatively linked to GFP (TRE3G promoter, SEQ ID NO:7). Figure 4A (Bottom). DOX indicates doxycycline. Figure 4B The protein imprints of liver samples from mice treated with the following criteria are shown: (1) absence of AAV; (2) presence of proteins containing AAV in the absence of doxycycline (DOX). Figure 4A AAV of the AAV vector containing the first nucleic acid, and AAV carrying the first nucleic acid of the AAV vector .... Figure 4A AAV of the AAV vector containing the second nucleic acid; or (3) AAV of (2) in the presence of doxycycline (DOX). mKate, GFP, and actin expression are shown.

[0116] Figures 5A-5B The tetracycline-on system using rtTA4 (SEQ ID NO:13) was shown to leak less than the same system using rtTA3. Figure 5A This is a schematic diagram depicting a Tet-On system reporter system employing rtTA4. The top of the diagram shows the nucleic acid, where the promoter sequence is operatively linked to the sequence encoding rtTA4. The bottom of the diagram shows a second nucleic acid, where the TRE3G promoter is operatively linked to the sequence encoding luciferase. Figure 5BThis is a graph showing the effect of doxycycline (DOX, ng / ml) on luciferase production in the Tet-On reporter system using rtTA3 compared to the Tet-On reporter system using rtTA4. Luciferase production is measured as luminescence / protein. Baseline levels of luminescence / protein are shown in the absence of any rtTA.

[0117] Figures 6A-6C Includes a series of graphs showing that the Tet-On system using rtTA4 shuts down more rapidly in response to doxycycline removal compared to the Tet-On system using rtTA3. A DOX-inducible luciferase reporter system was used, and luciferase yield was measured as luminescence / protein. +DOX indicates doxycycline treatment, -DOX indicates the absence of doxycycline treatment, and +-DOX indicates doxycycline removal after doxycycline treatment. Figure 6A This is a graph showing the effect of doxycycline on expression in the Tet-On reporter system, where rtTA3 expression is driven by the desmin promoter and luciferase expression is controlled by the TRE3G promoter. Figure 6B This is a graph showing the effect of doxycycline on expression in the Tet-On reporter system, where rtTA4 expression is driven by the desmin promoter and luciferase expression is controlled by the TRE3G promoter. Figure 6C This graph shows the time required for doxycycline withdrawal to reduce luciferase expression in the Tet-On reporter system using rtTA3 compared to the Tet-On reporter system using rtTA4. In both reporter systems, rtTA expression is driven by the UBC promoter, and luciferase expression is controlled by the TRE3G promoter.

[0118] Figure 7 This is a proteomic imprint comparing the effects of doxycycline treatment and withdrawal on transgene expression induced by rtTA3 (SEQ ID NO:11) and rtTA4 (SEQ ID NO:13) in 293T cells. The nucleic acid sequence encoding rtTA3 (SEQ ID NO:10) is operatively linked to the UBC promoter (SEQ ID NO:18), while rtTA4 is operatively linked to either the UBC promoter (SEQ ID NO:18) or the desmin promoter (SEQ ID NO:29).

[0119] Figures 8A-8C This includes data showing that the Tet-On system containing rtTA4 (SEQ ID NO:13) exhibits low permeability in mouse liver. Figure 8A These are a series of immunofluorescence images showing KLF4 levels after administration of doxycycline (DOX-carrying) in the absence of doxycycline (DOX-free) and in the presence of doxycycline (DOX-containing). Figure 8BThe nucleic acid AAV is shown to be expressed in the liver of mice. DAPI is a nuclear dye used to visualize cells. Figure 8B This is a schematic diagram depicting two nucleic acids in the AAV9 virus administered to mice. Figure 8C It comes from acceptance Figure 8B Western blots were performed on liver samples from mice that were neither treated with nor without doxycycline. OCT4, KLF4, and SOX2 levels were detected using antibodies as indicated. Actin is shown as a loading control.

[0120] Figure 9 This is a vector map depicting a non-limiting example of an inducible AAV vector (TRE3G-OSK-SV40pA, SEQ ID NO:16) encoding Oct4, SOX2, and KLF4, which can be used in combination with any rtTA4 vector described herein.

[0121] Figure 10 This is a schematic diagram illustrating a non-limiting example of an inducible expression system combining rtTA (e.g., rtTA4) and a tetracycline repressor (tetR, e.g., tetRKRAB). Triangles represent tetR (e.g., tetRKRAB) proteins, and circles represent rtTA4 proteins.

[0122] Figure 11 This is a vector map of pAAV2_CMV_rtTA(VP16) (SEQ ID NO:31). This vector is a non-limiting example of a vector encoding rtTA.

[0123] Figure 12 This is a vector map of pAAV-MCS-tTA2 (or CAG-tTA) (SEQ ID NO:32). This vector is a non-limiting example of a vector encoding tTA under the CAG promoter.

[0124] Figure 13 This is a vector map of p-AAV-TetO-OSK-WPRE3-SV50LpA (TRE2-OSK, pAAV-TRE2-OSK-SV40LpA, or TRE2-OSK) (SEQ ID NO:33). This vector is a non-limiting example of an AAV vector containing a nucleic acid (e.g., engineered nucleic acid) sequence greater than 4.7 kb between two ITRs in the vector.

[0125] Figure 14 This demonstrates that partial reprogramming of the polycistronic OSK delivered by AAV under rtTA4 control is non-toxic. Figure 14 The figures show WT mice, OSK transgenic mice, and AAV-mediated OSK expression mice (1.0 × 10⁻⁶) induced with or without doxycycline during the first 4 weeks.12 The body weight of (n = 5, 3, 6, 4, 6, 3) gene copies.

[0126] Figures 15A-15E This shows rtTA4, which can be used to control OSK expression in vivo. Figure 15A The expression of AAV9 in the liver is shown compared to that in transgenic mice. Figure 15B The body weights of WT mice and AAV-mediated OSK-expressing mice (total 1.0 × 10^12 gene copies) with or without doxycycline administration during the first 4 weeks and 9 months are shown (n = 5, 3, 6, 4, respectively). Figure 15C The AAV-UBC-rtTA and AAV-TRE-Luc vectors used to measure tissue distribution are shown. Figure 15D The image shows luciferase imaging in WT mice 2 months after retroorbital injection of AAV9-UBC-rtTA and AAV9-TRE-Luc (total 1.0 × 10^12 gene copies). As shown on the right, doxycycline was delivered to mice in drinking water (1 mg / mL) for 7 days. Figure 15E This image shows luciferase imaging of the eye (Ey), brain (Br), pituitary gland (Pi), heart (He), thymus (Th), lung (Lu), liver (Li), kidney (Ki), spleen (Sp), pancreas (Pa), testis (Te), fat (Ad), muscle (Mu), spinal cord (SC), stomach (St), small intestine (In), and cecum (Ce) 2 months after retroorbital injection of AAV9-UBC-rtTA and AAV9-TRE-Luc, followed by doxycycline treatment for 7 days. Luciferase signaling was predominantly observed in the liver. Imaging of the same tissues with longer exposure times was also shown. Figure 15E The lower group showed lower levels of luciferase signaling in the pancreas (liver was removed).

[0127] Detailed description of certain embodiments of the present invention

[0128] This disclosure is based, at least in part, on unexpected results showing that four mutations corresponding to residues G72, G12, F67, and R171 in rtTA3 (SEQ ID NO:11) produce a mutant rtTA (referred to herein as rtTA4) with lower leakage and increased sensitivity to tetracycline withdrawal compared to rtTA3. The existing rtTA3 Tet-On system is leaky in vivo (e.g., in mouse liver). Figure 4B The rtTA4 system described in this paper did not induce detectable transgene expression (e.g., in mouse liver).

[0129] As demonstrated herein, the rtTA4 sequence (e.g., SEQ ID NO:13) exhibits less leakage than rtTA3 (SEQ ID NO:11), and in the absence of tetracycline (e.g., doxycycline), rtTA4 does not induce detectable transgene expression in the liver, whereas rtTA3 induces transgene expression even in the absence of tetracycline (e.g., doxycycline). Furthermore, the Tet-On system employing rtTA4 shuts down 4–12 times faster than rtTA3. In some embodiments, the rtTA4 Tet-On system described herein is further regulated by tetR (tetRKRAB), which binds to the TRE promoter in the absence of tetracycline (e.g., doxycycline), thereby preventing rtTA binding and thus further suppressing gene expression.

[0130] Therefore, in some embodiments, this document provides mutant rtTA (e.g., rtTA4), engineered nucleic acids (e.g., expression vectors), recombinant viruses, systems, kits, and compositions comprising them, as well as methods for regulating gene expression using them. Any mutant rtTA, nucleic acids comprising mutant rtTA (e.g., engineered nucleic acids, including expression vectors), recombinant viruses, systems, kits, and compositions comprising them can be used to regulate gene expression, induce cell reprogramming, tissue repair, tissue regeneration, organ regeneration, reverse aging, treat diseases (e.g., acute injury, neurodegenerative diseases, chronic diseases, proliferative diseases, cardiovascular diseases, genetic diseases, inflammatory diseases, autoimmune diseases, neurological diseases, hematological diseases, pain conditions, mental disorders, metabolic disorders, cancer, aging, age-related diseases, and diseases affecting any tissue in a subject), or any combination thereof.

[0131] Mutant reverse tetracycline transactivator (rtTA)

[0132] This disclosure provides a mutant reverse tetracycline transactivator (rtTA) that can activate gene expression in the presence of tetracycline (e.g., doxycycline) via an operatively linked tetracycline response element (TRE) (e.g., TRE3G, TRE2, or Ptight promoter).

[0133] The mutant rtTA disclosed herein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 transactivation domains. Non-limiting examples of transactivation domains include VP64, P65, RTA, and MPH MS2-P65-HSF1. In some embodiments, the nucleotide sequence encoding VP64 comprises at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) the same sequence as SEQ ID NO:34. In some embodiments, the amino acid sequence encoding VP64 comprises at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) the same as the amino acid sequence encoded by SEQ ID NO:34. In some embodiments, the nucleotide sequence encoding P65 comprises at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) the same as the amino acid sequence encoded by SEQ ID NO:35. In some embodiments, the amino acid sequence encoding P65 comprises at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) the same as the amino acid sequence encoded by SEQ ID NO:35. In some embodiments, the nucleotide sequence encoding the RTA comprises at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) the same sequence as SEQ ID NO:36. In some embodiments, the amino acid sequence encoding the RTA comprises at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) the same sequence as the amino acid sequence encoded by SEQ ID NO:36. In some embodiments, the nucleotide sequence encoding MPH MS2-P65-HSF1 comprises at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) the same sequence as SEQ ID NO:37. In some embodiments, the amino acid sequence encoding MPH MS2-P65-HSF1 contains at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) the same sequence as the amino acid sequence encoded by SEQ ID NO:37.

[0134] The mutant rtTA (e.g., rtTA4) disclosed herein contains mutations at positions corresponding to the following residues in SEQ ID NO:11: G72; G12; F67; and R171. SEQ ID NO:11 is a non-limiting example of the rtTA3 sequence. Non-limiting examples of mutations include point mutations, truncated mutations, deletions, or insertions.

[0135] Non-restrictive mutations at the positions corresponding to residue G12 in SEQ ID NO:11 include alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), histidine (H), isoleucine (I), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

[0136] Non-restrictive mutations at the positions corresponding to residue G72 in SEQ ID NO:11 include alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), histidine (H), isoleucine (I), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

[0137] Non-restrictive mutations at the position corresponding to residue F67 in SEQ ID NO:11 include alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), lysine (K), methionine (M), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

[0138] Non-restrictive mutations at the position corresponding to residue F67 in SEQ ID NO:11 include alanine (A), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), lysine (K), methionine (M), proline (P), phenylalanine (F), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

[0139] Non-restrictive mutations at the position corresponding to residue F67 in SEQ ID NO:11 include alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), lysine (K), methionine (M), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

[0140] Mutations at G72, G12, F67, and / or R171 can be mutations to amino acids with charged side chains (e.g., arginine, histidine, lysine, aspartic acid, or glutamic acid). Amino acids may contain negatively charged side chains (e.g., aspartic acid or glutamic acid). Amino acids may contain positively charged side chains (arginine, histidine, or lysine).

[0141] Mutations at G72; G12; F67; and / or R171 can be mutations to amino acids containing polar, uncharged side chains (e.g., serine, threonine, asparagine, glutamine) or amino acids containing hydrophobic side chains (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan).

[0142] Mutations at G72; G12; F67; and / or R171 can be mutations to amino acids containing an aromatic ring (e.g., phenylalanine, tyrosine, or tryptophan).

[0143] In some embodiments, the nucleic acid encoding rtTA4 comprises 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%) the same sequence as SEQ ID NO:12, and / or encodes a protein having mutations at the following residues corresponding to the positions in rtTA3 (SEQ ID NO:11): G72; G12; F67; and R171. The rtTA4 nucleic acid sequence may consist of SEQ ID NO:12.

[0144] In some embodiments, the amino acid sequence encoding rtTA4 contains 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%) the same sequence as SEQ ID NO:13, and / or encodes a protein having mutations at the following residues corresponding to the positions in rtTA3 (SEQ ID NO:11): G72; G12; F67; and R171.

[0145] In some embodiments, in addition to the mutations corresponding to residues G72, G12, F67, and R171 in SEQ ID NO:11, the mutant rtTA also contains at least one other mutation at the position corresponding to rtTA3 (SEQ ID NO:11). In some embodiments, the mutant rtTA contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, or 240 additional mutations corresponding to positions that are not residues G72, G12, F67, and R171 in SEQ ID NO:11.

[0146] It should be understood that any rtTA (e.g., rtTA3 or M2-rtTA) can be used as a reference sequence to be mutated. For example, the mutant rtTA of this disclosure may contain mutations corresponding to residues G72; G12; F67; and R171 of SEQ ID NO:11, but may also contain amino acid sequences present in M2-rtTA. In some embodiments, in addition to the mutations corresponding to residues G72, G12, F67, and R171 of SEQ ID NO:11, the rtTA of this disclosure may also contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, or 240 additional mutations corresponding to positions that are not residues G72, G12, F67, and R171 of SEQ ID NO:11.

[0147] In some embodiments, the amino acid sequence of rtTA4, relative to rtTA3, includes the following mutations: a valine (V) or proline (P) mutation at the residue corresponding to position G72 in SEQ ID NO:11, a serine (S) mutation at the residue corresponding to position G12 in SEQ ID NO:11, a serine (S) mutation at the residue corresponding to position F67 in SEQ ID NO:11, and a lysine (K) mutation at the residue corresponding to position R171 in SEQ ID NO:11. The amino acid sequence of rtTA4 may also consist of SEQ ID NO:13.

[0148] Any suitable sequence alignment algorithm can be used to align two sequences of interest (e.g., rtTA4 of this disclosure with rtTA3 as shown in SEQ ID NO:11) to identify corresponding residue positions in rtTA3. As a non-limiting example, Clustal Omega can be used to align SEQ ID NO:13 (amino acid sequence of rtTA4) with SEQ ID NO:11 (amino acid sequence of rtTA3) (see, for example, Larkin et al., Bioinformatics. 1 November 2007; 23(21):2947-8). In the following exemplary alignment, position 12 (glycine) in SEQ ID NO:11 is mutated to serine in SEQ ID NO:13, position 72 (glycine) in SEQ ID NO:11 is mutated to valine in SEQ ID NO:13, position 67 (phenylalanine) in SEQ ID NO:11 is mutated to serine in SEQ ID NO:13, and position 171 (arginine) in SEQ ID NO:11 is mutated to lysine in SEQ ID NO:13.

[0149]

[0150] It should be understood that this disclosure covers rtTA4 variants containing mutations at the following residues corresponding to the positions in rtTA3 (SEQ ID NO:11): G72; G12; F67; and R171. Such rtTA4 variants may contain sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% identical to the sequence in SEQ ID NO:13.

[0151] As is known in the art, the term "sequence identity" refers to the relationship between the sequences of two polypeptides or two polynucleotides as determined by sequence comparison (alignment). In some embodiments, sequence identity is determined over the full length of the mutant rtTA (e.g., rtTA4) sequence. In some embodiments, sequence identity is determined over a region (e.g., a fragment of an amino acid or nucleotide chain, such as 10, 20, 30, 40, 50, etc.) of the mutant rtTA (e.g., rtTA4).

[0152] Identity can also refer to the degree of sequence correlation between two sequences, such as by measuring the number of matches between chains of two or more residues (e.g., nucleotide or amino acid residues). Identity is determined by resolving vacancy alignments (if any) using a specific mathematical model or computer program (e.g., an "algorithm") to measure the percentage of identical matches between the smaller of two or more sequences.

[0153] The identity of related polypeptide or nucleic acid sequences can be readily calculated using any method known to those skilled in the art. The “percentage identity” of two sequences (e.g., nucleic acid or amino acid sequences) can be determined, for example, using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990 (as modified in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993). Such algorithms are incorporated into the algorithm of Altschul et al., J. Mol. Biol. 215:403-10, 1990. and In the program (version 2.0). For example, the XBLAST program (score = 50, byte length = 3) can be used. Protein search is performed to obtain amino acid sequences homologous to the protein molecule of this invention. In cases where a gap exists between two sequences, gapped sequences can be used, for example. As described by Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using and Gapped When using a program, you can use various programs (e.g.) and The default parameters, or the parameters can be adjusted appropriately as understood by those skilled in the art.

[0154] For example, another available local alignment technique is based on the Smith-Waterman algorithm (Smith, TF & Waterman, MS (1981) "Identification of common molecular subsequences." J. Mol. Biol. 147: 195-197). Another available general global alignment technique is the Needleman–Wunsch algorithm (Needleman, SB & Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48: 443-453), which is based on dynamic programming.

[0155] Recently, a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, which is claimed to generate global alignments of nucleic acid and amino acid sequences faster than other optimal global alignment methods, including the Needleman–Wunsch algorithm. In some implementations, the identity of two polypeptides is determined by aligning two amino acid sequences, calculating the number of identical amino acids, and dividing by the length of one amino acid sequence. In some implementations, the identity of two nucleic acids is determined by aligning two nucleotide sequences, calculating the number of identical nucleotides, and dividing by the length of one nucleic acid.

[0156] For multiple sequence alignment, computer programs including Clustal Omega can be used (Sievers et al., MolSyst Biol. 2011 Oct 11; 7:539).

[0157] In some implementations, the protein encoding the mutant rtTA (e.g., rtTA4) is fused with a protein transduction domain. Without specific theoretical constraints, protein transduction domains facilitate the delivery of carriers (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. See, for example, Zhou et al., Cell Stem Cell. May 8, 2009; 4(5):381-4; Zahid et al., Curr Gene Ther. October 2012; 12(5):374-80.

[0158] In some embodiments, the protein is formulated in nanoparticles for delivery. In some embodiments, the chitosan polymer nanoparticles are loaded with a mutant rtTA protein (e.g., rtTA4). See, for example, Tamammam et al., Oncotarget. 21 June 2016; 7(25):37728-37739.

[0159] Nucleic acid encoding the mutant rtTA (e.g., engineered nucleic acid)

[0160] Any nucleic acid sequence encoding recombinant rtTA (e.g., rtTA4) described herein can be cloned into an expression vector. The nucleic acids disclosed herein (e.g., engineered nucleic acids) can be present on viral or non-viral vectors. Suitable non-viral vectors include, but are not limited to, plasmid DNA or RNA (e.g., mRNA). In some embodiments, plasmid DNA can be introduced into nanoparticles and / or fused to a protein transduction domain (PTD). See above.

[0161] As a non-limiting example, the engineered nucleic acids of this disclosure (e.g., expression vectors) (e.g., the RNA of this disclosure, including mRNA, or DNA (e.g., plasmid DNA)) can be formulated in nanoparticles for delivery. See, for example, Dong et al., NanoLett. 2016 Feb 10; 16(2):842-8. In some embodiments, the nanoparticles contain acetylated galactose. See, for example, Lozano-Torres et al., J Am Chem Soc. 2017 Jul 5; 139(26):8808-8811. In some embodiments, the engineered nucleic acids (e.g., expression vectors) (e.g., RNA, including mRNA, or DNA) are electroporated or transfected into cells. In some embodiments, the engineered nucleic acids are delivered as naked nucleic acids (e.g., naked DNA or naked RNA).

[0162] Non-limiting examples of viral vectors include lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, and AAV vectors. Any recombinant rtTA using methods known in the art. Expression vectors containing essential expression elements are commercially available and known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, 2012). See also, for example, the following conventional techniques. Non-limiting examples of elements on nucleic acids (e.g., engineered nucleic acids, including expression vectors) include promoters, nucleic acid sequences operatively linked to promoters (e.g., open reading frames), termination sequences, spacer sequences, or WPRE sequences. Vectors may contain one or more of these elements.

[0163] In some embodiments, the nucleic acid sequence encoding the mutant rtTA is codon-optimized for expression in specific host cells. In some embodiments, the sequence encoding the mutant rtTA is codon-optimized for expression in mammalian cells. In some embodiments, the sequence encoding the mutant rtTA is codon-optimized for expression in human cells. In some embodiments, the sequence encoding the mutant rtTA is at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:12.

[0164] The engineered nucleic acid (e.g., expression vector) encoding the mutant rtTA (e.g., rtTA4) of this disclosure comprises a promoter operatively linked to the nucleic acid encoding rtTA. The promoter may be a constitutive promoter (e.g., CP1, CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, Ac5, polyhedrone protein, TEF1, GDS, CaM3 5S, Ubi, H1, or U6 promoter). The Ubc promoter may contain at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) of the sequence identical to SEQ ID NO:18.

[0165] To allow tissue-specific expression of the mutant rtTA, the promoter may be tissue-specific (e.g., an eye-specific promoter, bone-specific promoter, lung-specific promoter, breast-specific promoter, pancreas-specific promoter, muscle-specific promoter, liver-specific promoter, skin-specific promoter, heart-specific promoter, brain-specific promoter, nerve tissue-specific promoter, kidney-specific promoter, testis-specific promoter, ovary-specific promoter, or intestinal-specific promoter). In some embodiments, the muscle-specific promoter is the desmin promoter. In some embodiments, the desmin promoter contains at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) of the sequence identical to SEQ ID NO:29.

[0166] As will be understood by those skilled in the art, promoters operatively linked to the nucleic acid encoding the mutant rtTA can be selected based on the host cell for expression. For example, mammalian promoters can be used to drive the expression of mutant rtTA in mammalian cells. Eukaryotic promoters can be used to drive the expression of rtTA in eukaryotic cells. Prokaryotic promoters can be used to drive the expression of rtTA in prokaryotic cells. Tissue-specific promoters can be used to express mutant rtTA in tissues of interest in a subject.

[0167] In some implementations, the nucleic acid (e.g., engineered nucleic acid) sequence encoding the mutant rtTA is codon-optimized for expression in a specific host cell. For example, the nucleic acid (e.g., engineered nucleic acid) encoding the mutant rtTA may be codon-optimized for expression in eukaryotic cells (e.g., mammalian cells) or prokaryotic cells.

[0168] The nucleic acid encoding the mutant rtTA (e.g., engineered nucleic acid, including expression vectors) may also contain a post-transcriptional regulatory element (WPRE) of marmot hepatitis B virus (WHP), which can be used to enhance gene expression of the transgene (e.g., derived from a viral vector). In some 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. In some embodiments, the WPRE sequence is located downstream of the nucleic acid encoding the mutant rtTA (e.g., rtTA4).

[0169] Termination sequences can be used to designate the ends of transcripts (causing transcription to terminate). Non-limiting examples of mammalian termination 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 some embodiments, the termination sequence is SV40 and contains at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) the same sequence as SEQ ID NO:8.

[0170] Nucleic acids encoding mutant rtTA (e.g., rtTA4) (e.g., engineered nucleic acids, including expression vectors) may also encode tetracycline repressors (TetR), which prevent gene expression containing a TRE promoter in the absence of tetracycline (e.g., doxycycline). In the presence of tetracycline, TetR cannot bind to a TRE-containing promoter, and TetR cannot prevent transcription. Non-limiting examples of TetR include TetR, TRSID, and tetRKRAB. In some embodiments, TetR (e.g., TetR) is encoded by a nucleic acid containing 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%) the same sequence as SEQ ID NO:25. In some embodiments, TetR (e.g., TetR) comprises 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%) of the sequence identical to SEQ ID NO:26. In some embodiments, TetR (e.g., tetRKRAB) is encoded by a nucleic acid comprising 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%) of the sequence identical to SEQ ID NO:27. In some embodiments, TetR (e.g., tetRKRAB) comprises 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%) of the sequence identical to SEQ ID NO:28. Compared to the presence of tetracycline, TetR reduces transgene expression at the promoter by 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 100% in the absence of tetracycline (e.g., doxycycline). Gene expression can be measured using any suitable method, including assessment of protein and RNA levels.

[0171] Nucleic acids (e.g., engineered nucleic acids, including expression vectors) may also contain spacer sequences (e.g., IRES or peptide cleavage signals). Exemplary peptide cleavage signals include 2A peptides (e.g., T2A, P2A, E2A, and F2A). 2A peptides may contain at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) of the sequence identical to SEQ ID NO:9. For expression vectors encoding more than one transgene (e.g., mutants rtTA and tetR (e.g., tetRKRAB)), each transgene may be operatively linked to a different promoter or the same promoter. Transgenes may be spaced apart on the expression vector (e.g., IRES or peptide cleavage signals). Expression of the nucleic acid results in the separation of amino acid sequences encoding each protein of interest.

[0172] In some embodiments, the nucleic acid encoding the mutant rtTA (e.g., rtTA4) (e.g., engineered nucleic acid, including an expression vector) may contain at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) of the same sequence as pAAV-UBC-rtTA4-WPRE3-SV40pA (SEQ ID NO:17). In some embodiments, the nucleic acid encoding the mutant rtTA (e.g., rtTA4) (e.g., engineered nucleic acid, including an expression vector) is composed of SEQ ID NO:17. In some embodiments, the nucleic acid encoding the mutant rtTA (e.g., rtTA4) (e.g., engineered nucleic acid, including an expression vector) may contain at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) of the same sequence as the desmin-rtTA4 vector (SEQ ID NO:30). In some implementations, the nucleic acid encoding the mutant rtTA (e.g., rtTA4) (e.g., engineered nucleic acid, including expression vectors) consists of SEQ ID NO:30.

[0173] The vectors of the present invention may also contain marker sequences for identifying cells that have been or have not been transformed or transfected by the vector. Markers include, for example, genes encoding proteins that enhance or weaken resistance or sensitivity to antibiotics (e.g., ampicillin resistance genes, kanamycin resistance genes, neomycin resistance genes, tetracycline resistance genes, and chloramphenicol resistance genes) or other compounds; genes encoding enzymes (e.g., β-galactosidase, luciferase, or alkaline phosphatase) with activities detectable by standard assays known in the art; and genes that significantly affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques (e.g., green fluorescent protein). In some embodiments, the vectors used herein are capable of autonomously replicating and expressing structural gene products present in DNA fragments operatively linked together.

[0174] In some implementations, the nucleic acid encoding rtTA (e.g., engineered nucleic acid, including expression vectors) is a viral vector (e.g., lentiviral vector, adenovirus vector, alphavirus vector, vaccinia virus vector, herpesvirus vector, adeno-associated virus (AAV) vector). As used herein, AAV vectors typically contain an ITR side-joined to an expression cassette (e.g., a nucleic acid containing a promoter sequence operatively linked to a sequence encoding mutant rtTA (including rtTA4).

[0175] In some embodiments, the number of base pairs between two ITRs in the AAV vector of this disclosure is less than 5 kilobits (kb) (e.g., less than 4.9 kb, less than 4.8 kb, less than 4.7 kb, less than 4.6 kb, less than 4.5 kb, less than 4.4 kb, less than 4.3 kb, less than 4.2 kb, less than 4.1 kb, less than 4 kb, less than 3.5 kb, less than 3 kb, less than 2.5 kb, less than 2 kb, less than 1.5 kb, less than 1 kb, or less than 0.5 kb). In some implementations, AAV vectors with a distance of less than 4.7 kb between two ITRs can be packaged into the virus at titers of at least 0.5 × 10^10 particle formation units / ml (pfu / ml), at least 1 × 10^10 pfu / ml, at least 5 × 10^10 pfu / ml, at least 1 × 10^11 pfu / ml, at least 5 × 10^11 pfu / ml, at least 1 × 10^12 pfu / ml, at least 2 × 10^12 pfu / ml, at least 3 × 10^12 pfu / ml, at least 4 × 10^12 pfu / ml, at least 5 × 10^12 pfu / ml, at least 6 × 10^12 pfu / ml, at least 7 × 10^12 pfu / ml, at least 8 × 10^12 pfu / ml, at least 9 × 10^12 pfu / ml, or at least 1 × 10^13 pfu / ml.

[0176] In some embodiments, the infection efficiency of recombinant viruses (e.g., lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAVs) carrying the mutant rtTA (e.g., rtTA4) vector of this disclosure in cells (e.g., animal cells, including mammalian cells) 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%).

[0177] In some embodiments, the nucleic acid of this disclosure (e.g., engineered nucleic acid, including expression vectors) is at least 1 kilobits (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 some embodiments, the nucleic acid of this disclosure (e.g., engineered nucleic acid, including expression vectors) 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).

[0178] Inducible expression vectors encoding transgenes

[0179] Any mutant rtTA described herein can be used to promote the expression of a transgene operatively linked to an inducible promoter. In some embodiments, the nucleic acid encoding the mutant rtTA (e.g., engineered nucleic acid, including an expression vector) also comprises a transgene operatively linked to an inducible promoter. In some embodiments, the nucleic acid encoding the mutant rtTA (e.g., engineered nucleic acid, including an expression vector) is separate from the nucleic acid encoding a transgene operatively linked to an inducible promoter (e.g., engineered nucleic acid, including an expression vector). The mutant rtTA can drive the expression of at least one inducible promoter containing a Tet-O sequence. The mutant rtTA can drive the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 inducible promoters containing Tet-O sequences.

[0180] It should be understood that an inducible promoter containing a Tet-O sequence can be operatively linked to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 transgenic sequences.

[0181] Suitable transgenic expression vectors used with the mutant rtTA of this disclosure contain an inducible promoter (e.g., a TRE promoter) containing at least one Tet-O sequence (e.g., an inducible promoter may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90 or all of the Tet-O sequences). The TRE promoter of this disclosure may contain 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 100%) of the sequence identical to SEQ ID NO:7. TRE promoters typically contain a minimal promoter that cannot promote transcription in the absence of an upstream enhancer present in the TRE promoter. The minimum promoter may be a minimum CMV promoter (e.g., a sequence identical to at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of SEQ ID NO:20). For example, the TRE promoter may be a TRE3G promoter (e.g., a sequence identical to at least 70% of SEQ ID NO:7 (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%)). For example, the TRE promoter may be a TRE3G promoter (e.g., a sequence identical to at least 70% of SEQ ID NO:7 (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%)). In some embodiments, the TRE promoter is a TRE2 or P-tight promoter. In some embodiments, the TRE promoter is the TRE2 promoter and contains at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same sequence as SEQ ID NO:23. In some embodiments, the TRE promoter is the P-tight promoter and contains at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same sequence as SEQ ID NO:24.

[0182] The inducible expression vectors disclosed herein comprise at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100) transgene. The transgene may encode any gene (e.g., a protein-coding gene, a gene-targeting nucleic acid, and / or a therapeutic gene). Non-limiting examples of genes include wild-type genes mutated in genetic diseases. In some embodiments, the gene is a transcription factor. In some embodiments, the transgene, alone or in combination, encodes OCT4, SOX2, KLF4, or their homologs or variants (e.g., functional variants). In some embodiments, the engineered nucleic acid encodes c-Myc. In some embodiments, the engineered nucleic acid does not encode c-Myc. In some embodiments, the engineered nucleic acid does not encode functional c-Myc because it lacks a c-Myc sequence. Assays for determining the activity of transcription factors (e.g., OCT4, SOX2, KLF4, c-Myc, or any combination thereof) are known in the art and include cell-based transcription assays and in vitro transcription assays. Other methods can also be used to determine transcription factor expression, including enzyme-linked immunosorbent assay (ELISA), Western blotting, and RNA quantification (e.g., using reverse transcription polymerase chain reaction).

[0183] Non-restricted examples of genes include Tet1, Tet2, Nanog, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, FGF21, GDF15, GDF11, NF-kb, PCSK9, mTERT, HAS2, PDE4By358c, sIGF1r-FC, sIGF2r-FC, Fat-1, mTOR, Klotho, TFEB, Grin2b, DNMT1, AMPK, NRF2, NEU1, NGF, Bcat-1, FoxP2, ZAG, adiponectin, and TFAM.

[0184] In some embodiments, the transgenic gene-encoding nucleic acid is complementary to the promoter and / or enhancer region of the gene's endogenous locus. In some embodiments, the gene-encoding nucleic acid is complementary to the protein-coding region of the gene. In some embodiments, the gene-encoding nucleic acid is complementary to RNA (e.g., mRNA). In some embodiments, the RNA (e.g., mRNA) encodes a protein.

[0185] In some implementations, the gene-targeting nucleic acid is a small interfering RNA (siRNA), a small hairpin RNA or short hairpin RNA (shRNA), a microRNA (miRNA), a guide RNA (gRNA), or an antisense RNA (asRNA). In some implementations, the siRNA, shRNA, miRNA, or asRNA can reduce the expression of the target gene by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0186] In some implementations, the guide RNA is a CRISPR guide RNA and can guide the Cas9 nuclease to an endogenous location in the genome. Guide RNAs and CRISPR systems can be used to knock out or activate gene expression. When used with a donor template, guide RNAs and CRISPR systems can also be used to knock in alleles or genes of interest. Methods using CRISPR systems and considerations for guide RNA design are known in the art. Many web-based tools are available for designing guide RNAs. For example, non-limiting examples may be found from MIT (e.g., http: / / crispr.mit.edu / ) and the Broad Institute (https: / / portals.broadinstitute.org / gpp / public / analysis-tools / sgrna-design). See also, for example, Hsu et al., Cell. June 5, 2014; 157(6):1262-78; Sander et al., Nat Biotechnol. April 2014; 32(4):347-55; Doench et al., Nat Biotechnol. February 2016; 34(2):184-191.

[0187] In some implementations, the transgene encodes a Cas9 and / or nuclease-deficient Cas9 fused to a transcriptional activation complex (e.g., containing VP64, P65, Rta, and / or MPH).

[0188] Generally, a CRISPR activation system comprises an enzyme-catalyzed Cas9 nuclease (or nuclease-deficient Cas9 (dCas9)) fused to a transcription activation complex (e.g., containing 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 may contain at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical sequences to any VP64, P65, Rta, and / or MPH sequence described herein. This Cas9 fusion protein may be referred to as a CRISPR activator. Guide RNAs targeting promoter and / or enhancer regions of the gene of interest are used in CRISPR activation systems to target the dCas9 transcription activation complex and drive the expression of endogenous genes.

[0189] In some embodiments, administration of an inducible nucleic acid (e.g., engineered nucleic acid, including an expression vector) results in a decrease in the expression of the target gene of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, wherein the inducible nucleic acid contains a TRE promoter operatively linked to a transgene encoding Cas9 and / or a guide RNA targeting an endogenous gene of interest. In some embodiments, Cas9 and the guide RNA are located on two separate expression vectors. In some implementations, the administration of an inducible nucleic acid (e.g., engineered nucleic acid, including an expression vector) results in an increase in the expression of an operatively linked gene by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, wherein the inducible nucleic acid contains a TRE promoter operatively linked to a transgene encoding dCas9 fused to the activation complex and / or a guide RNA targeting a promoter and / or enhancer region of an endogenous gene of interest.

[0190] The transgenes disclosed herein (e.g., transgenes encoding proteins, therapeutic sequences, gene-targeting nucleic acids, nucleic acids encoding OCT4, SOX2, KLF4, c-Myc, transcription factors or their homologs or variants, including mammalian OCT4, mammalian SOX2, and mammalian KLF4) may be encoded by a single nucleic acid, or a single nucleic acid may encode two or more transgenes (e.g., each operatively linked to a different promoter, or all operatively linked to the same promoter). For example, in some embodiments, the nucleic acid may be able to encode OCT4, SOX2, c-Myc, KLF4, OCT4, and SOX2 in any order; OCT4 and KLF4; SOX2 and KLF4; or OCT4, SOX2, and KLF4; OCT4, c-Myc, and SOX2; OCT4, c-Myc, and KLF4; SOX2, c-Myc, and KLF4; or OCT4, SOX2, c-Myc, and KLF4.

[0191] In some implementations, the nucleic acid (e.g., engineered nucleic acid, including expression vectors) encodes at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100) transgenes.

[0192] In some embodiments, the transgene encodes a protein. In some embodiments, the protein is a human protein. In some embodiments, the protein is a non-human protein (e.g., mammalian (e.g., primates (e.g., macaques, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and / or turkeys)). If two or more transgenes are on a vector, they can be in any order. The terms "first," "second," and "third" do not imply an order of genes on the vector.

[0193] In some embodiments, the transgene encodes a therapeutic sequence. The therapeutic sequence may also be referred to as a gene suitable for gene therapy in the art. Non-limiting examples of therapeutic proteins include antibodies, enzymes, kinases, hormones, growth factors, cytokines, plasma proteins, fusion proteins, membrane-lysing proteins, and coagulation factors. In some embodiments, the therapeutic protein is an inflammatory cytokine. In some embodiments, the therapeutic protein is an anti-inflammatory agent. In some embodiments, the therapeutic protein is an immunomodulator. In some embodiments, the therapeutic protein is an anticancer agent. In some embodiments, the therapeutic protein is a metabolite. In some embodiments, the therapeutic protein is an antiviral agent / viricidal agent. In some embodiments, the therapeutic protein is an antibacterial agent / bactericidal agent.

[0194] The transgenic structures described herein (e.g., therapeutic sequences, gene-targeting nucleic acids) may contain one or more amino acid substitutions. Variants may be prepared according to methods known to those skilled in the art for altering polypeptide sequences, such as those seen in references compiling such methods, e.g., *Molecular Cloning: A Laboratory Manual*, edited by J. Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or *Current Protocols in Molecular Biology*, edited by F. MAusubel et al., John Wiley & Sons, Inc., New York. Conserved substitutions of amino acids include substitutions between amino acids within the following group: (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.

[0195] Expression vectors containing essential expression elements are commercially available and are known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition, Cold SpringHarbor Laboratory Press, 2012).

[0196] In some embodiments, nucleic acids (e.g., engineered nucleic acids, including expression vectors) containing transgenes (e.g., protein-coding genes, gene-targeting nucleic acids, and / or therapeutic genes) are present on viral vectors (e.g., AAV vectors). As used herein, AAV vectors typically contain an ITR side-attached to an expression cassette (e.g., a nucleic acid containing a TRE promoter operatively linked to the transgene (e.g., a therapeutic sequence, gene-targeting nucleic acid, and / or protein-coding sequence)).

[0197] In some implementations, nucleic acids (e.g., engineered nucleic acids, including expression vectors) containing transgenes (e.g., protein-coding genes, gene-targeting nucleic acids, and / or therapeutic genes) are present on non-viral vectors (e.g., plasmids used for transient transfection).

[0198] In some embodiments, the number of base pairs between two ITRs in the AAV vector of this disclosure is less than 5 kilobits (kb) (e.g., less than 4.9 kb, less than 4.8 kb, less than 4.7 kb, less than 4.6 kb, less than 4.5 kb, less than 4.4 kb, less than 4.3 kb, less than 4.2 kb, less than 4.1 kb, less than 4 kb, less than 3.5 kb, less than 3 kb, less than 2.5 kb, less than 2 kb, less than 1.5 kb, less than 1 kb, or less than 0.5 kb). In some implementations, AAV vectors with a distance of less than 4.7 kb between two ITRs can be packaged into the virus at titers of at least 0.5 × 10^10 particle formation units / ml (pfu / ml), at least 1 × 10^10 pfu / ml, at least 5 × 10^10 pfu / ml, at least 1 × 10^11 pfu / ml, at least 5 × 10^11 pfu / ml, at least 1 × 10^12 pfu / ml, at least 2 × 10^12 pfu / ml, at least 3 × 10^12 pfu / ml, at least 4 × 10^12 pfu / ml, at least 5 × 10^12 pfu / ml, at least 6 × 10^12 pfu / ml, at least 7 × 10^12 pfu / ml, at least 8 × 10^12 pfu / ml, at least 9 × 10^12 pfu / ml, or at least 1 × 10^13 pfu / ml.

[0199] In some embodiments, the nucleic acid of this disclosure (e.g., engineered nucleic acid, including expression vectors) is at least 1 kilobits (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 some embodiments, the nucleic acid of this disclosure (e.g., engineered nucleic acid, including expression vectors) 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).

[0200] Without being limited by specific theories, nucleic acids encoding multiple transgenes (e.g., protein-coding sequences, gene-targeting nucleic acids, and / or therapeutic sequences) (e.g., engineered nucleic acids, including expression vectors) (e.g., AAV vectors) under a single promoter result in more efficient transduction of all transgenes in vivo compared to single nucleic acids encoding one or two transgenes. In some embodiments, the infection efficiency of recombinant viruses (e.g., lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAVs) carrying the transgene vectors of this disclosure in cells (e.g., eukaryotic or prokaryotic cells) 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%).

[0201] Recombinant virus

[0202] This disclosure provides recombinant viruses (e.g., lentiviruses, adenoviruses, herpesviruses, alphaviruses, vaccinia viruses, retroviruses, or AAVs). The recombinant viruses (e.g., lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAVs) may carry nucleic acids encoding mutant rtTA (e.g., engineered nucleic acids, including expression vectors), inducible nucleic acids containing transgenes (e.g., therapeutic sequences, gene-targeting nucleic acids, and / or protein-coding sequences) (e.g., engineered nucleic acids, including expression vectors), or combinations thereof.

[0203] In some embodiments, the recombinant virus is a recombinant AAV. In some embodiments, the recombinant AAV has tissue-specific targeting capability, such that the transgene of the AAV will be specifically delivered to one or more predetermined tissues. Typically, the AAV capsid is the relevant factor determining the tissue-specific targeting capability of the AAV. The AAV capsid may contain amino acid sequences derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and their variants. Non-limiting examples of tissue specificity of AAV serotypes are provided in Table 1. “x” indicates that the specified AAV serotype is capable of delivering the transgene to a specific tissue.

[0204] Table 1. Non-limiting examples of AAV serotypes and their utility in specific tissues:

[0205]

[0206] Recombinant AAVs containing a specific capsid protein can be produced using any suitable method. See, for example, U.S. Patent Application Publication US 2003 / 0138772, which is incorporated herein by reference. AAV capsid protein sequences are also known in the art. See, for example, published PCT Patent Application WO2010 / 138263, which is incorporated herein by reference. Typically, recombinant AAVs are produced in host cells having the following components: (1) a nucleic acid sequence or fragment thereof encoding the AAV capsid protein, (2) a nucleic acid encoding a functional rep gene, (3) a recombinant AAV vector containing an AAV inverted terminal repeat sequence side-attached to a transgene (e.g., a protein-coding sequence, a gene-targeting nucleic acid, and / or a therapeutic sequence), and (4) an accessory function that allows the recombinant AAV vector to be packaged into the AAV capsid protein. In some embodiments, the accessory function is introduced via an accessory vector known in the art.

[0207] In some cases, suitable host cell lines (e.g., HEK293T cells) can be used to generate the recombinant AAV disclosed herein, in accordance with standard practice. One or more expression vectors encoding one or more of the aforementioned components can be introduced into host cells via exogenous nucleic acids, which can be cultured under suitable conditions allowing for the production of AAV particles. Helper vectors can be used, when needed, to promote replication, to promote the assembly of AAV particles, or any combination thereof. In some embodiments, the recombinant AAV vector is present on nucleic acids separate from other components (e.g., nucleic acid sequences or fragments thereof encoding AAV capsid proteins, nucleic acids encoding functional rep genes, and helper functionalities that allow the recombinant AAV vector to be packaged into the AAV capsid protein). In some embodiments, the host cells can stably express one or more components required for the production of AAV virus. In this case, the remaining components can be introduced into the host cells. The supernatant of the cell culture can be collected, and the viral particles contained therein can be collected via standard methodologies.

[0208] The compositions disclosed herein may comprise any recombinant virus described herein (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV), alone or in combination with one or more other recombinant viruses (e.g., encoding a second AAV having one or more different transgenes). In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV), each having one or more different transgenes.

[0209] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. Those skilled in the art can readily select a suitable carrier considering the indications targeted by the recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV). For example, a 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 this disclosure.

[0210] Optionally, in addition to recombinant viruses (e.g., lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAVs) and one or more carriers, the compositions disclosed herein may also contain other pharmaceutical ingredients, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0211] Recombinant viruses (e.g., lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAVs) are administered in an amount sufficient to transfect cells in the desired tissue (e.g., ocular tissue, such as corneal tissue) and provide adequate levels of gene transfer and expression without adverse side effects. Examples of pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., intramammary delivery to the eye), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parenteral routes. Combined routes of administration may be used as needed.

[0212] The dosage of recombinant viral (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) virion required to achieve a specific therapeutic effect, for example, in units of genome copy number per kilogram of body weight (GC / kg), will vary based on several factors, including but not limited to: the route of administration of the recombinant viral (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) virion, the gene or RNA expression level required to achieve the therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. Based on the above and other factors, those skilled in the art can readily determine the dosage range of recombinant viral (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV virion) for treating patients with a specific disease or disorder.

[0213] An effective amount of recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) is sufficient to target and infect an animal and target the desired tissue. In some embodiments, an effective amount of recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) is sufficient to produce a stable somatic transgenic animal model. The effective amount will depend primarily on factors such as species, age, weight, the health status of the subject, and the tissue to be targeted, and therefore can vary between animals and tissues. For example, an effective amount of recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) is typically from about 1 ml to about 100 ml containing about 10 9 Up to 10 16 A solution containing one copy of the genome. In some cases, approximately 10 11 Up to 10 13 A dose of 10 copies of the genome of a recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) is appropriate. In some embodiments, 10 10 Or 10 11A single copy of the genome of a recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) can effectively target eye tissues (e.g., retinal tissue). In some cases, stable transgenic animals are produced by multiple doses of recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV).

[0214] In some embodiments, the dose of recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) administered to the subject is not more than once per calendar day (e.g., within a 24-hour period). In some embodiments, the dose of recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) administered to the subject is not more than once every 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, the dose of recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) administered to the subject is not more than once per calendar week (e.g., every 7 calendar days). In some embodiments, the dose of recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) administered to the subject is not more than once every two weeks (e.g., once every two calendar weeks). In some implementations, the dose of recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) administered to the subject is administered no more than once per calendar month (e.g., once every 30 calendar days). In some implementations, the dose of recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) administered to the subject is administered no more than once every six calendar months. In some implementations, the dose of recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) administered to the subject is administered no more than once per calendar year (e.g., 365 days or 366 days in a leap year).

[0215] In some embodiments, the recombinant viral (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) composition is formulated to reduce the aggregation of AAV particles in the composition, particularly in the presence of high concentrations of recombinant viral (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) (e.g., ~10). 13 In cases of GC / ml or higher, appropriate methods for reducing aggregation can be used, including, for example, the addition of surfactants, pH adjustment, salt concentration adjustment, etc. (see, for example, Wright FR et al., Molecular Therapy (2005) 12, 171–178, the contents of which are incorporated herein by reference).

[0216] In some embodiments, the nucleic acid is delivered in a non-viral manner (e.g., not on a viral vector and / or not in a virus). In some embodiments, the nucleic acid (e.g., RNA or DNA) encoding a transgene and / or mutant rtTA (e.g., rtTA4) operatively linked to a TRE promoter is administered via liposomes. In some embodiments, the nucleic acid is RNA (e.g., mRNA). In some embodiments, the nucleic acid (e.g., RNA or DNA) encoding a transgene and / or mutant rtTA (e.g., rtTA4) operatively linked to a TRE promoter is administered via nanoparticles.

[0217] Systems and recombinant cells containing mutant rtTA or its nucleic acids (e.g., engineered nucleic acids, including expression vectors)

[0218] This disclosure also provides systems or cells comprising mutant rtTA (e.g., rtTA protein) and / or nucleic acids encoding mutant rtTA (e.g., engineered nucleic acids, including expression vectors). Cells may be eukaryotic or prokaryotic and may originate from any tissue (e.g., ear, nose, mouth (including gums and tooth roots), bone, lung, mammary gland, breast, pancreas, stomach, esophagus, muscle (including myocardium), liver, blood vessels, skin (including hair), heart, brain, nerve tissue, kidney, testis, prostate, penis, cloaca, fins, ovary, or intestine).

[0219] In some embodiments, the system or cell comprises a nucleic acid (e.g., engineered nucleic acid, including an expression vector) encoding both a mutant rtTA and a transgene operatively linked to an inducible promoter containing a Tet-O sequence. In some embodiments, the system or cell comprises a nucleic acid (e.g., engineered nucleic acid, including an expression vector) encoding the mutant rtTA and a second nucleic acid (e.g., engineered nucleic acid, including an expression vector) containing a transgene operatively linked to an inducible promoter containing a Tet-O sequence. In some embodiments, the system or cell comprises multiple (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 transgenes) encoding one or more transgenes (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 transgenes).

[0220] In some embodiments, nucleic acids encoding a mutant rtTA (e.g., rtTA4) and / or a TRE promoter (e.g., TRE3G, TRE2, and / or P tight) are integrated into the genome of a cell. In some embodiments, nucleic acids encoding a mutant rtTA (e.g., rtTA4) and / or a TRE promoter (e.g., TRE3G, TRE2, and / or P tight) are integrated into the genome of a subject (e.g., to produce a transgenic subject). Any suitable method can be used to integrate nucleic acids encoding a mutant rtTA (e.g., rtTA4) and / or a TRE promoter. See, for example, Cho et al., Curr Protoc Cell Biol. March 2009; CHAPTER: Unit–19.11. In some embodiments, CRISPR is used to knock the mutant rtTA and / or TRE promoter into cells or a subject. See, for example, Aida et al., Genome Biol. April 29, 2015; 16:87. As a non-limiting example, rtTA can be integrated into the rosa 26 locus in mice, and / or TRE can be integrated into the Col1a1 locus.

[0221] In some implementations, recombinant cells are generated in vitro and administered to subjects who require them.

[0222] Composition (e.g., pharmaceutical composition)

[0223] This disclosure provides compositions comprising any mutant rtTA, recombinant cells, nucleic acids encoding rtTA (e.g., engineered nucleic acids, expression vectors, plasmid DNA and / or RNA), inducible vectors encoding transgenes, recombinant viruses encoding rtTA and / or transgenes, or combinations thereof. Any mutant rtTA, recombinant cells, nucleic acids encoding rtTA (e.g., engineered nucleic acids, expression vectors, plasmid DNA and / or RNA), inducible vectors encoding transgenes, recombinant viruses encoding rtTA and / or transgenes, or combinations thereof, can be formulated into a pharmaceutical composition together with pharmaceutically acceptable excipients. The formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as well as the development of suitable dosing and treatment regimens for use of the specific compositions described herein in a variety of therapeutic regimens. Typically, these formulations may contain at least about 0.1% or more of the active compound, but the percentage of one or more active ingredients can, of course, vary and can conveniently be about 1% or 2% to about 70% or 80% or more of the total formulation weight or volume. Naturally, the amount of active compound in a composition useful for various treatments can be prepared in such a manner that a suitable dose is obtained at any given unit dose of the compound. Those skilled in the art preparing such pharmaceutical formulations should consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations; therefore, multiple dosages and treatment regimens may be desirable.

[0224] In some implementations, the mutant rtTA, recombinant cells, nucleic acids encoding rtTA (e.g., engineered nucleic acids, expression vectors, plasmid DNA and / or RNA), inducible vectors encoding transgenes, and / or recombinant viruses described herein are appropriately formulated, and the pharmaceutical compositions disclosed herein are delivered directly to the target tissues and / or organs of the subject, such as the eye, ear, nose, mouth (including gums and tooth roots), bone, lung, mammary gland, breast, pancreas, stomach, esophagus, muscle (including myocardium), liver, blood vessels, skin (including hair), heart, brain, nerve tissue, kidney, testis, prostate, penis, cloaca, fins, ovary, or intestine.

[0225] However, in some cases, it may be necessary to deliver recombinant viruses, recombinant cells, nucleic acids (e.g., engineered nucleic acids), and / or mutant rtTA (e.g., rtTA4) alone or additionally via another route, such as intravenous, intradermal, intraarticular, intralesional, intracranial, intra-articular, intraprostatic, intrapleural, intranasal, intravitreal, intravaginal, intrarectal, local, intratumoral, intramuscular, intraperitoneal, subcutaneous, subconjunctival, intrabladder, mucosa, intraperitoneal, intraumbilical, intraocular, oral, topical, local, systemic, by injection, infusion, continuous infusion, direct local perfusion of target cells, via catheter, in cream, or in lipid composition (e.g., liposomes).

[0226] In some embodiments, the administration methods described in U.S. Patent 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., lentiviruses, adenoviruses, alphaviruses, vaccinia virus, retroviruses, herpesviruses, or AAVs). In some embodiments, a preferred administration method is intramammary injection.

[0227] Suitable drug forms for injection include sterile aqueous solutions or dispersions, as well as sterile powders for the ad-hoc formulation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and oils. Under normal storage and use conditions, these formulations contain preservatives to inhibit microbial growth. In many cases, the form is sterile and is a fluid readily injectable. It must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. For example, in the case of dispersions, appropriate flowability can be maintained by using a coating such as lecithin, by maintaining the desired particle size, and by using surfactants. Microbial action can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars or sodium chloride, are preferred. Extended absorption of injectable compositions can be achieved by using delayed-absorption agents such as aluminum monostearate and gelatin in the composition.

[0228] For administration of injectable aqueous solutions, the solution may be appropriately buffered as needed, and the liquid diluent may first be isotonicized with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. For this purpose, a suitable sterile aqueous medium may be used. For example, a dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion or injected at the designated infusion site (see, for example, Remington's Pharmaceutical Sciences, 15th edition, pp. 1035-1038 and 1570-1580). Depending on the host's condition, some dosage variations may be necessary. The person responsible for administration will determine the appropriate dose for each host in any given event.

[0229] Sterile injectable solutions are prepared by introducing the desired amount of active recombinant virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV) along with various other components listed herein into a suitable solvent as needed, followed by filtration sterilization. Typically, dispersions are prepared by introducing various sterilized active ingredients into a sterile carrier containing a basic dispersion matrix and the desired other components from those listed above. For the preparation of sterile powders for sterile injectable solutions, a preferred method of preparation is vacuum drying and lyophilization, which yields a powder containing the active ingredient plus any additional desired components from its previously sterile filtered solution.

[0230] The recombinant viral compositions (e.g., lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAVs) disclosed herein can also be formulated into neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (forming with the free amino group of a protein) and those formed with inorganic or organic acids, such as hydrochloric acid or phosphoric acid, and organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived, for example, from inorganic bases such as hydroxides of sodium, potassium, ammonium, calcium, or iron, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. After formulation, the solution is administered in a manner compatible with dosage forms and at a therapeutically effective amount. The formulation is readily administered in various dosage forms, such as injectable solutions, drug-release capsules, etc.

[0231] Carriers include any and all solvents, dispersion media, mediators, coatings, diluents, antibacterial and antifungal agents, isotonic and delayed absorption agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and reagents for pharmaceutically active substances is well known in the art. Additional active ingredients may also be incorporated into the composition.

[0232] Delivery media such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc., can be used to introduce the compositions of this disclosure into suitable host cells. As a non-limiting example, transgenes delivered by recombinant viral vectors (e.g., lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAVs) can be formulated for delivery encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles.

[0233] Such formulations are preferably used to introduce pharmaceutically acceptable formulations containing nucleic acids (e.g., engineered nucleic acids, including expression vectors) or recombinant viral constructs (e.g., lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAVs) disclosed herein. The formation and application of liposomes are generally known to those skilled in the art. Recently, liposomes have been developed to have improved serum stability and circulating half-life (US Patent No. 5,741,516). Furthermore, various methods for using liposomes and liposome-like formulations as potential drug delivery systems have been described (US Patent Nos. 5,567,434; 5,552,157; ​​5,565,213; 5,738,868 and 5,795,587; each of which is incorporated herein by reference).

[0234] Liposomes have been successfully used in many cell types that typically develop resistance to transfection by other methods. Furthermore, liposomes have no DNA length limitations, a characteristic typical of virus-based delivery systems. Liposomes have been effectively used to introduce genes, drugs, radiotherapy agents, viruses, transcription factors, and allosteric effectors into a wide variety of cultured cell lines and animals. In addition, several successful clinical trials have been completed to test the effectiveness of liposome-mediated drug delivery.

[0235] Liposomes are formed from phospholipids dispersed in an aqueous medium and spontaneously form multilayered concentric bilayered vesicles (also known as multilayered liposomes (MLVs)). MLVs typically have a diameter of 25 nm to 4 μm. Sonication of MLVs leads to the formation of small monolayered vesicles (SUVs) with a diameter of 200 to 500 nm, containing an aqueous solution in the core.

[0236] Alternatively, nanocapsule formulations of recombinant viruses (e.g., lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAVs) can be used. Nanocapsules typically encapsulate material in a stable and reproducible manner. To avoid side effects caused by intracellular polymerization overload, such ultrafine particles (approximately 0.1 μm in size) should be designed using polymers that are biodegradable in vivo. The use of biodegradable polyalkyl cyanoacrylate nanoparticles that meet these requirements is considered.

[0237] Kits and related compositions

[0238] In some embodiments, the reagents described herein may be assembled into pharmaceutical, diagnostic, or research kits to facilitate their use in therapeutic, diagnostic, or research applications. The kit may include one or more containers containing the components of this disclosure and instructions for use. Specifically, such kits may include one or more reagents described herein, along with instructions describing the intended application and appropriate use of these reagents. In some embodiments, the reagents in the kit may be pharmaceutical formulations and dosages suitable for a particular application and method of administration of the reagent. Kits for research purposes may contain appropriate concentrations or amounts of components for running various experiments.

[0239] In some embodiments, this disclosure relates to a kit containing a nucleic acid (e.g., engineered nucleic acid, including an expression vector) encoding a mutant rtTA (e.g., rtTA4), which can be used, for example, to generate recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV). The kit may include a container holding a nucleic acid (e.g., engineered nucleic acid, including an expression vector) encoding a mutant rtTA (e.g., rtTA4). The kit may also include a second container holding a nucleic acid (e.g., engineered nucleic acid, including an expression vector) encoding a transgene (e.g., a gene associated with a disease such as retinal disease). In some embodiments, the transgene is a sequence encoding a protein, a gene-targeting nucleic acid, and / or a therapeutic sequence. In some embodiments, the kit also includes instructions for generating recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpesvirus, or AAV).

[0240] In some embodiments, this disclosure relates to kits comprising containers housing any engineered nucleic acid (e.g., expression vector) or recombinant virus described herein. For example, the kit may contain a nucleic acid encoding an inducer (e.g., an engineered nucleic acid, including an expression vector) or a recombinant virus. In some embodiments, the nucleic acid encoding mutant rtTA4 (e.g., an engineered nucleic acid, including an expression vector) comprises at least 70% of the sequence identical to SEQ ID NO:17. In some embodiments, the nucleic acid encoding an inducer (e.g., an engineered nucleic acid, including an expression vector) consists of at least 70% of the sequence identical to SEQ ID NO:17. In some embodiments, the nucleic acid encoding mutant rtTA4 (e.g., an engineered nucleic acid, including an expression vector) comprises at least 70% of the sequence identical to desmin-rtTA4 (SEQ ID NO:30). In some embodiments, the nucleic acid encoding an inducer (e.g., an engineered nucleic acid, including an expression vector) consists of at least 70% of the sequence identical to desmin-rtTA4 (SEQ ID NO:30). The kit may also contain nucleic acids (e.g., engineered nucleic acids, including expression vectors) or recombinant viruses encoding any transgene (e.g., therapeutic sequences, gene-targeting nucleic acids, and / or sequences encoding proteins) operatively linked to a Tet-O-containing promoter (e.g., a TRE promoter). Non-limiting examples of vectors encoding transgenes (e.g., multiple transgenes) are provided in SEQ ID NO:16. In some embodiments, the nucleic acid encoding the transgene (e.g., engineered nucleic acids, including expression vectors) comprises SEQ ID NO:16.

[0241] The kit may be designed to facilitate use by researchers using the methods described herein and may take various forms. Where applicable, each composition of the kit may be provided in liquid form (e.g., in solution) or solid form (e.g., dry powder). In some cases, some compositions may be composable or otherwise processable (e.g., in an active form), for example by adding a suitable solvent or other class (e.g., water or cell culture medium), which may or may not be provided with the kit. As used herein, “instructions” may define the components that are described and / or promoted, and typically refers to written instructions on or associated with the packaging of this disclosure. Instructions may also include any oral or electronic instructions provided in any manner that will make it clear to the user that the instructions will be associated with the kit, such as audiovisual (e.g., videotape, DVD, etc.), Internet and / or web-based communications, etc. Written instructions may be in the form of those prepared by government agencies regulating the manufacture, use or sale of pharmaceutical or biological products, and may also reflect agency approval for manufacture, use or sale for animal administration.

[0242] The kit may contain any one or more of the components described herein in one or more containers. For example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and / or separating and mixing samples for application to a subject. The kit may include a container holding the reagents described herein. The reagents may be in liquid, gel, or solid (powder) form. The reagents may be aseptically prepared, packaged in syringes, and shipped refrigerated. Alternatively, they may be contained in vials or other containers for storage. A second container may contain other aseptically prepared reagents. Alternatively, the kit may include an active agent premixed and shipped in syringes, vials, tubes, or other containers. The kit may have one or more or all of the components required to administer the reagents to animals, such as syringes, local application devices, or intravenous (iv) syringes and bags, particularly in the case of kits used to generate specific animal models.

[0243] The kit may be available in various forms, such as blister packs, shrink wraps, vacuum-sealed bags, sealable thermoformed trays, or similar bag or tray forms, as well as loosely packaged accessories, one or more tubes, containers, boxes, or bags within the bag. The kit may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to unfold in other ways. The kit may be sterilized using any suitable sterilization technique, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kit may also include other components, depending on the specific application, such as containers, cell media, salts, buffers, reagents, syringes, needles, fabrics such as gauze for applying or removing disinfectants, disposable gloves, and supports for these reagents prior to application.

[0244] Therapeutic applications

[0245] Any composition (e.g., a pharmaceutical composition) comprising the mutant rtTA described herein (e.g., rtTA4), a nucleic acid encoding the mutant rtTA (e.g., engineered nucleic acid, including an expression vector), an inducible nucleic acid encoding a transgene (e.g., engineered nucleic acid, including an expression vector), a recombinant virus encoding the mutant rtTA, or a transgene operatively linked to an inducible promoter, may be used to regulate (e.g., inhibit or induce) cell reprogramming, tissue repair, tissue regeneration, disease treatment, organ regeneration, aging reversal, or any combination thereof. The composition may be used to regulate cell reprogramming, tissue repair, tissue survival, tissue regeneration, tissue growth, tissue function, organ regeneration, organ survival, organ function, or any combination thereof. Regulation may include in vivo or in vitro induction of cell reprogramming, reversal of aging, improvement of tissue function, improvement of organ function, tissue repair, tissue survival, tissue regeneration, tissue growth, angiogenesis, scar formation, aging appearance, organ regeneration, organ survival, alteration of the taste and quality of agricultural products derived from animals, treatment of disease, or any combination thereof, and may be administered to cells, tissues, or organs in vivo (e.g., a portion of a subject) or to cells, tissues, or organs ex vivo. As used in this article, regulation can refer to any type of regulation, including induction or promotion, inhibition, and / or cessation. Angiogenesis refers to the growth of new blood vessels.

[0246] In some cases, viral vectors (such as lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAV vectors) are administered as recombinant viruses (such as lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAV vectors).

[0247] Without specific theoretical constraints, transient expression of one or more transgenic genes (e.g., OCT4, SOX2, KLF4, any transcription factor, any protein-coding sequence, any gene-targeting nucleic acid, and / or any therapeutic sequence) can lead to partial reprogramming of cells. For example, partial reprogramming can induce fully differentiated cells to become pluripotent. In some cases, prolonged expression of one or more transcription factors (e.g., any transcription factor, including OCT4, SOX2, KLF4, and / or c-MYC) (e.g., sustained expression for at least 1 day, at least 5 days, at least 1 week, or at least 1 month) leads to complete reprogramming of cells. For example, cells can be completely reprogrammed into pluripotent cells (e.g., induced pluripotent cells).

[0248] To implement this embodiment, an effective amount of mutant rtTA (e.g., rtTA4) may be administered to cells, tissues, or subjects together with a nucleic acid (e.g., engineered nucleic acid, including an expression vector) containing a transgene to be expressed (protein-coding sequence, gene-targeting nucleic acid, and / or therapeutic sequence) operably linked to a TRE promoter (e.g., TRE2, P-tight, or TRE3G promoter). In some embodiments, mutant rtTA (e.g., rtTA4) is administered as a protein. In some embodiments, mutant rtTA (e.g., rtTA4) is administered as a nucleic acid (e.g., engineered nucleic acid, including an expression vector).

[0249] In some embodiments, the nucleic acid (e.g., engineered nucleic acid) encoding the mutant rtTA4 and / or operatively linked to a TRE promoter (e.g., TRE3G, Ptight, or TRE2 promoter) is not a viral vector. For example, the nucleic acid may be a plasmid (e.g., plasmid DNA) or RNA (e.g., mRNA). As a non-limiting example, the engineered nucleic acid (e.g., RNA, including mRNA, or DNA) of this disclosure may be formulated into nanoparticles for delivery. See, for example, Dong et al., NanoLett. 2016 Feb 10; 16(2):842-8. In some embodiments, the nanoparticles contain acetylated galactose. See, for example, 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, including mRNA, or DNA) is electroporated or transfected into cells. In some embodiments, engineered nucleic acids are delivered as naked nucleic acids (e.g., naked DNA or naked RNA). In some embodiments, the naked nucleic acid is plasmid DNA. In some embodiments, nucleic acids (e.g., engineered nucleic acids) are administered via liposomes.

[0250] In some implementations, the nucleic acid encoding the mutant rtTA4 and / or the transgenic nucleic acid (e.g., engineered nucleic acid) operatively linked to a TRE promoter (e.g., TRE3G, Ptight, or TRE2 promoter) is a viral vector. Non-limiting examples of viral vectors include lentiviral vectors, adenovirus vectors, alphavirus vectors, vaccinia virus vectors, herpesvirus vectors, and adenovirus (AAV) vectors.

[0251] In some embodiments, nucleic acids (e.g., engineered nucleic acids encoding mutant rtTA (e.g., rtTA4)) are administered as viruses (e.g., lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAVs). In some embodiments, nucleic acids (e.g., engineered nucleic acids, including expression vectors) comprising a transgene operatively linked to a TRE promoter (e.g., TRE3G, P-tight, or TRE2 promoter) are administered as viruses (e.g., lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAVs).

[0252] An appropriate amount of tetracycline (e.g., doxycycline) may be added to drive the expression of a TRE promoter (e.g., TRE3G, P-tight, or TRE2 promoter). The appropriate amount of tetracycline to be added can be determined by those skilled in the art and may depend on a variety of factors, including the type of drug excipient (if any), cell type, tissue type, or any characteristics of the subject (e.g., weight, medical history, genetics, etc.).

[0253] In some implementations, tetracycline is administered in the following ways: intravenously, intradermally, intraarterially, intralesionally, intracranially, intra-articularly, intraprostatically, intrapleurally, intranasally, intravitreally, intravaginally, intrarectally, locally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intrabladderly, mucosally, intraperitoneally, intraumbilically, intraocularly, orally, topically, locally, systemically, by injection, infusion, continuous infusion, direct local perfusion of target cells, via catheter, as a cream, or as a lipid composition.

[0254] In some embodiments, a recombinant virus and / or expression vector encoding the mutant rtTA and / or a transgene operably linked to a TRE promoter is administered systemically. In some embodiments, a recombinant virus and / or expression vector encoding the mutant rtTA and / or a transgene operably linked to a TRE promoter is administered locally (e.g., directly to the tissue or organ of interest, including the eye, ear, nose, mouth (including gums and tooth roots), bone, lung, mammary gland, breast, pancreas, stomach, esophagus, muscle (including myocardium), liver, blood vessels, skin (including hair), heart, brain, nervous tissue, kidney, testis, prostate, penis, cloaca, fins, ovary, or intestine).

[0255] In some embodiments, the virus and / or expression vector are administered together with a tetracycline (e.g., doxycycline). In some embodiments, the virus and / or expression vector containing the TRE promoter are administered separately from the tetracycline (e.g., doxycycline). For example, any virus and / or expression vector containing the TRE promoter described herein can be administered systemically, and the tetracycline can be administered locally (e.g., to the organ or tissue of interest). In some embodiments, any virus and / or expression vector containing the TRE promoter described herein can be administered locally (e.g., directly to the tissue or organ of interest, including the eye, ear, nose, mouth (including gums and tooth roots), bone, lung, mammary gland, breast, pancreas, stomach, esophagus, muscle (including myocardium), liver, blood vessels, skin (including hair), heart, brain, nervous tissue, kidney, testis, prostate, penis, cloaca, fins, ovary, or intestine), and the tetracycline and / or mutant rtTA (e.g., mutant rtTA protein or nucleic acid encoding mutant rtTA) can be administered systemically. As non-limiting examples, viruses and / or expression vectors containing the TRE promoter are administered directly (e.g., injected) into the eye of a subject, and tetracycline (e.g., doxycycline) and / or mutant rtTA (e.g., mutant rtTA protein or nucleic acid encoding mutant rtTA) are administered systemically (e.g., orally as a pill). In some embodiments, nucleic acids containing the TRE promoter (e.g., engineered nucleic acids) are administered to a subject via the same route as mutant rtTA (e.g., mutant rtTA protein or nucleic acid encoding mutant rtTA). In some embodiments, nucleic acids containing the TRE promoter (e.g., engineered nucleic acids) are administered to a subject via a different route than mutant rtTA (e.g., mutant rtTA protein or nucleic acid encoding mutant rtTA).

[0256] In some embodiments of this disclosure, the method further includes withdrawing tetracycline (e.g., doxycycline) from cells, tissues, or subjects after administration of tetracycline, which can be used to terminate transgene expression. Tetracycline may be withdrawn at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, or at least 1 year after tetracycline administration. In some embodiments, withdrawal of tetracycline results in a detectable reduction in transgene expression (e.g., 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%, or 100%). For example, a decrease in transgene expression can be detected at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, or at least 1 year after tetracycline withdrawal.

[0257] Without specific theoretical limitations, compared to rtTA3 (SEQ ID NO:11), the four mutations in rtTA4 (mutations at positions corresponding to G12, F67, R171, or G72 in SEQ ID NO:11, such as G12S, G72V or G72P, F67S, and R171K) result in lower leakage because these mutations reduce the binding affinity of rtTA4 to the promoter in the absence of tetracycline. In some embodiments, in the absence of tetracycline (e.g., doxycycline), the amount of transgene expression using rtTA4 (e.g., SEQ ID NO:13) is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% lower than the amount of transgene expression detected using rtTA3 (e.g., SEQ ID NO:11). Without specific theoretical limitations, compared to rtTA3 (SEQ ID NO:11), four mutations in rtTA4 (mutations at positions corresponding to G12, F67, R171, or G72 in SEQ ID NO:11, such as G12S, G72V or G72P, F67S, and R171K) result in greater sensitivity to tetracycline withdrawal because these mutations reduce the binding affinity of rtTA to tetracycline. In some embodiments, compared to the amount of transgene expression using rtTA3 (e.g., SEQ ID NO:11), the amount of transgene expression using rtTA4 (e.g., SEQ ID NO:13) is reduced at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster when tetracycline (e.g., doxycycline) is withdrawn for a given time.

[0258] The nucleic acid encoding the mutant rtTA (e.g., rtTA4) (e.g., engineered nucleic acid, including expression vectors), the nucleic acid containing a TRE promoter (e.g., TRE3G, TRE2, or P tight promoter) operatively linked to the transgene (e.g., a therapeutic sequence, gene-targeting nucleic acid, and / or protein-coding sequence) (e.g., a P-tight promoter) (e.g., engineered nucleic acid, including expression vectors), and the administration of tetracycline result in an increase in transgene expression in cells of 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 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1,000%. Gene expression can be determined by conventional methods, including enzyme-linked immunosorbent assay (ELISA), Western blotting, and RNA quantification (e.g., reverse transcription polymerase chain reaction).

[0259] The pharmaceutical compositions described herein may be administered to subjects in need of them. Non-limiting examples of subjects include any animal (e.g., mammals, including humans). Subjects may be suspected of having a condition, be at risk of having a condition, or have a condition. For example, a condition may be an injury or disease, and a condition may affect any tissue (e.g., ear, nose, mouth (including gums and tooth roots), bone, lungs, mammary glands, breast, pancreas, stomach, esophagus, muscles (including myocardium), liver, blood vessels, skin (including hair), heart, brain, nerve tissue, kidneys, testes, prostate, penis, cloaca, fins, ovaries, or intestines). Non-limiting examples of conditions, diseases, and disorders include acute injuries, neurodegenerative diseases, chronic diseases, proliferative diseases, cardiovascular diseases, genetic diseases, inflammatory diseases, autoimmune diseases, neurological diseases, hematological diseases, pain conditions, mental disorders, metabolic disorders, cancer, aging, age-related diseases, and diseases affecting any tissue in the subject. In some embodiments, the disease is an eye disease.

[0260] In some implementations, the condition is aging. All animals typically experience a period of growth and maturation, followed by a progressive and irreversible physiological decline, ending in death. The length of time from birth to death is called the lifespan of an organism, and each organism has a characteristic average lifespan. Aging is the physical manifestation of change over time, as measured as a percentage of average lifespan.

[0261] In some cases, the signs of aging can be quite pronounced. For example, signs of aging in older individuals include wrinkled skin, gray hair, hair loss, and cataracts, as well as hyperpigmentation, osteoporosis, cerebral cortical atrophy, lymphatic failure, thymic atrophy, and an increased incidence of type II diabetes, atherosclerosis, cancer, and heart disease. (Nehlin et al. (2000), Annals NY Acad Sci 980:176-79). Other aspects of aging in mammals include weight loss, kyphosis (hunchback), lack of vitality, lymphatic atrophy, decreased bone density, thickened dermis and subcutaneous adipose tissue, reduced tolerance to stress (including heat or cold, trauma, anesthesia, and ablation of hematopoietic progenitor cells), liver pathology, atrophy of intestinal villi, skin ulcers, amyloid deposits, and joint disease. (Tyner et al. (2002), Nature 415:45-53).

[0262] Those skilled in the art recognize that the aging process also manifests at the cellular level and in mitochondria. Cellular senescence is characterized by loss of proliferative capacity, increased levels of apoptosis, altered differentiation phenotypes, and metabolic changes, such as decreased protein synthesis and turnover.

[0263] Given the programmed nature of cellular and biological aging, the “biological age” of a cell or organism can be assessed using phenotypic characteristics associated with aging. For example, biological age can be derived from patterns such as gene expression, stress resistance (e.g., oxidative or genotoxic stress), cell proliferation rate, and cellular metabolic characteristics (e.g., protein synthesis and turnover rates, mitochondrial function, ubiquinone biosynthesis, cholesterol biosynthesis, intracellular ATP levels, intracellular Krebs cycle intermediate levels, glucose metabolism, nucleic acid metabolism, ribosome translation rate, etc.). As used herein, “biological age” is a measure of the age of a cell or organism based on its molecular characteristics. Biological age differs from “chronological age,” which refers to the age of a cell or organism as measured in days, months, and years.

[0264] The rate of aging in an organism, such as an invertebrate (e.g., a worm or fly) or a vertebrate (e.g., a rodent, such as a mouse), can be determined by a variety of methods, such as by one or more of the following: (a) assessing the lifespan of a cell or organism; (b) assessing the presence or abundance of gene transcripts or gene products in a cell or organism exhibiting a biologically age-dependent expression pattern; (c) evaluating the resistance of a cell or organism to stress, such as genotoxic stress (e.g., etoposide, ultraviolet radiation, exposure to mutagens, etc.) or oxidative stress; (d) evaluating one or more metabolic parameters of a cell or organism; (e) evaluating the proliferative capacity of a cell or group of cells present in the organism; and (f) evaluating the physical appearance or behavior of a cell or organism. In one example, assessing the rate of aging involves directly measuring the mean lifespan of a group of animals (e.g., a group of genetically matched animals) and comparing the resulting mean with the mean lifespan of a control group of animals (e.g., a group of animals that did not receive the test compound but were genetically matched to a group of animals that received the test compound). Alternatively, the rate of aging in an organism can be determined by measuring age-related parameters. Examples of age-related parameters include: appearance, such as visible signs of aging; expression of one or more genes or proteins (e.g., genes or proteins with age-related expression patterns); resistance to oxidative stress; metabolic parameters (e.g., protein synthesis or degradation, ubiquinone biosynthesis, cholesterol biosynthesis, ATP levels, glucose metabolism, nucleic acid metabolism, ribosome translation rate, etc.); and cell proliferation (e.g., proliferation of retinal cells, osteocytes, leukocytes, etc.).

[0265] The methods described can be used to prevent or mitigate neurodegeneration and related peripheral neuropathy. Neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Huntington's disease, and muscular dystrophy. Neurodegeneration can be quantified using any method known in the art. For example, an individual's executive function can be determined (Moreira et al., Front Aging Neurosci. 2017 Nov 9; 9:369).

[0266] Other age-related conditions that can be treated include heart failure, stroke, diabetes, osteoporosis, arthritis, hearing loss (partial or complete), eye-related conditions (such as vision loss or retinal disease), glaucoma, and cancer. In some embodiments, the disease is a retinal disease (such as macular degeneration). The condition may be a retinal disease, cancer, aging, age-related disease, injury, or neurodegenerative disease. In some embodiments, the cells or tissues originate from the eye, ear, nose, mouth (including gums and tooth roots), bone, lung, breast, mammary gland, pancreas, stomach, esophagus, muscle (including myocardium), liver, blood vessels, skin (including hair), heart, brain, nerve tissue, kidney, testis, prostate, penis, cloaca, fins, ovary, or intestine. In some embodiments, the tissue is damaged (e.g., due to injury, accident, or iatrogenic injury) and / or is aging tissue. In some implementations, an organization may be considered healthy, but its performance or survival is not optimal under current or future conditions (e.g., in agriculture or adverse conditions, including toxic therapy, sunlight exposure, or travel outside the Earth's atmosphere).

[0267] For example, a condition can be an injury or disease, and it can affect any tissue (e.g., eye, ear, bone, lung, breast, pancreas, muscle, heart, liver, skin, brain, nerve tissue, or intestine). Non-limiting examples of conditions, diseases, and disorders include acute injuries, neurodegenerative diseases, chronic diseases, cancer, aging, age-related diseases, and diseases affecting any tissue in the subject.

[0268] In some embodiments, any mutant rtTA4, the nucleic acid encoding rtTA4 (e.g., engineered nucleic acid), the nucleic acid containing a TRE promoter operatively linked to the transgenic sequence, recombinant viruses, and / or recombinant cells can be used to treat diseases affecting non-human subjects (e.g., diseases affecting livestock, domesticated pets, and / or other non-human animals). For example, diseases can be bovine diseases, primate diseases (e.g., rhesus monkeys), diseases affecting commercially relevant animals (such as cattle, pigs, horses, sheep, goats, cats, and / or dogs), and / or diseases affecting birds (e.g., commercially relevant birds such as chickens, ducks, geese, and / or turkeys). For example, any mutant rtTA4 described herein, the nucleic acid encoding rtTA4 (e.g., engineered nucleic acid), the nucleic acid containing a TRE promoter operatively linked to the transgenic sequence, recombinant viruses, and / or recombinant cells can be used to promote wound healing, treat injuries (e.g., fractures, hemorrhagic gunshot wounds, and / or reduce scarring during surgery). In some embodiments, the surgery includes a cesarean section.

[0269] Methods for identifying subjects suspected of having a condition may include physical examination, the subject's family medical history, the subject's medical history, biopsy, genetic testing, or a variety of imaging techniques such as ultrasound, computed tomography, magnetic resonance imaging, magnetic resonance spectroscopy, or positron emission tomography.

[0270] As those skilled in the art will recognize, the effective amount of nucleic acids (e.g., engineered nucleic acids, including expression vectors), viruses (e.g., lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, retroviruses, herpesviruses, or AAVs), or combinations thereof, varies depending on the route of administration, the use of excipients, and the co-administration with other active agents. The amount to be administered depends on the subject to be treated, including, for example, the subject's age, the severity of the condition, the subject's weight, the subject's genetics, the cells, tissues, or organs to be targeted, or any combination thereof.

[0271] Using the mutant rtTA disclosed herein, expression of one or more transgenes (e.g., therapeutic sequences, gene-targeting nucleic acids, and / or protein-coding sequences) can lead to cell reprogramming, tissue repair, tissue regeneration, organ regeneration, reversal of aging, infectious diseases, disease prevention, disease treatment, or any combination thereof. Cell reprogramming can be determined by identifying the degree of cell differentiation (e.g., by identifying the expression of one or more lineage markers or pluripotent markers, including OCT4, KLF4, SOX2, NANOG, ESRRB, NR4A2, and C / EBPα). The differentiation potential of cells can also be determined using conventional differentiation assays. Tissue repair can be determined by tissue turnover and tissue regeneration assays. For example, tissue turnover assays include wound healing assays. Tissue regeneration can be determined by quantifying specific cell types after transgene expression (e.g., expression of one or more transcription factors, including OCT4, KLF4, and SOX2) compared to before transgene expression. In some cases, the methods described herein promote organ regeneration.

[0272] These and other aspects of the invention will be further understood by considering the following embodiments, which are intended to illustrate certain specific embodiments of the invention but are not intended to limit its scope as defined by the claims.

[0273] General technology

[0274] Unless otherwise stated, the practice of this invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art. MolecularCloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (edited by MJ Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by JECellis, 1998) Academic Press; Animal Cell Culture (edited by RI Freshney, 1987); Introduction to Cell and Tissue Culture (JPMather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, JBGriffiths and DG Newell, 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (edited by DMWeir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (edited by J.M. Miller and M.C. Alos, 1987); Current Protocols in Molecular Biology (edited by F.M. Usubel et al., 1987); PCR: The Polymerase Chain Reaction (edited by Mullis et al., 1994); Current Protocols in Immunology (edited by J.E. Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (edited by J.A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997; Antibodies: a practical approach (edited by D. Catty, IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (edited by M. Zanetti and JD Capra, Harwood Academic Publishers, 1995). Without further detailed description, it is believed that those skilled in the art can utilize the invention to the fullest extent based on the foregoing description. The following specific embodiments are therefore to be construed as merely illustrative and in no way intended to limit the remainder of the disclosure. All publications cited herein for the purposes or subject matter of this reference are incorporated herein by reference.

[0275] These and other aspects of the invention will be further understood by considering the following embodiments, which are intended to illustrate certain specific embodiments of the invention but are not intended to limit its scope as defined by the claims. Example

[0276] To provide a more complete understanding of this disclosure, the following embodiments are illustrated. The synthetic and biological embodiments described in this application are provided to illustrate the compounds, pharmaceutical compositions, and methods provided herein, and are not to be construed as limiting their scope in any way.

[0277] Example 1: Development of a mutant reverse tetracycline transactivator (rtTA) with low leakage and improved reaction time in the in vitro Tet-On system upon doxycycline withdrawal.

[0278] The mutant inverse tetracycline transactivator (rtTA) was engineered using conventional cloning techniques. Compared to rtTA3 (SEQ ID NO:11), the exemplary mutant rtTA (rtTA4) contains four mutations (G12S, F67S, G72V, and R171K). The rtTA containing the four mutations at positions G12, F67, G72, and R171 of rtTA3 (SEQ ID NO:11) is referred to herein as rtTA4. rtTA4 (SEQ ID NO:13) contains three VP16 transactivation domains, while rtTA3 (SEQ ID NO:11) contains two VP16 transactivation domains.

[0279] The nucleic acid sequence encoding rtTA4 (SEQ ID NO:13) (SEQ ID NO:12) was cloned into the AAV vector using conventional methods. The pAAV-UBC-rtTA4-WPRE3-SV40pA (SEQ ID NO:17) vector is shown in [image / description missing]. Figure 1 , Figure 2A-2M and Figure 3 middle. Figure 1 This is a vector map showing the characteristics of the AAV vector encoding rtTA4. UBC is a constitutive promoter operatively linked to the nucleic acid encoding rtTA4. SV40pA is an SV40-derived termination sequence. Figure 2A-2M Including Figure 1 The diagram shows a series of schematic representations of the features mapped onto the nucleic acid sequence of the vector encoding rtTA4. Figure 3 It shows Figure 2A-2M The location and size of each feature depicted.

[0280] The pAAV-UBC-rtTA4-WPRE3-SV40 pA vector (SEQ ID NO:17) contains two inverted terminal repeat (ITR) sequences side-attached to a sequence encoding the UBC promoter, which is operatively linked to a nucleic acid sequence encoding rtTA4 (SEQ ID NO:12). The AAV vector also contains a WPRE3 sequence (SEQ ID NO:21) and an SV40 termination sequence (SEQ ID NO:8). The locations of the restriction enzyme digestion sites are shown in Table 2 below.

[0281] Table 2. Restriction enzyme digestion sites in pAAV-UBC-rtTA4-WPRE3-SV40pA (SEQ ID NO:17)

[0282]

[0283]

[0284] like Figures 4A-4B As shown, even in the absence of doxycycline, rtTA3 (SEQ ID NO:11) leaks and induces the expression of a transgene operatively linked to a tetracycline-inducible promoter. The nucleic acid sequence encoding rtTA3 is provided as SEQ ID NO:10. Figure 4A This is a schematic diagram depicting the two nucleic acids used in this experiment. One nucleic acid encodes a UBC promoter operatively linked to (1) a nucleic acid encoding rtTA4 (SEQ ID NO:13) (SEQ ID NO:12), (2) a nucleic acid encoding a 2A peptide, and (3) a nucleic acid encoding mKate (a far-infrared fluorescent protein). mKate expression was used as the readout for rtTA3 expression. Figure 4B The second nucleic acid encodes GFP under the control of the TRE3G (SEQ ID NO:7) promoter. GFP should only be expressed in the presence of doxycycline (DOX), such as... Figure 4A As shown.

[0285] Next, tests were conducted in vivo. Figure 4A The carrier shown. Will carry Figure 4A The AAV and carrying UBC-rtTA3 vector depicted at the top Figure 4B The TRE3G-GFP promoter, depicted at the bottom, was administered AAV to mice. No AAV administration served as a control. Mice were treated with or without doxycycline (DOX). Liver samples were then analyzed using Western blotting with relevant antibodies to determine mKate expression (readout of rtTA3 expression), GFP expression, and actin expression. Figure 4B As shown, GFP expression was detected even in the absence of doxycycline. Therefore, rtTA3 leaks in vivo.

[0286] To determine whether rtTA4 leaks less and is more sensitive to doxycycline compared to rtTA3, the Tet-On luciferase reporter system was used. Figure 5A This is a schematic diagram of the two nucleic acids used in this experiment. The first nucleic acid contains the promoter that drives rtTA expression ( Figure 5A (Top). rtTA4 is shown in Figure 5A However, rtTA3 is used instead of rtTA4 in the Tet-On luciferase reporter system used to test rtTA3. The second nucleic acid (TRE3G-Luc) contains a TRE3G promoter operatively linked to the nucleic acid sequence encoding luciferase (luc). Figure 5A (Bottom). Tet-On reporter systems encoding rtTA4 or rtTA3 were introduced into 293T cells using separate vectors, and the cells were treated with doxycycline at increasing concentrations. Luciferase expression was determined by measuring luminescence / protein. (See bottom). Figure 5BAs shown, when doxycycline levels were low (0.01 ng / ml to 1 ng / ml), luminescence / protein levels were also low. At these low doxycycline levels, the luminescence / protein levels using rtTA4 were the same as the baseline levels in the absence of any rtTA (baseline was...). Figure 5B (Indicated by dashed lines). In the Tet-On system using rtTA4, the luminescence / protein level increased with increasing doxycycline level ( Figure 5B In contrast, even at low levels of doxycycline (e.g., 0.01 ng / ml to 1 ng / ml DOX), the luminescence / protein levels with rtTA3 were significantly higher than baseline. These results suggest that rtTA4 leaks less than rtTA3 (does not induce detectable transgene expression in the absence of doxycycline), but is able to induce transgene expression with increased doxycycline concentrations.

[0287] To compare the sensitivity of rtTA4 and rtTA3 to doxycycline withdrawal, the following methods were used: Figure 5A A similar Tet-On luciferase reporter system described in [the text]. For Figure 6A The results shown indicate that the TRE3G-luc vector and the vector encoding rtTA3 under the control of the desmin promoter were introduced into 293T cells. Cells received either the empty vector or the TRE3G-luc vector as controls. Cells carrying both the TRE3G-luc vector and the vector encoding rtTA3 under the control of the desmin promoter were treated as follows: (1) without doxycycline (-DOX), (2) with doxycycline (+DOX), or (3) with doxycycline followed by withdrawal of doxycycline (+-DOX). Figure 6A As shown, the luminescence / protein levels detected using treatment (3) were significantly higher than those detected using treatment (1). These results indicate that rtTA3 is leaking. In contrast, as Figure 6B As shown, when the same experiment was performed with rtTA4, doxycycline withdrawal significantly reduced the amount of detected luminescent / protein to levels comparable to those detected in cells that had never received doxycycline. Therefore, rtTA4 is more sensitive to doxycycline withdrawal than rtTA3.

[0288] To compare the time required to shut down transgene expression by withdrawing doxycycline using rtTA4 versus rtTA3, a Tet-On luciferase reporter system containing the TRE3G-luc vector and a vector encoding an rtTA protein was introduced into 293T cells. The TRE3G-luc vector was introduced into the cells alone as a control. The luminescence / protein levels of the rtTA3 and rtTA4 systems in the absence of any doxycycline treatment (-DOX) were also determined. Figure 6CSubsequently, cells with either the rtTA3 Tet-On system or the rtTA4 Tet-On system were treated with doxycycline (+DOX), or with doxycycline followed by withdrawal (+-) for a specified number of hours. Figure 6C rtTA4 can shut down transgene expression faster than rtTA3. Figure 6C ).like Figures 6A-6C As shown, using the desmin promoter and the Ubc promoter, rtTA4 shuts down 4-12 times faster than rtTA3.

[0289] rtTA4 was further tested in the Tet-On system in mammalian 293T cells. The TRE3G-GFP-SV40pAAAV vector and a second AAV vector containing (1) a sequence encoding rtTA3 operatively linked to the UBC promoter, (2) a sequence encoding rtTA4 operatively linked to the UBC promoter, or (3) a sequence encoding rtTA4 operatively linked to the desmin promoter were introduced into 293T cells. Figure 7 Then, the cells were treated in the absence of doxycycline (-DOX), in the presence of doxycycline (+DOX), or in the presence of DOX (+DOX) followed by the removal of doxycycline (-DOX). Figure 7 As shown, rtTA3 induced GFP expression even in the absence of doxycycline, while rtTA4 did not. Furthermore, compared to the UBC-rtTA3 system, removing DOX seven days after one day of doxycycline treatment resulted in lower GFP expression in the UBC-rtTA4 system. Therefore, the Tet-On system containing an AAV vector encoding rtTA4 successfully induced transgene expression in mammalian cells, exhibiting lower leakage compared to the same system using rtTA3, and demonstrating faster transgene expression inhibition after doxycycline removal.

[0290] Example 2: The AAV vector encoding the mutant reverse tetracycline transactivator (rtTA) showed low osmosis in mouse liver.

[0291] The Tet-On system containing rtTA4 (SEQ ID NO:13) was also tested in vivo using a recombinant AAV9 virus. Figure 8B The components shown are two AAV vectors. An AAV virus encoding rtTA4 operatively linked to the UBC promoter (pAAV-UBC-rtTA4-WPRE3-SV40pA vector provided as SEQ ID NO:17) and an AAV vector encoding rtTA4 operatively linked to the UBC promoter are shown. Figure 9The AAV virus of the AAVTRE3G-OSK-SV40pA vector (SEQ ID NO:16) depicted in the vector map was administered to mice. Liver samples were collected from mice, with or without doxycycline treatment. Figure 8A As shown in the immunofluorescence images, KLF4 expression was undetectable in the liver in the absence of doxycycline. KLF4 expression was detected in the liver when mice were treated with doxycycline via their drinking water. Figure 8A Using antibodies against OCT4, KLF4, and SOX2, the expression of these proteins was determined by Western blotting, and the results were also evident. Figure 8C Actin was used as a loading control. Figure 8C When mice were treated with doxycycline, OCT4, KLF4, and SOX2 were detected only in the liver. Figure 8C ).

[0292] Example 3: Development of the Tet-On system combining tetracycline repressor and rtTA4.

[0293] To further reduce the background binding of the rtTA and TRE operators in the absence of DOX, we have developed a dual-insurance system of rtTA and tetR (tetRKRAB). Figure 10 tetR or tetRKRAB can bind to the TRE operon in the absence of DOX, preventing rtTA binding and inhibiting TRE activity. When DOX is added to the system, tetR or tetRKRAB leaves the TRE element and exposes it to rtTA binding to activate expression downstream of the TRE gene. Placing this rtTA-IRES-tetRKRAB or rtTA-IRES-tetR under a sequential expression promoter such as UBC, CAG, or a tissue-specific promoter allows for tight control of protein expression throughout the animal or in a specific tissue.

[0294] Example 4. Controlling gene expression in vivo using rtTA4.

[0295] To determine whether the rtTA4 system functions in vivo, two AAVs (UBC-rtTA4 and TRE-Luc or TRE-OSK) were delivered to mice via retroorbital injection. Five-month-old C57BL / 6J mice were infected with rtTA4 and TRE-OSK AAV9. In the absence of DOX treatment, TRE promoter expression was not detected. Figure 15A However, strong expression was observed in tissues such as the liver and pancreas when treated with DOX, resulting in induced levels comparable to those in transgenic mice. Figure 15ASurprisingly, continuous OSK induction for over a year had no noticeable negative effects on mice over that period. Figure 14 and Figure 15B Not subject to specific theoretical constraints, it has no perceptible negative effects on the surface of mice because it avoids high-level expression in the intestine. Figure 15C-15E This avoids developmental abnormalities and weight loss seen in other studies, including Abad et al., Nature 502, 340-345, doi:10.1038 / nature12586 (2013). Therefore, the rtTA4 system allows for spatial and temporal control of gene expression in vivo.

[0296] method

[0297] mouse strains

[0298] C57BL6 / J wild-type mice were purchased from Jackson Laboratory (000664) for optic nerve compression and glaucoma model experiments. For aging experiments, females from the NIA aged rodent population were used (https: / / www.nia.nih.gov / research / dab / aged-rodent-colonies-handbook). The Col1a1-tetOP-OKS-mCherry / Rosa26-M2rtTA alleles are described in Bar-Nur et al., Nat Methods, 2014.11(11): 1170–6. All animal work was approved by Harvard Medical School, Boston Children's Hospital, and the Mass Eye and Ear Institutional Animal Care and Use Committees.

[0299] AAV generation

[0300] The AAV-TRE-OSK vector was prepared by cloning mouse Oct4, Sox2, and Klf4 cDNAs into an AAV plasmid composed of a Tet response element (TRE3G promoter) and an SV40 element. Other vectors were synthesized directly by chemical synthesis. All pAAVs listed in Table 6 were then packaged into serotypes 2 / 2 or 2 / 9 AAVs (titer: >5 × 10⁻⁶). 12 (Number of genome copies / mL). Adeno-associated virus is produced by the Virus Core at Boston Children's Hospital.

[0301] AAV9 delivers systemic delivery to internal organs

[0302] AAV9 (3×10) was injected retroorbitally. 11TRE-OSK plus 7×10 11 OSK expression was achieved in visceral organs via UBC-rtTA4. OSK expression was induced by continuous treatment with 1 mg / mL doxycycline for 3 weeks following injection.

[0303] Example 5. Non-limiting example of a sequence.

[0304] The nucleotide sequence encoding OCT4 (without a stop codon): (SEQ ID NO:1):

[0305]

[0306] The amino acid sequence encoding OCT4 is as follows: (SEQ ID NO:2):

[0307] MAGHLASDFAFSPPPGGGDGSAGLEPGWVDPRTWLSFQGPPGGPGIGPGSEVLGISPCPPAYEFCGGMAYCGPQVGLGLVPQVGVETLQPEGQAGARVESNSEGTSSEPCADRPNAVKLEKVEPTPEESQDMKALQKELEQFAKLLKQKRITLGYTQADVGLTLGVLFGKVFSQTT ICRFEALQLSLKNMCKLRPLLEKWVEEADNNENLQEICKSETLVQARKRKRTSIENRVRWSLETMFLKCPKPSLQQITHIANQLGLEKDVVRVWFCNRRQKGKRSSIEYSQREEYEATGTPFPGGAVSFPLPPGPHFGTPGYGSPHFTTLYSVPFPEGEAFPSVPVTALGSPMHSN

[0308] Nucleotide sequence encoding SOX2 (without a stop codon): (SEQ ID NO:3):

[0309] ATGTATAACATGATGGAGACGGAGCTGAAGCCGCCGGGCCCGCAGCAAGCTTCGGGGGGCGGCGGCGGAGGAGGCAACGCCACGGCGGCGGCGACCGGCGGCAACCAGAAGAACAGCCCGGACCGCGTCAAGAGGCCCATGAACGCCTTCATGGTATGGTCCCGGGGGCAGCGGCGTAAGATGGCCCAGGAGAACCCCAAGATGCACAACTCGGAGATCAGCAAGCGCCTGGGCGCGGAGTGGAAACTTTTGTCCGAGACCGAGAAGCGGCCGTTCATCGACGAGGCCAAGCGGCTGCGCGCTCTGCACATGAAGGAGCACCCGGATTATAAATACCGGCCGCGGCGGAAAACCAAGACGCTCATGAAGAAGGATAAGTACACGCTTCCCGGAGGCTTGCTGGCCCCCGGCGGGAACAGCATGGCGAGCGGGGTTGGGGTGGGCGCCGGCCTGGGTGCGGGCGTGAACCAGCGCATGGACAGCTACGCGCACATGAACGGCTGGAGCAACGGCAGCTACAGCATGATGCAGGAGCAGCTGGGCTACCCGCAGCACCCGGGCCTCAACGCTCACGGCGCGGCACAGATGCAACCGATGCACCGCTACGACGTCAGCGCCCTGCAGTACAACTCCATGACCAGCTCGCAGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAGCAGGGCACCCCCGGTATGGCGCTGGGCTCCATGGGCTCTGTGGTCAAGTCCGAGGCCAGCTCCAGCCCCCCCGTGGTTACCTCTTCCTCCCACTCCAGGGCGCCCTGCCAGGCCGGGGACCTCCGGGACATGATCAGCATGTACCTCCCCGGCGCCGAGGTGCCGGAGCCCGCTGCGCCCAGTAGACTGCACATGGCCCAGCACTACCAGAGCGGCCCGGTGCCCGGCACGGCCATTAACGGCACACTGCCCCTGTCGCACATG

[0310] The amino acid sequence encoding SOX2 (translated): (SEQ ID NO:4)

[0311] MYNMMETELKPPGPQQASGGGGGGGNATAAATGGNQKNSPDRVKRPMNAFMVWSRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRM DSYAHMNGWSNGSYSMMQEQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVVKSEASSSPPVVTSSHSRAPCQAGDLRDMISMYLPGAEVPEPAAPSRLHMAQHYQSGPVPGTAINGTLPLSHM

[0312] The nucleotide sequence encoding KLF4 (without a stop codon): (SEQ ID NO:5):

[0313]

[0314] Amino acid sequence of encoded KLF4 (translated): (SEQ ID NO:6):

[0315] MRQPPGESDMAVSDALLPSFSTFASGPAGREKTLRPAGAPTNRWREELSHMKRLPPLPGRPYDLAATVATDLESGGAGAACSSNNPALLARRETEEFNDLLDLDFILSNSLTHQESVAATVTTSASASSSSSPASSGPASAPSTCSFSYPIRAGGDPGVAASNTGGGLLYSRESAPPPTAPFNLADINDVSPSGGFVAELLRPELDPVYIPPQQPQPPGGGLMGKFVLKASLTTPGSEYSSPSVISVSKGSPDGSHPVVVAPYSGGPPRMCPKIKQEAVPSCTVSRSLEAHLSAGPQLSNGHRPNTHDFPLGRQLPTRTTPTLSPEELLNSRDCHPGLPLPPGFHPHPGPNYPPFLPDQMQSQVPSLHYQELMPPGSCLPEEPKPKRGRRSWPRKRTATHTCDYAGCGKTYTKSSHLKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQCQKCDRAFSRSDHLALHMKRH

[0316] TRE3G promoter sequence (non-limiting example of TRE promoter): (SEQ ID NO:7): TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTGATAGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGTTTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGAGAACGTATCTACAGTTTAC TCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTCCCTATCAGTGATAGAGAACGTATAAGCTTTTAGGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTTGTCTTATAACCAACTTTCCGTACCACTTCCTACCCTCGTAAA

[0317] SV40-derived termination sequence: (SEQ ID NO:8):

[0318] TGCGCGCAGCGGCCGACCATGGCCCAACTTGTTTATTGCAGCTTAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTCGGTACCG

[0319] T2A sequence (SEQ ID NO:9): GSGEGRGSLLTCGDVEENPGP

[0320] The nucleotide sequence encoding rtTA3 (with two VP16 domains at the 3' end): (SEQ ID NO:10):

[0321] ATGTCTAGGCTGGACAAGAGCAAAGTCATAAACGGAGCTCTGGAATTACTCAATGGTGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCTGCCAATCGAGATGCTGGACAGGCATCATACCCACTTCTGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGCTCTCCTCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCGATTCTATGCCCCCACTTCTGAGACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCTGCCTTCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAAGCGGCGGGCCGACCGACGCCCTTGACGATTTTGACTTAGACATGCTCCCAGCCGATGCCCTTGACGATTTTGACCTTGACATGCTCCCCGGGTAA

[0322] Amino acid sequence encoding rtTA3: (SEQ ID NO:11):

[0323] MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRPTEKQYETLENQL AFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPADALDDFDLDMLPG

[0324] The nucleotide sequence encoding rtTA4 (with three VP16 domains at the 3' end): (SEQ ID NO:12):

[0325] ATGTCCCGCTTGGATAAGAGCAAGGTAATAAATAGCGCACTCGAACTCCTCAACGGCGTGGGCATCGAAGGTCTGACTACTCGAAAGCTCGCCCAGAAATTGGGTGTGGAGCAACCTACATTGTATTGGCATGTCAAGAACAAAAGAGCCCTGCTGGACGCTCTTCCTATTGAAATGCTTGACAGGCATCACACTCATTCCTGCCCCCTTGAGGTCGAGAGTTGGCAAGATTTTCTCCGAAACAATGCAAAGTCCTACCGCTGCGCACTTTTGTCCCATAGGGATGGAGCAAAAGTGCACCTGGGAACCAGGCCAACAGAGAAACAATACGAGACTCTCGAGAACCAGTTGGCTTTCTTGTGCCAACAGGGGTTCTCACTTGAAAATGCCCTTTACGCACTGTCAGCCGTTGGACATTTTACCCTGGGGTGCGTTCTTGAGGAGCAAGAACATCAGGTTGCTAAGGAGGAGCGCGAGACTCCAACCACTGATTCTATGCCACCTTTGCTGAAACAGGCCATTGAACTTTTCGATAGACAGGGTGCTGAACCTGCCTTTCTCTTCGGGTTGGAGCTGATTATTTGTGGTCTCGAAAAACAGCTGAAATGTGAAAGTGGTGGCCCTACTGACGCCCTCGATGATTTCGACCTGGATATGCTGCCAGCCGATGCACTTGATGATTTCGATTTGGATATGCTTCCAGCCGACGCACTGGACGACTTCGATTTGGACATGCTTCCCGGTTAA

[0326] Amino acid sequence encoding rtTA4: (SEQ ID NO:13):

[0327] MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPIEMLDRHHTHSCPLEVESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG

[0328] The nucleotide sequence encoding M2-rtTA (SEQ ID NO: 14):

[0329]

[0330] Amino acid sequence of encoded M2-rtTA (SEQ ID NO:15):

[0331] MPLYHAIASRMAFIFSSLYKSWLLSLYEELWPVVRQRGVVCTVFADATPTGWGIATTCQLLSGTFAFPLPIATAELIAACLARCWTGARLLGTDNSVVLSGKSSSFPWLLACVATWILRGTSFCYVPSALNPADLPSRGLLPALRPLPRLRLRPQTSRISLWAASPHRYRRPRDLEKHGAITSSNTAATNADCAWLEAQEEEEVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQGYFPDWQNYTPGPGIRYPLTFGWCYKLVPVEQEKVEEANEGENTRLLHPVSLHGMDDPEREVLEWRFDSRLAFHHMARELHPDCTGSLWLDQI

[0332] Nucleic acid sequence of pAAV-TRE3G-OSK-SV40pA or TRE3G-OSK-SV40pA vector (SEQ ID NO:16):

[0333]

[0334] The nucleic acid sequence of the pAAV-UBC-rtTA4-WPRE3-SV40pA vector (SEQ ID NO:17):

[0335]

[0336] UBC promoter sequence (SEQ ID NO:18):

[0337]

[0338] Tet-O sequence (SEQ ID NO:19): TCCCTATCAGTGATAGAGA

[0339] Nucleic acid sequence encoding the minimal CMV promoter (SEQ ID NO:20):

[0340] GCTTTAGGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGA

[0341] Nucleic acid sequence encoding WPRE (SEQ ID NO:21):

[0342] AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTT

[0343] Nucleic acid sequence encoding terminal inverted repeat (SEQ ID NO:22):

[0344] CCTTAATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

[0345] Nucleic acid sequence of TRE2 promoter (non-limiting example of TRE promoter) (SEQ ID NO:23):

[0346] AATTCGTACACGCCTACCTCGACCCATCAAGTGCCACCTGACGTCTCCCTATCAGTGATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACGTCTAGAACGTCTCCCTATCAGTGATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACGTCTAGAACGTCTCCCTATCAGTGATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACGTCTAGAACGTCTCCCTATCAGTGATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACCCCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTGGATCGC

[0347] Nucleic acid sequence of the P tight promoter (non-limiting example of the TRE promoter) (SEQ ID NO: 24):

[0348] GAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTATCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCC

[0349] Nucleic acid sequence encoding TetR (SEQ ID NO: 25):

[0350] ATGGCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGG

[0351] Amino acid sequence encoding TetR (SEQ ID NO:26):

[0352] MARLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG

[0353] Nucleic acid sequence encoding TetR-Krab (SEQ ID NO:27)

[0354]

[0355] Amino acid sequence encoding TetR-KRAB (SEQ ID NO:28):

[0356] MARLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSPKKKRKVDGGGALSPQHSAVTQGSIIKNKEGMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSV

[0357] Desmin promoter (SEQ ID NO:29):

[0358] ACCTTGCTTCCTAGCTGGGCCTTTCCTTCTCCTCTATAAATACCAGCTCTGGTATTTCGCCTTGGCAGCTGTTGCTGCTAGGGAGACGGCTGGCTTGACATGCATCTCCTGACAAAACACAAACCCGTGGTGTGAGTGGGTGTGGGCGGTGTGAGTAGGGGGATGAATCAGAGAGGGGGCGAGGGAGACAGGGGCGCAGGAGTCAGGCAAAGGCGATGCGGGGGTGCGACTACACGCAGTTGGAAACAGTCGTCAGAAGATTCTGGAAACTATCTTGCTGGCTATAAACTTGAGGGAAGCAGAAGGCCAACATTCCTCCCAAGGGAAACTGAGGCTCAGAGTTAAAACCCAGGTATCAGTGATATGCATGTGCCCCGGCCAGGGTCACTCTCTGACTAACCGGTACCTACCCTACAGGCCTACCTAGAGACTCTTTTGAAAGGATGGTAGAGACCTGTCCGGGCTTTGCCCACAGTCGTTGGAAACCTCAGCATTTTCTAGGCAACTTGTGCGAATAAAACACTTCGGGGGTCCTTCTTGTTCATTCCAATAACCTAAAACCTCTCCTCGGAGAAAATAGGGGGCCTCAAACAAACGAAATTCTCTAGCCCGCTTTCCCCAGGATAAGGCAGGCATCCAAATGGAAAAAAAGGGGCCGGCCGGGGGTCTCCTGTCAGCTCCTTGCCCTGTGAAACCCAGCAGGCCTGCCTGTCTTCTGTCCTCTTGGGGCTGTCCAGGGGCGCAGGCCTCTTGCGGGGGAGCTGGCCTCCCCGCCCCCTCGCCTGTGGCCGCCCTTTTCCTGGCAGGACAGAGGGATCCTGCAGCTGTCAGGGGAGGGGCGCCGGGGGGTGATGTCAGGAGGGCTACAAATAGTGCAGACAGCTAAGGGGCTCCGTCACCCATCTTCACATCCACTCCAGCCGGCTGCCCGCCCGCTGCCTCCTCTGTGCGTCCGCCCAGCCAGCCTCGTCCACGCC

[0359] Desmin-rtTA4 vector (SEQ ID NO:30):

[0360]

[0361] pAAV2_CMV_rtTA(V16)(SEQ ID NO:31):

[0362]

[0363] CAG-tTA(SEQ ID NO:32):

[0364]

[0365] pAAV-Tet-O-OSK-SV40LpA (or pAAV-TRE2-OSK-SV40LpA) (SEQ ID NO:33):

[0366]

[0367] VP64, a repeating sequence of four VP16 (SEQ ID NO:34) (a non-limiting example of an inverse activation domain):

[0368] GAGGCCAGCGGTTCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGATTAACTCTAGA

[0369] P65 (SEQ ID NO:35) (a non-restrictive example of an inverse activation domain):

[0370] AGCCAGTACCTGCCCGACACCGACGACCGGCACCGGATCGAGGAAAAGCGGAAGCGGACCTACGAGACATTCAAGAGCATCATGAAGAAGTCCCCCTTCAGCGGCCCCACCGACCCTAGACCTCCACCTAGAAGAATCGCCGTGCCCAGCAGATCCAGCGCCAGCGTGCCAAAACCTGCCCCCCAGCCTTACCCCTTCACCAGCAGCCTGAGCACCATCAACTACGACGAGTTCCCTACCATGGTGTTCCCCAGCGGCCAGATCTCTCAGGCCTCTGCTCTGGCTCCAGCCCCTCCTCAGGTGCTGCCTCAGGCTCCTGCTCCTGCACCAGCTCCAGCCATGGTGTCTGCACTGGCTCAGGCACCAGCACCCGTGCCTGTGCTGGCTCCTGGACCTCCACAGGCTGTGGCTCCACCAGCCCCTAAACCTACACAGGCCGGCGAGGGCACACTGTCTGAAGCTCTGCTGCAGCTGCAGTTCGACGACGAGGATCTGGGAGCCCTGCTGGGAAACAGCACCGATCCTGCCGTGTTCACCGACCTGGCCAGCGTGGACAACAGCGAGTTCCAGCAGCTGCTGAACCAGGGCATCCCTGTGGCCCCTCACACCACCGAGCCCATGCTGATGGAATACCCCGAGGCCATCACCCGGCTCGTGACAGGCGCTCAGAGGCCTCCTGATCCAGCTCCTGCCCCTCTGGGAGCACCAGGCCTGCCTAATGGACTGCTGTCTGGCGACGAGGACTTCAGCTCTATCGCCGATATGGATTTCTCAGCCTTGCTG

[0371] RTA (SEQ ID NO:36) (Non-limiting example of the transactivation domain):

[0372] CGGGATTCCAGGGAAGGGATGTTTTTGCCGAAGCCTGAGGCCGGCTCCGCTATTAGTGACGTGTTTGAGGGCCGCGAGGTGTGCCAGCCAAAACGAATCCGGCCA

[0373] TTTCATCCTCCAGGAAGTCCATGGGCCAACCGCCCACTCCCCGCCAGCCTCGCACCAACACCAACCGGTCCAGTACATGAGCCAGTCGGGTCACTGACCCCGGCACCAGTCCCTCAGCCACTGGATCCAGCGCCCGCAGTGACTCCCGAGGCCAGTCACCTGTTGGAGGATCCCGATGAAGAGACGAGCCAGGCTGTCAAAGCCCTTCGGGAGATGGCCGATACTGTGATTCCCCAGAAGGAAGAGGCTGCAATCTGTGGCCAAATGGACCTTTCCCATCCGCCCCCAAGGGGCCATCTGGATGAGCTGACAACCACACTTGAGTCCA

[0374] TGACCGAGGATCTGAACCTGGACTCACCCCTGACCCCGGAATTGAACGAGATTCTGGATACCTTCCTGAACGACGAGTGCCTCTTGCATGCCATGCATATCAGCACAGGAC TGTCCA TCTTCGACACA TCTCTGTTT

[0375] MPH MS2-P65-HSF1 (SEQ ID NO:37) (Non-limiting example of transactivation domain):

[0376]

[0377] Equivalents and scope

[0378] In the claims, articles such as “a,” “an,” and “the” may mean one or more, unless otherwise indicated or otherwise apparent from the context. A claim or description including “or” among one or more members of the group is considered satisfactory if one, more than one, or all members of the group are present, used, or otherwise associated with a given product or method, unless otherwise indicated or otherwise apparent from the context. This disclosure includes embodiments in which exactly one member of the group is present, used, or otherwise associated with a given product or method. This disclosure also includes embodiments in which more than one or all members of the group are present, used, or otherwise associated with a given product or method.

[0379] Furthermore, this disclosure covers all variations, combinations, and arrangements in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are incorporated into another claim. For example, any claim attached to another claim may be modified to include one or more limitations seen in any other claim attached to the same basic claim. Where elements are presented as a list in Markush group format, each subgroup of elements is also disclosed, and any element may be removed from the group. It should be understood that, generally, where a disclosure or aspect described herein is referred to as containing a particular element and / or feature, certain embodiments or aspects described herein consist of or are substantially composed of such elements and / or features. For simplicity, those embodiments with respect to this aspect are not specifically stated herein. It should also be noted that the terms “comprising” and “containing” are intended to be open and allow for the inclusion of additional elements or steps. When a scope is given, the endpoints are included. Furthermore, unless otherwise specified or otherwise apparent from the context and understanding of one of ordinary skill in the art, values ​​expressed as scope may assume any specific value or subscope within the scope described in the different embodiments described herein, up to one-tenth of the unit of the lower limit of the scope, unless the context explicitly indicates otherwise.

[0380] This application relates to various granted patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of any conflict between any introduced reference and this specification, this specification shall prevail. Furthermore, any particular embodiment of this disclosure falling within the prior art may be expressly excluded from any one or more of the claims. Because such embodiments are considered known to one of ordinary skill in the art, they may be excluded even if such exclusion is not expressly stated herein. Any particular embodiment described herein may be excluded from any claim for any reason, whether or not related to the existence of prior art.

[0381] Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments described herein using no more than conventional experiments. The scope of the embodiments of the invention described herein is not intended to be limited to the above description, but rather as set forth in the appended claims. Those skilled in the art will understand that various changes and modifications can be made to this specification without departing from the spirit or scope of this disclosure as defined in the following claims. sequence list <110> The president and members of Harvard University <120> Mutant reverse tetracycline transactivator used for gene expression <130> H0824.70300WO00 <140> Not yet allocated <141> 2019-09-27 <150> US 62 / 738,894 <151> 2018-09-28 <160> 37 <170> PatentIn version 3.5 <210> 1 <211> 1056 <212> DNA <213> House mouse <400> 1 atggctggac acctggcttc agacttcgcc ttctcacccc caccaggtgg gggtgatggg 60 tcagcagggc tggagccggg ctgggtggat cctcgaacct ggctaagctt ccaagggcct 120 ccaggtgggc ctggaatcgg accaggctca gaggtattgg ggatctcccc atgtccgccc 180 gcatacgagt tctgcggagg gatggcatac tgtggacctc aggttggact gggcctagtc 240 ccccaagttg gcgtggagac tttgcagcct gagggccagg caggagcacg agtggaaagc 300 aactcagagg gaacctctc tgagccctgt gccgaccgcc ccaatgccgt gaagttggag 360 aaggtggaac caactcccga ggagtcccag gacatgaaag ccctgcagaa ggagctagaa 420 cagtttgcca agctgctgaa gcagaagagg atcaccttgg ggtacaccca ggccgacgtg 480 gggctcacc tgggcgttct ctttggaaag gtgttcagcc agaccaccat ctgtcgcttc 540 gagggccttgc agctcagcct taagaacatg tgtaagctgc ggcccctgct gggaagtgg 600 gtggaggaag ccgacaacaa tgagaacctt caggagatat gcaaatcgga gaccctggtg 660 caggcccgga agaaagcg aactagcatt gagaaccgtg tgaggtggag tctggagacc 720 780 gggctagaga aggatgtggt tcgagtatgg ttctgtaacc ggcgccagaa gggcaaaaga 840 tcaagtattg agtattccca acgagaagag tatgaggcta cagggacacc tttcccaggg 900 ggggctgtat cctttcctct gcccccaggt ccccactttg gcaccccagg ctatggaagc 960 ccccacttca ccacactcta ctcagtccct tttcctgagg gcgaggcctt tccctctgtt 1020 cccgtcactg ctctgggctc tcccatgcat tcaaac 1056 <210> 2 <211> 352 <212> PRT <213> Mus musculus <400> 2 Met Ala Gly His Leu Ala Ser Asp Phe Ala Phe Ser Pro Pro Pro Gly 1 5 10 15 Gly Gly Asp Gly Ser Ala Gly Leu Glu Pro Gly Trp Val Asp Pro Arg 20 25 30 Thr Trp Leu Ser Phe Gln Gly Pro Pro Gly Gly Pro Gly Ile Gly Pro 35 40 45 Gly Ser Glu Val Leu Gly Ile Ser Pro Cys Pro Pro Ala Tyr Glu Phe 50 55 60 Cys Gly Gly Met Ala Tyr Cys Gly Pro Gln Val Gly Leu Gly Leu Val 65 70 75 80 Pro Gln Val Gly Val Glu Thr Leu Gln Pro Glu Gly Gln Ala Gly Ala 85 90 95[[ID=Arg Val Glu Ser Asn Ser Glu Gly Thr Ser Ser Glu Pro Cys Ala Asp 100 105 110 Arg Pro Asn Ala Val Lys Leu Glu Lys Val Glu Pro Thr Pro Glu Glu 115 120 125 Ser Gln Asp Met Lys Ala Leu Gln Lys Glu Leu Glu Gln Phe Ala Lys 130 135 140 Leu Leu Lys Gln Lys Arg Ile Thr Leu Gly Tyr Thr Gln Ala Asp Val 145 150 155 160 Gly Leu Thr Leu Gly Val Leu Phe Gly Lys Val Phe Ser Gln Thr Thr 165 170 175 Ile Cys Arg Phe Glu Ala Leu Gln Leu Ser Leu Lys Asn Met Cys Lys 180 185 190 Leu Arg Pro Leu Leu Glu Lys Trp Val Glu Glu Ala Asp Asn Asn Glu 195 200 205 Asn Leu Gln Glu Ile Cys Lys Ser Glu Thr Leu Val Gln Ala Arg Lys 210 215 220 Arg Lys Arg Thr Ser Ile Glu Asn Arg Val Arg Trp Ser Leu Glu Thr 225 230 235 240 Met Phe Leu Lys Cys Pro Lys Pro Ser Leu Gln Gln Ile Thr His Ile 245 250 255 Ala Asn Gln Leu Gly Leu Glu Lys Asp Val Val Arg Val Trp Phe Cys 260 265 270 Asn Arg Arg Gln Lys Gly Lys Arg Ser Ser Ile Glu Tyr Ser Gln Arg 275 280 285 Glu Glu Tyr Glu Ala Thr Gly Thr Pro Phe Pro Gly Gly Ala Val Ser 290 295 300 Phe Pro Leu Pro Pro Gly Pro His Phe Gly Thr Pro Gly Tyr Gly Ser 305 310 315 320 Pro His Phe Thr Thr Leu Tyr Ser Val Pro Phe Pro Glu Gly Glu Ala 325 330 335 Phe Pro Ser Val Pro Val Thr Ala Leu Gly Ser Pro Met His Ser Asn 340 345 350 <210> 3 <211> 957 <212> DNA <213> Mus musculus <400> 3 atgtataaca tgatggagac ggagctgaag ccgccgggcc cgcagcaagc ttcggggggc 60 ggcggcggag gaggcaacgc cacggcggcg gcgaccggcg gcaaccagaa gaacagcccg 120 gaccgcgtca agaggcccat gaacgccttc atggtatggt cccgggggca gcggcgtaag 180 atggcccagg agaaccccaa gatgcacaac tcggagatca gcaagcgcct gggcgcggag 240 tggaacttt tgtccgagac cgagaagcgg ccgttcatcg acgaggccaa gcggctgcgc gctctgcaca tgaaggagca cccggattat aaataccggc cgcggcgga aaccaagacg ctcatgaaga aggataagta cacgcttccc ggaggcttgc tggcccccgg cgggaacagc 420 atggcgagcg gggttggggt gggcgccggc ctgggtgcgg gcgtgaacca gcgcatggac 480 agctacgcgc acatgaacgg ctggagcaac ggcagctaca gcatgatgca ggagcagctg ggctacccgc agcaccggg cctcaacgct cacggcgcgg cacagatgca accgatgcac cgctacgacg tcagcgccct gcagtacaac tccatgacca gctcgcagac ctacatgaac ggctcgccca cctacagcat gtcctactcg cagcagggca cccccggtat ggcgctgggc 720 tccatgggct ctgtggtcaa gtccgaggcc agctccagcc cccccgtggt tacctcttcc 780 tcccactcca gggcgccctg ccaggccggg gacctccggg acatgatcag catgtacctc 840 cccggcgccg aggtgccgga gcccgctgcg cccagtagac tgcacatggc ccagcactac cagagcggcc cggtgcccgg cacggccatt aacggcacac tgcccctgtc gcacatg 957 <210> 4 <211> 319 <212> PRT <213> Mus musculus <400> 4 Met Tyr Asn Met Met Glu Thr Glu Leu Lys Pro Pro Gly Pro Gln Gln 1 5 10 15 Ala Ser Gly Gly Gly Gly Gly Gly Gly Asn Ala Thr Ala Ala Ala Thr 20 25 30 Gly Gly Asn Gln Lys Asn Ser Pro Asp Arg Val Lys Arg Pro Met Asn 35 40 45 Ala Phe Met Val Trp Ser Arg Gly Gln Arg Arg Lys Met Ala Gln Glu 50 55 60 Asn Pro Lys Met His Asn Ser Glu Ile Ser Lys Arg Leu Gly Ala Glu 65 70 75 80 Trp Lys Leu Leu Ser Glu Thr Glu Lys Arg Pro Phe Ile Asp Glu Ala 85 90 95 Lys Arg Leu Arg Ala Leu His Met Lys Glu His Pro Asp Tyr Lys Tyr 100 105 110 Arg Pro Arg Arg Lys Thr Lys Thr Leu Met Lys Lys Asp Lys Tyr Thr 115 120 125 Leu Pro Gly Gly Leu Leu Ala Pro Gly Gly Asn Ser Met Ala Ser Gly 130 135 140 Val Gly Val Gly Ala Gly Leu Gly Ala Gly Val Asn Gln Arg Met Asp 145 150 155 160 Ser Tyr Ala His Met Asn Gly Trp Ser Asn Gly Ser Tyr Ser Met Met 165 170 175 Gln Glu Gln Leu Gly Tyr Pro Gln His Pro Gly Leu Asn Ala His Gly 180 185 190 Ala Ala Gln Met Gln Pro Met His Arg Tyr Asp Val Ser Ala Leu Gln 195 200 205 Tyr Asn Ser Met Thr Ser Ser Gln Thr Tyr Met Asn Gly Ser Pro Thr 210 215 220 Tyr Ser Met Ser Tyr Ser Gln Gln Gly Thr Pro Gly Met Ala Leu Gly 225 230 235 240 Ser Met Gly Ser Val Val Lys Ser Glu Ala Ser Ser Ser Pro Pro Val 245 250 255 Val Thr Ser Ser Ser His Ser Arg Ala Pro Cys Gln Ala Gly Asp Leu 260 265 270 Arg Asp Met Ile Ser Met Tyr Leu Pro Gly Ala Glu Val Pro Glu Pro 275 280 285 Ala Ala Pro Ser Arg Leu His Met Ala Gln His Tyr Gln Ser Gly Pro 290 295 300 Val Pro Gly Thr Ala Ile Asn Gly Thr Leu Pro Leu Ser His Met 305 310 315 <210> 5 <211> 1446 <212> DNA <213> Mus musculus <400> 5 atgaggcagc cacctggcga gtctgacatg gctgtcagcg acgctctgct cccgtccttc 60 tccacgttcg cgtccggccc ggcgggaagg gagaagacac tgcgtccagc aggtgccccg 120 actaaccgtt ggcgtgagga actctctcac atgaagcgac ttcccccact tcccggccgc 180 ccctacgacc tggcggcgac ggtggccaca gacctggaga gtggcggagc tggtgcagct 240 tgcagcagta acaacccggc cctcctagcc cggagggaga ccgaggagtt caacgacctc 300 ctggacctag actttatcct ttccaactcg ctaacccacc aggaatcggt ggccgccacc 360 gtgaccacct cggcgtcagc ttcatcctcg tcttccccag cgagcagcgg ccctgccagc 420 gcgccctcca cctgcagctt cagctatccg atccgggccg ggggtgaccc gggcgtggct 480 gccagcaaca caggtggagg gctcctctac agccgagaat ctgcgccacc tcccacggcc 540 cccttcaacc tggcggacat caatgacgtg agcccctcgg gcggcttcgt ggctgagctc 600 ctgcggccgg agttggaccc agtatacatt ccgccacagc agcctcagcc gccaggtggc 660 gggctgatgg gcaagtttgt gctgaaggcg tctctgacca cccctggcag cgagtacagc 720 agcccttcgg tcatcagtgt tagcaaagga agcccagacg gcagccaccc cgtggtagtg 780 gcgccctaca gcggtggccc gccgcgcatg tgccccaaga ttaagcaaga ggcggtcccg 840 tcctgcacgg tcagccggtc cctagaggcc catttgagcg ctggacccca gctcagcaac 900 ggccaccggc ccaacacaca cgacttcccc ctggggcggc agctccccac caggactacc 960 cctacactga gtcccgagga actgctgaac agcagggact gtcaccctgg cctgcctctt 1020 cccccaggat tccatcccca tccggggccc aactaccctc ctttcctgcc agaccagatg 1080 cagtcacaag tcccctctct ccattatcaa gagctcatgc caccgggttc ctgcctgcca 1140 gaggagccca agccaaagag gggaagaagg tcgtggcccc ggaaaagaac agccacccac 1200 acttgtgact atgcaggctg tggcaaaacc tataccaaga gttctcatct caaggcacac 1260 ctgcgaactc acacaggcga gaaaccttac cactgtgact gggacggctg tgggtggaaa 1320 ttcgcccgct ccgatgaact gaccaggcac taccgcaaac acacagggca ccggcccttt 1380 cagtgccaga agtgcgacag ggccttttcc aggtcggacc accttgcctt acacatgaag 1440 aggcac 1446 <210> 6 <211> 482 <212> PRT <213> Mus musculus <400> 6 Met Arg Gln Pro Pro Gly Glu Ser Asp Met Ala Val Ser Asp Ala Leu 1 5 10 15 Leu Pro Ser Phe Ser Thr Phe Ala Ser Gly Pro Ala Gly Arg Glu Lys 20 25 30 Thr Leu Arg Pro Ala Gly Ala Pro Thr Asn Arg Trp Arg Glu Glu Leu 35 40 45 Ser His Met Lys Arg Leu Pro Pro Leu Pro Gly Arg Pro Tyr Asp Leu 50 55 60 Ala Ala Thr Val Ala Thr Asp Leu Glu Ser Gly Gly Ala Gly Ala Ala 65 70 75 80 Cys Ser Ser Asn Asn Pro Ala Leu Leu Ala Arg Arg Glu Thr Glu Glu 85 90 95 Phe Asn Asp Leu Leu Asp Leu Asp Phe Ile Leu Ser Asn Ser Leu Thr 100 105 110 His Gln Glu Ser Val Ala Ala Thr Val Thr Thr Ser Ala Ser Ala Ser 115 120 125 Ser Ser Ser Ser Pro Ala Ser Ser Gly Pro Ala Ser Ala Pro Ser Thr 130 135 140 Cys Ser Phe Ser Tyr Pro Ile Arg Ala Gly Gly Asp Pro Gly Val Ala 145 150 155 160 Ala Ser Asn Thr Gly Gly Gly Leu Leu Tyr Ser Arg Glu Ser Ala Pro 165 170 175 Pro Pro Thr Ala Pro Phe Asn Leu Ala Asp Ile Asn Asp Val Ser Pro 180 185 190 Ser Gly Gly Phe Val Ala Glu Leu Leu Arg Pro Glu Leu Asp Pro Val 195 200 205 Tyr Ile Pro Pro Gln Gln Pro Gln Pro Pro Gly Gly Gly Leu Met Gly 210 215 220 Lys Phe Val Leu Lys Ala Ser Leu Thr Thr Pro Gly Ser Glu Tyr Ser 225 230 235 240 Ser Pro Ser Val Ile Ser Val Ser Lys Gly Ser Pro Asp Gly Ser His 245 250 255 Pro Val Val Val Ala Pro Tyr Ser Gly Gly Pro Pro Arg Met Cys Pro 260 265 270 Lys Ile Lys Gln Glu Ala Val Pro Ser Cys Thr Val Ser Arg Ser Leu 275 280 285 Glu Ala His Leu Ser Ala Gly Pro Gln Leu Ser Asn Gly His Arg Pro 290 295 300 Asn Thr His Asp Phe Pro Leu Gly Arg Gln Leu Pro Thr Arg Thr Thr 305 310 315 320 Pro Thr Leu Ser Pro Glu Glu Leu Leu Asn Ser Arg Asp Cys His Pro 325 330 335 Gly Leu Pro Leu Pro Pro Gly Phe His Pro His Pro Gly Pro Asn Tyr 340 345 350 Pro Pro Phe Leu Pro Asp Gln Met Gln Ser Gln Val Pro Ser Leu His 355 360 365 Tyr Gln Glu Leu Met Pro Pro Gly Ser Cys Leu Pro Glu Glu Pro Lys 370 375 380 Pro Lys Arg Gly Arg Arg Ser Trp Pro Arg Lys Arg Thr Ala Thr His 385 390 395 400 Thr Cys Asp Tyr Ala Gly Cys Gly Lys Thr Tyr Thr Lys Ser Ser His 405 410 415 Leu Lys Ala His Leu Arg Thr His Thr Gly Glu Lys Pro Tyr His Cys 420 425 430 Asp Trp Asp Gly Cys Gly Trp Lys Phe Ala Arg Ser Asp Glu Leu Thr 435 440 445 Arg His Tyr Arg Lys His Thr Gly His Arg Pro Phe Gln Cys Gln Lys 450 455 460 Cys Asp Arg Ala Phe Ser Arg Ser Asp His Leu Ala Leu His Met Lys 465 470 475 480 Arg His <210> 7 <211> 376 <212> DNA <213> Synthetic sequence <220> <223> Synthetic polynucleotide <400> 7 tttactccct atcagtgata gagaacgtat gaagagttta ctccctatca gtgatagaga 60 acgtatgcag actttactcc ctatcagtga tagagaacgt ataaggagtt tactccctat 120 cagtgataga gaacgtatga ccagtttact ccctatcagt gatagagaac gtatctacag 180 tttactccct atcagtgata gagaacgtat atccagttta ctccctatca gtgatagaga 240 acgtataagc tttaggcgtg tacggtgggc gcctataaaa gcagagctcg tttagtgaac 300 cgtcagatcg cctggagcaa ttccacaaca cttttgtctt ataccaactt tccgtaccac 360 ttcctaccct cgtaaa 376 <210> 8 <211> 169 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 8 tgcgcgcagc ggccgaccat ggcccaactt gtttattgca gcttataatg gttacaaata 60 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 120 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc tcggtaccg 169 <210> 9 <211> 21 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 9 Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu 1 5 10 15 Glu Asn Pro Gly Pro 20 <210> 10 <211> 708 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <400> 10 atgtctaggc tggacaagag caaagtcata aacggagctc tggaattact caatggtgtc 60 ggtatcgaag gcctgacgac aaggaaactc gctcaaaagc tgggagttga gcagcctacc 120 ctgtactggc acgtgaagaa caagcgggcc ctgctcgatg ccctgccaat cgagatgctg 180 gacaggcatc atacccactt ctgccccctg gaaggcgagt catggcaaga ctttctgcgg 240 aacaacgcca agtcataccg ctgtgctctc ctctcacatc gcgacggggc taaagtgcat 300 ctcggcaccc gcccaacaga gaaacagtac gaaaccctgg aaaatcagct cgcgttcctg 360 tgtcagcaag gcttctccct ggagaacgca ctgtacgctc tgtccgccgt gggccacttt 420 acactgggct gcgtattgga ggaacaggag catcaagtag caaaagagga aagagagaca 480 cctaccaccg attctatgcc cccacttctg agacaagcaa ttgagctgtt cgaccggcag 540 ggagccgaac ctgccttcct tttcggcctg gaactaatca tatgtggcct ggagaaacag 600 ctaaagtgcg aaagcggcgg gccgaccgac gcccttgacg attttgactt agacatgctc 660 ccagccgatg cccttgacga ttttgacctt gacatgctcc ccgggtaa 708 <210> 11 <211> 235 <212> PRT <213> Synthetic sequence <220> <223> Synthetic polypeptide <400> 11 Met Ser Arg Leu Asp Lys Ser Lys Val Ile Asn Gly Ala Leu Glu Leu 1 5 10 15 Leu Asn Gly Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln 20 25 30 Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys 35 40 45 Arg Ala Leu Leu Asp Ala Leu Pro Ile Glu Met Leu Asp Arg His His 50 55 60 Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg 65 70 75 80 Asn Asn Ala Lys Ser Tyr Arg Cys Ala Leu Leu Ser His Arg Asp Gly 85 90 95 Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr 100 105 110 Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu 115 120 125 Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys 130 135 140 Val Leu Glu Glu Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr 145 150 155 160 Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu 165 170 175 Phe Asp Arg Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu 180 185 190 Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro 195 200 205 Thr Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala 210 215 220 Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Gly 225 230 235 <210> 12 <211> 747 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 12 atgtcccgct tggataagag caaggtaata aatagcgcac tcgaactcct caacggcgtg 60 ggcatcgaag gtctgactac tcgaaagctc gcccagaaat tgggtgtgga gcaacctaca 120 ttgtattggc atgtcaagaa caaaagagcc ctgctggacg ctcttcctat tgaaatgctt 180 gacaggcatc acactcattc ctgccccctt gaggtcgaga gttggcaaga ttttctccga 240 aacaatgcaa agtcctaccg ctgcgcactt ttgtcccata gggatggagc aaaagtgcac 300 ctgggaacca ggccaacaga gaaacaatac gagactctcg agaaccagtt ggctttcttg 360 tgccaacagg ggttctcact tgaaaatgcc ctttacgcac tgtcagccgt tggacatttt 420 accctggggt gcgttcttga ggagcaagaa catcaggttg ctaaggagga gcgcgagact 480 ccaaccactg attctatgcc acctttgctg aaacaggcca ttgaactttt cgatagacag 540 ggtgctgaac ctgcctttct cttcgggttg gagctgatta tttgtggtct cgaaaaacag 600 ctgaaatgtg aaagtggtgg ccctactgac gccctcgatg atttcgacct ggatatgctg 660 ccagccgatg cacttgatga tttcgatttg gatatgcttc cagccgacgc actggacgac 720 ttcgatttgg acatgcttcc cggttaa 747 <210> 13 <211> 248 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 13 Met Ser Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu 1 5 10 15 Leu Asn Gly Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln 20 25 30 Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys 35 40 45 Arg Ala Leu Leu Asp Ala Leu Pro Ile Glu Met Leu Asp Arg His His 50 55 60 Thr His Ser Cys Pro Leu Glu Val Glu Ser Trp Gln Asp Phe Leu Arg 65 70 75 80 Asn Asn Ala Lys Ser Tyr Arg Cys Ala Leu Leu Ser His Arg Asp Gly 85 90 95 Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr 100 105 110 Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu 115 120 125 Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys 130 135 140 Val Leu Glu Glu Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr 145 150 155 160 Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Lys Gln Ala Ile Glu Leu 165 170 175 Phe Asp Arg Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu 180 185 190 Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro 195 200 205 Thr Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala 210 215 220 Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp 225 230 235 240 Phe Asp Leu Asp Met Leu Pro Gly 245 <210> 14 <211> 1050 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 14 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa 60 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg 120 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc 180 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc 240 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg 300 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg 360 acgtccttct gccgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg 420 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc 480 ctttgggccg cctccccgca tcgataccgt cgacctcgag acctagaaaa acatggagca 540 atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac 780. tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga actacacacc agggccaggg atcagatatc cactgacctt tggatggtgc 840 tacaagctag taccagttga gcaagagaag gtagaagaag ccaatgaagg agagaacacc 900 cgcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga agtattagag 960 tggaggtttg acagccgcct agcatttcat cacatggccc gagagctgca tccggactgt 1020 actgggtctc tctggttaga ccagatctga 1050 <210> 15 <211> 349 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 15 Met Pro Leu Tyr His Ala Ile Ala Ser Arg Met Ala Phe Ile Phe Ser 1 5 10 15 Ser Leu Tyr Lys Ser Trp Leu Leu Ser Leu Tyr Glu Glu Leu Trp Pro 20 25 30 Val Val Arg Gln Arg Gly Val Val Cys Thr Val Phe Ala Asp Ala Thr 35 40 45 Pro Thr Gly Trp Gly Ile Ala Thr Thr Cys Gln Leu Leu Ser Gly Thr 50 55 60 Phe Ala Phe Pro Leu Pro Ile Ala Thr Ala Glu Leu Ile Ala Ala Cys 65 70 75 80 Leu Ala Arg Cys Trp Thr Gly Ala Arg Leu Leu Gly Thr Asp Asn Ser 85 90 95 Val Val Leu Ser Gly Lys Ser Ser Ser Phe Pro Trp Leu Leu Ala Cys 100 105 110 Val Ala Thr Trp Ile Leu Arg Gly Thr Ser Phe Cys Tyr Val Pro Ser 115 120 125 Ala Leu Asn Pro Ala Asp Leu Pro Ser Arg Gly Leu Leu Pro Ala Leu 130 135 140 Arg Pro Leu Pro Arg Leu Arg Leu Arg Pro Gln Thr Ser Arg Ile Ser 145 150 155 160 Leu Trp Ala Ala Ser Pro His Arg Tyr Arg Arg Pro Arg Asp Leu Glu 165 170 175 Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala Thr Asn Ala Asp 180 185 190 Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu Glu Val Gly Phe Pro Val 195 200 205 Thr Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Ala Ala Val Asp 210 215 220 Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile His 225 230 235 240 Ser Gln Arg Arg Gln Asp Ile Leu Asp Leu Trp Ile Tyr His Thr Gln 245 250 255 Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Ile Arg 260 265 270 Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys Leu Val Pro Val Glu Gln 275 280 285 Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn Thr Arg Leu Leu His 290 295 300 Pro Val Ser Leu His Gly Met Asp Asp Pro Glu Arg Glu Val Leu Glu 305 310 315 320 Trp Arg Phe Asp Ser Arg Leu Ala Phe His His Met Ala Arg Glu Leu 325 330 335 His Pro Asp Cys Thr Gly Ser Leu Trp Leu Asp Gln Ile 340 345 <210> 16 <211> 7408 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 16 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 60 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 120 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 180 atgcctgtag taatggtaac aacgttgcgc aaactattaa ctggcgaact acttactcta 240 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 300 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 360 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 420 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 480 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 540 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 600 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 660 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 720 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 780 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 840 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 900 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 960 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 1020 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 1080 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 1140 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 1200 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 1260 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 1320 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 1380 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 1440 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 1500 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 1560 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 1620 1680 tttaattaag gccttaatta ggctgcgcgc tcgctcgctc actgaggccg cccgggcaaa 1740 gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga 1800 gggagtggcc aactccatca ctaggggttc cttgtagtta atgattaacc cgccatgcta 1860 cttatctacg tagccatgct ctaggaagat cggaattctt tactccctat cagtgataga 1920 gaacgtatga agagtttact ccctatcagt gatagagaac gtatgcagac tttactccct 1980 atcagtgata gagaacgtat aaggagttta ctccctatca gtgatagaga acgtatgacc 2040 agtttactcc ctatcagtga tagagaacgt atctacagtt tactccctat cagtgataga 2100 2160 cggtgggcgc ctataaaagc agagctcgtt tagtgaaccg tcagatcgcc tggagcaatt 2220 ccacaacact tttgtcttat accaactttc cgtaccactt cctaccctcg taaagcggcc 2280 gcgccaccat ggctggacac ctggcttcag acttcgcctt ctcaccccca ccaggtgggg 2340 gtgatgggtc agcagggctg gagccgggct gggtggatcc tcgaacctgg ctaagcttc 2400 aagggcctcc aggtgggcct ggaatcggac caggctcaga ggtattgggg atctccccat 2460 gtccgcccgc atacgagttc tgcggaggga tggcatactg tggacctcag gttggactgg 2520 gcctagtccc ccaagttggc gtggagactt tgcagcctga gggccaggca ggagcacgag 2580 tggaaagcaa ctcagaggga acctcctctg agccctgtgc cgaccgcccc aatgccgtga 2640 agttggagaa ggtggaacca actcccgagg agtcccagga catgaaagcc ctgcagaagg 2700 agctagaaca gtttgccaag ctgctgaagc agaagaggat caccttgggg tacacccagg 2760 ccgacgtggg gctcaccctg ggcgttctct ttggaaaggt gttcagccag accaccatct 2820 gtcgcttcga ggccttgcag ctcagcctta agaacatgtg taagctgcgg cccctgctgg 2880 agaagtgggt ggaggaagcc gacaacaatg agaaccttca ggagatatgc aaatcggaga 2940 ccctggtgca ggcccggaag agaaagcgaa ctagcattga gaaccgtgtg aggtggagtc 3000 tggagaccat gtttctgaag tgcccgaagc cctccctaca gcagatcact cacatcgcca atcagcttgg gctagagaag gatgtggttc gagtatggtt ctgtaaccgg cgccagaagg gcaaaagatc aagtattgag tattcccaac gagaagagta tgaggctaca gggacacctt tcccaggggg ggctgtatcc tttcctctgc ccccaggtcc ccctttggc accccaggct 3240 atggaagccc ccacttcacc acactctact cagtcccttt tcctgagggc gaggcctttc 3300. cctctgttcc cgtcactgct ctgggctctc ccatgcattc aaacgctagc ggcagcggcg 3360 cccgaactt ctctctgtta aagcaagcag gagatgttga agaaaacccc gggcctgcat gcatgtataa catgatggag acggagctga agccgccggg cccgcagcaa gcttcggggg 3480 gcggcggcgg aggaggcac gccacggcgg cggcgaccgg cggcaaccag aagaacagcc 3540 cggaccgcgt caagaggccc atgaacgcct tcatggtatg gtcccggggg cagcggcgta 3600. agatggccca ggagaacccc aagatgcaca actcggagat cagcaagcgc ctggggcgcgg agtggaaact tttgtccgag accgagaagc ggccgttcat cgacgaggcc aagcggctgc 3720. gcgctctgca catgaaggag cacccggatt ataaataccg gccgcggcgg aaaaccaaga 3780 cgctcatgaa gaaggataag tacacgcttc ccggaggctt gctggccccc ggcgggaaca 3840 gcatggcgag cggggttggg gtgggcgccg gcctgggtgc gggcgtgaac cagcgcatgg 3900 acagctacgc gcacatgaac ggctggagca acggcagcta cagcatgatg caggagcagc 3960 tgggctaccc gcagcacccg ggcctcaacg ctcacggcgc ggcacagatg caaccgatgc 4020 accgctacga cgtcagcgcc ctgcagtaca actccatgac cagctcgcag acctacatga 4080 acggctcgcc cacctacagc atgtcctact cgcagcaggg cacccccggt atggcgctgg 4140 gctccatggg ctctgtggtc aagtccgagg ccagctccag cccccccgtg gttacctctt 4200 cctcccactc cagggcgccc tgccaggccg gggacctccg ggacatgatc agcatgtacc 4260 tccccggcgc cgaggtgccg gagcccgctg cgcccagtag actgcacatg gcccagcact 4320 accagagcgg cccggtgccc ggcacggcca ttaacggcac actgcccctg tcgcacatgg 4380 catgcggctc cggcgagggc aggggaagtc ttctaacatg cggggacgtg gaggaaaatc 4440 ccggcccact cgagatgagg cagccacctg gcgagtctga catggctgtc agcgacgctc 4500 tgctcccgtc cttctccacg ttcgcgtccg gcccggcggg aagggagaag acactgcgtc 4560 cagcaggtgc cccgactaac cgttggcgtg aggaactctc tcacatgaag cgacttcccc 4620 cacttcccgg ccgcccctac gacctggcgg cgacggtggc cacagacctg gagagtggcg 4680 gagctggtgc agcttgcagc agtaacaacc cggccctcct agcccggagg gagaccgagg 4740 agttcaacga cctcctggac ctagacttta tcctttccaa ctcgctaacc caccaggaat 4800 cggtggccgc caccgtgacc acctcggcgt cagcttcatc ctcgtcttcc ccagcgagca 4860 gcggccctgc cagcgcgccc tccacctgca gcttcagcta tccgatccgg gccgggggtg 4920 acccgggcgt ggctgccagc aacacaggtg gagggctcct ctacagccga gaatctgcgc 4980 cacctcccac ggcccccttc aacctggcgg acatcaatga cgtgagcccc tcgggcggct 5040 tcgtggctga gctcctgcgg ccggagttgg acccagtata cattccgcca cagcagcctc 5100 agccgccagg tggcgggctg atgggcaagt ttgtgctgaa ggcgtctctg accacccctg 5160 gcagcgagta cagcagccct tcggtcatca gtgttagcaa aggaagccca gacggcagcc 5220 accccgtggt agtggcgccc tacagcggtg gcccgccgcg catgtgcccc aagattaagc 5280 aagaggcggt cccgtcctgc acggtcagcc ggtccctaga ggcccatttg agcgctggac 5340 cccagctcag caacggccac cggcccaaca cacagactt ccccctgggg cggcagctcc 5400 ccaccaggac tacccctaca ctgagtcccg aggaactgct gaacagcagg gactgtcacc 5460 ctggcctgcc tcttccccca ggattccatc cccatccggg gcccaactac cctcctttcc 5520 tgccagacca gatgcagtca caagtcccct ctctccatta tcaagagctc atgccaccgg 5580 gttcctgcct gccagaggag cccaagccaa agagggggaag aaggtcgtgg ccccggaaaa 5640 gaacgccac ccacacttgt gactatgcag gctgtggcaa aacctatacc aagagttctc 5700 atctcaaggc acacctgcga actcacacag gcgagaaacc ttaccactgt gactgggacg 5760 gctgtgggtg gaaattcgcc cgctccgatg aactgaccag gcactaccgc aaacacag 5820 ggcaccggcc ctttcagtgc cagaagtgcg acagggcctt ttccaggtcg gaccaccttg 5880 ccttacacat gaagaggcac taaatgacta gtgcgcgcag cggccgacca tggcccaact 5940 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 6000 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc 6060 atgtctggat ctcggtaccg gatccaaatt cccgataagg atcttcctag agcatggcta 6120 cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag tgatggagtt 6180 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg 6240 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagcc ttaattaacc 6300 taattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact 6360 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac 6420 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg 6480 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 6540 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 6600 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 6660 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac 6720 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 6780 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat 6840 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa 6900 aatattaacg tttataattt caggtggcat ctttcgggga aatgtgcgcg gaacccctat 6960 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 7020 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct 7080 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 7140 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa 7200 tagtggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt 7260 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg 7320 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca 7380 tcttacggat ggcatgacag taagagaa 7408 <210> 17 <211> 5657 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 17 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 60 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 120 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 180 atgcctgtag taatggtaac aacgttgcgc aaactattaa ctggcgaact acttactcta 240 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 300 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 360 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 420 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 480 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 540 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 600 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 660 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 720 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 780 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 840 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 900 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 960 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 1020 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 1080 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 1140 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 1200 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 1260 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 1320 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 1380 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 1440 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 1500 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 1560 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 1620 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaga 1680 tttaattaag gccttaatta ggctgcgcgc tcgctcgctc actgaggccg cccgggcaaa 1740 gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga 1800 gggagtggcc aactccatca ctaggggttc cttgtagtta atgattaacc cgccatgcta 1860 cttatctacg tagccatgct ctaggaagat cggaattcct gatctggcct ccgcgccggg 1920 ttttggcgcc tcccgcgggc gcccccctcc tcacggcgag cgctgccacg tcagacgaag 1980 ggcgcagcga gcgtcctgat ccttccgccc ggacgctcag gacagcggcc cgctgctcat 2040 aagactcggc cttagaaccc cagtatcagc agaaggacat tttaggacgg gacttgggtg 2100 actctagggc actggttttc tttccagaga gcggaacagg cgaggaaaag tagtcccttc 2160 tcggcgattc tgcggaggga tctccgtggg gcggtgaacg ccgatgatta tataaggacg 2220 cgccgggtgt ggcacagcta gttccgtcgc agccgggatt tgggtcgcgg ttcttgtttg 2280 tggatcgctg tgatcgtcac ttggtgagta gcgggctgct gggctggccg gggctttcgt 2340 ggccgccggg ccgctcggtg ggacggaagc gtgtggagag accgccaagg gctgtagtct 2400 gggtccgcga gcaaggttgc cctgaactgg gggttggggg gagcgcagca aaatggcggc 2460 tgttcccgag tcttgaatgg aagacgcttg tgaggcgggc tgtgaggtcg ttgaaacaag 2520 gtggggggca tggtgggcgg caagaaccca aggtcttgag gccttcgcta atgcgggaaa 2580 gctcttattc gggtgagatg ggctggggca ccatctgggg accctgacgt gaagtttgtc 2640 actgactgga gaactcggtt tgtcgtctgt tgcgggggcg gcagttatgc ggtgccgttg 2700 ggcagtgcac ccgtaccttt gggagcgcgc gcctcgtcgt gtcgtgacgt cacccgttct 2760 gttggcttat aatgcagggt ggggccacct gccggtaggt gtgcggtagg cttttctccg 2820 tcgcaggacg cagggttcgg gcctagggta ggctctcctg aatcgacagg cgccggacct 2880 ctggtgaggg gagggataag tgaggcgtca gtttctttgg tcggttttat gtacctatct 2940 tcttaagtag ctgaagctcc ggttttgaac tatgcgctcg gggttggcga gtgtgttttg 3000 tgaagttttt taggcacctt ttgaaatgta atcatttggg tcaatatgta attttcagtg 3060 ttagactagt aaattgtccg ctaaattctg gccgtttttg gcttttttgt tagacgaagc 3120 ggccgcatta aacgccacca tgtcccgctt ggataagagc aaggtaataa atagcgcact 3180 cgaactcctc aacggcgtgg gcatcgaagg tctgactact cgaaagctcg cccagaaatt 3240 gggtgtggag caacctacat tgtattggca tgtcaagaac aaaagagccc tgctggacgc 3300 tcttcctatt gaaatgcttg acaggcatca cactcattcc tgcccccttg aggtcgagag 3360 ttggcaagat ttctccgaa acaatgcaaa gtcctaccgc tgcgcacttt tgtcccatag 3420 ggatggagca aaagtgcacc tgggaaccag gccaacagag aaacaatacg agactctcga 3480 gaaccagttg gctttcttgt gccaacaggg gttctcactt gaaaatgccc tttacgcact 3540 gtcagccgtt ggacatttta ccctggggtg cgttcttgag gagcaagaac atcaggttgc 3600 taaggaggag cgcgagactc caaccactga ttctatgcca cctttgctga aacaggccat 3660 tgaacttttc gatagacagg gtgctgaacc tgccttctc ttcgggttgg agctgattat 3720 ttgtggtctc gaaaaacagc tgaaatgtga aagtggtggc cctactgacg ccctcgatga 3780 ttcgacctg gatatgctgc cagccgatgc acttgatgat ttcgatttgg atatgcttc 3840 agccgacgca ctggacgact tcgatttgga catgcttccc ggttaaacta gtctagcaat 3900 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct 3960 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg 4020 gctttcattt tctcctcctt gtataaatcc tggttagttc ttgccacggc ggaactcatc 4080 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg 4140 gtgtttattt gtgaaatttg tgatgctatt gctttatttg taaccattct agctttattt 4200 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta 4260 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt 4320 aaagcgggg atccaaattc ccgataagga tcttcctaga gcatggctac gtagataagt 4380 agcatggcgg gttaatcatt aactacaagg aacccctagt gatggagttg gccactccct 4440 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct 4500 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagcct taattaacct aattcactgg 4560 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg 4620 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt 4680 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg 4740 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 4800 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 4860 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 4920 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 4980 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacacac 5040 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 5100 ggttaaaaaa tgagctgatt taaaaaaat ttaacggaa ttttaacaaa atattaacgt 5160 ttataatttc aggtggcatc tttcgggggaa atgtgcgcgg aacccctatt tgtttatttt 5220 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 5280 aatattgaaa aaagaagagt atgagtattc aacatttccg tgtcgccctt attcccttt 5340 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 5400 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaat agtggtaaga 5460 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 5520 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 5580 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 5640 gcatgacagt aagagaa 5657 <210> 18 <211> 1215 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 18 gatctggcct ccgcgccggg ttttggcgcc tcccgcgggc gcccccctcc tcacggcgag 60 cgctgccacg tcagacgaag ggcgcagcga gcgtcctgat ccttccgccc ggacgctcag 120 gacagcggcc cgctgctcat aagactcggc cttagaaccc cagtatcagc agaaggacat 180 tttaggacgg gacttgggtg actctagggc actggttttc tttccagaga gcggaacagg 240 cgaggaaaag tagtcccttc tcggcgattc tgcggaggga tctccgtggg gcggtgaacg 300 ccgatgatta tataaggacg cgccgggtgt ggcacagcta gttccgtcgc agccgggatt 360 tgggtcgcgg ttcttgtttg tggatcgctg tgatcgtcac ttggtgagta gcgggctgct 420 gggctggccg gggctttcgt ggccgccggg ccgctcggtg ggacggaagc gtgtggagag 480 accgccaagg gctgtagtct gggtccgcga gcaaggttgc cctgaactgg gggttggggg 540 gagcgcagca aaatggcggc tgttcccgag tcttgaatgg aagacgcttg tgaggcgggc 600 tgtgaggtcg ttgaaacaag gtggggggca tggtgggcgg caagaaccca aggtcttgag 660 gccttcgcta atgcgggaaa gctcttattc gggtgagatg ggctggggca ccatctgggg 720 accctgacgt gaagtttgtc actgactgga gaactcggtt tgtcgtctgt tgcgggggcg 780 gcagttatgc ggtgccgttg ggcagtgcac ccgtaccttt gggagcgcgc gcctcgtcgt 840 gtcgtgacgt cacccgttct gttggcttat aatgcagggt ggggccacct gccggtaggt 900 gtgcggtagg cttttctccg tcgcaggacg cagggttcgg gcctagggta ggctctcctg 960 aatcgacagg cgccggacct ctggtgaggg gagggataag tgaggcgtca gtttctttgg 1020 tcggttttat gtacctatct tcttaagtag ctgaagctcc ggttttgaac tatgcgctcg 1080 gggttggcga gtgtgttttg tgaagttttt taggcacctt ttgaaatgta atcatttggg 1140 tcaatatgta attttcagtg ttagactagt aaattgtccg ctaaattctg gccgtttttg 1200 gcttttttgt tagac 1215 <210> 19 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 19 tccctatcag tgatagaga 19 <210> 20 <211> 68 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 20 gctttaggcg tgtacggtgg gcgcctataa aagcagagct cgtttagtga accgtcagat 60 cgcctgga 68 <210> twenty one <211> 248 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> twenty one aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120 atggctttca ttttctcctc cttgtataaa tcctggttag ttcttgccac ggcggaactc 180 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc 240 gtggtgtt 248 <210> twenty two <211> 141 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 22 ccttaattag gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120 actccatcac taggggttcc t 141 <210> 23 <211> 438 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 23 aattcgtaca cgcctacctc gacccatcaa gtgccacctg acgtctccct atcagtgata 60 gagaagtcga cacgtctcga gctccctatc agtgatagag aaggtacgtc tagaacgtct 120 ccctatcagt gatagagaag tcgacacgtc tcgagctccc tatcagtgat agagaaggta 180 cgtctagaac gtctccctat cagtgataga gaagtcgaca cgtctcgagc tccctatcag 240 tgatagagaa ggtacgtcta gaacgtctcc ctatcagtga tagagaagtc gacacgtctc 300 gagctcccta tcagtgatag agaaggtacc ccctatataa gcagagctcg tttagtgaac 360 cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac 420 cgatccagcc tggatcgc 438 <210> 24 <211> 315 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 24 gagtttactc cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag 60 agaacgatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct 120 atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga acgtatgtcg 180 agtttatccc tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag 240 aacgtatgtc gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg 300 aaccgtcaga tcgcc 315 <210> 25 <211> 618 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 25 atggctagat tagataaaag taaagtgatt aacagcgcat tagagctgct taatgaggtc 60 ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc taggtgtaga gcagcctaca 120 ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg ccttagccat tgagatgtta 180 gataggcacc atactcactt ttgcccttta gaaggggaaa gctggcaaga ttttttacgt 240 aataacgcta aaagttttag atgtgcttta ctaagtcatc gcgatggagc aaaagtacat 300 ttaggtacac ggcctacaga aaaacagtat gaaactctcg aaaatcaatt agccttttta 360 tgccaacaag gtttttcact agagaatgca ttatatgcac tcagcgctgt ggggcatttt 420 actttaggtt gcgtattgga agatcaagag catcaagtcg ctaaagaaga aagggaaaca 480 cctactactg atagtatgcc gccattatta cgacaagcta tcgaattatt tgatcaccaa 540 ggtgcagagc cagccttctt attcggcctt gaattgatca tatgcggatt agaaaaacaa 600 cttaaatgtg aaagtggg 618 <210> 26 <211> 206 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 26 Met Ala Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu 1 5 10 15 Leu Asn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln 20 25 30 Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys 35 40 45 Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His 50 55 60 Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg 65 70 75 80 Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly 85 90 95 Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr 100 105 110 Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu 115 120 125 Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys 130 135 140 Val Leu Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr 145 150 155 160 Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu 165 170 175 Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu 180 185 190 Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly 195 200 205 <210> 27 <211> 1008 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <400> 27 atggctagat tagataaaag taaagtgatt aacagcgcat tagagctgct taatgaggtc 60 ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc taggtgtaga gcagcctaca 120 ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg ccttagccat tgagatgtta 180 gataggcacc atactcactt ttgcccttta gaaggggaaa gctggcaaga ttttttacgt 240 aataacgcta aaagttttag atgtgcttta ctaagtcatc gcgatggagc aaaagtacat 300 ttaggtacac ggcctacaga aaaacagtat gaaactctcg aaaatcaatt agccttttta 360 tgccaacaag gtttttcact agagaatgca ttatatgcac tcagcgctgt ggggcatttt 420 actttaggtt gcgtattgga agatcaagag catcaagtcg ctaaagaaga aagggaaaca 480 cctactactg atagtatgcc gccattatta cgacaagcta tcgaattatt tgatcaccaa 540 ggtgcagagc cagccttctt attcggcctt gaattgatca tatgcggatt agaaaaacaa 600 cttaaatgtg aaagtgggtc gccaaaaaag aagagaaagg tcgacggcgg tggtgctttg 660 tctcctcagc actctgctgt cactcaagga agtatcatca agaacaagga gggcatggat 720 gctaagtcac taactgcctg gtcccggaca ctggtgacct tcaaggatgt atttgtggac 780 ttcaccaggg aggagtggaa gctgctggac actgctcagc agatcgtgta cagaaatgtg 840 atgctggaga actataagaa cctggtttcc ttgggttatc agcttactaa gccagatgtg 900 atcctccggt tggagaaggg agaagagccc tggctggtgg agagagaaat tcaccaagag 960 acccatcctg attcagagac tgcatttgaa atcaaatcat cagtttaa 1008 <210> 28 <211> 335 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 28 Met Ala Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu 1 5 10 15 Leu Asn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln 20 25 30 Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys 35 40 45 Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His 50 55 60 Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg 65 70 75 80 Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly 85 90 95 Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr 100 105 110 Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu 115 120 125 Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys 130 135 140 Val Leu Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr 145 150 155 160 Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu 165 170 175 Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu 180 185 190 Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Ser Pro 195 200 205 Lys Lys Lys Arg Lys Val Asp Gly Gly Gly Ala Leu Ser Pro Gln His 210 215 220 Ser Ala Val Thr Gln Gly Ser Ile Ile Lys Asn Lys Glu Gly Met Asp 225 230 235 240 Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu Val Thr Phe Lys Asp 245 250 255 Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys Leu Leu Asp Thr Ala 260 265 270 Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu Asn Tyr Lys Asn Leu 275 280 285 Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp Val Ile Leu Arg Leu 290 295 300 Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg Glu Ile His Gln Glu 305 310 315 320 Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile Lys Ser Ser Val 325 330 335 <210> 29 <211> 977 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 29 accttgcttc ctagctgggc ctttccttct cctctataaa taccagctct ggtatttcgc 60 cttggcagct gttgctgcta gggagacggc tggcttgaca tgcatctcct gacaaaacac 120 aaacccgtgg tgtgagtggg tgtgggcggt gtgagtaggg ggatgaatca gagagggggc 180 gagggagaca ggggcgcagg agtcaggcaa aggcgatgcg ggggtgcgac tacacgcagt 240 tggaaacagt cgtcagaaga ttctggaaac tatcttgctg gctataaact tgagggaagc 300 agaaggccaa cattcctccc aagggaaact gaggctcaga gttaaaaccc aggtatcagt 360 gatatgcatg tgccccggcc agggtcactc tctgactaac cggtacctac cctacaggcc 420 tacctagaga ctcttttgaa aggatggtag agacctgtcc gggctttgcc cacagtcgtt 480 ggaaacctca gcattttcta ggcaacttgt gcgaataaaa cacttcgggg gtccttcttg 540 ttcattccaa taacctaaaa cctctcctcg gagaaaatag ggggcctcaa acaaacgaaa 600 ttctctagcc cgctttcccc aggataaggc aggcatccaa atggaaaaaa aggggccggc 660 cgggggtctc ctgtcagctc cttgccctgt gaaacccagc aggcctgcct gtcttctgtc 720 ctcttggggc tgtccagggg cgcaggcctc ttgcggggga gctggcctcc ccgccccctc 780 gcctgtggcc gcccttttcc tggcaggaca gagggatcct gcagctgtca ggggaggggc 840 gccggggggt gatgtcagga gggctacaaa tagtgcagac agctaagggg ctccgtcacc 900 catcttcaca tccactccag ccggctgccc gcccgctgcc tcctctgtgc gtccgcccag 960 ccagcctcgt ccacgcc 977 <210> 30 <211> 5428 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <400> 30 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 60 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 120 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 180 atgcctgtag taatggtaac aacgttgcgc aaactattaa ctggcgaact acttactcta 240 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 300 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 360 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 420 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 480 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 540 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 600 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 660 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 720 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 780 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 840 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 900 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 960 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 1020 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 1080 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 1140 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 1200 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 1260 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 1320 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 1380 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 1440 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 1500 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 1560 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 1620 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaga 1680 tttaattaag gccttaatta ggctgcgcgc tcgctcgctc actgaggccg cccgggcaaa 1740 gccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga 1800 gggagtggcc aactccatca ctaggggttc cttgtagtta atgattaacc cgccatgcta 1860 cttatctacg tagccatgct ctaggaagat cggaattcct agatctacct tgcttcctag 1920 ctgggcctt ccttctcctc tataaatacc agctctggta ttcgccttg gcagctgttg 1980 ctgctaggga gacggctggc ttgacatgca tctcctgaca aaacacaaac ccgtggtgtg 2040 agtgggtgtg ggcggtgtga gtagggggat gaatcagaga gggggcgagg gagacagggg 2100 cgcaggagtc aggcaaaggc gatgcggggg tgcgactaca cgcagttgga aacgtcgtc 2160 agaagattct ggaaactatc ttgctggcta taaacttgag ggaagcagaa ggccaacatt 2220 cctcccaagg gaaactgagg ctcagagtta aaacccaggt atcagtgata tgcatgtgcc 2280 ccggccaggg tcactctctg actaaccggt acctacccta caggcctacc tagagactct 2340 tttgaaagga tggtagagac ctgtccgggc ttgcccaca gtcgttggaa acctcagcat 2400 tttctaggca acttgtgcga ataaaacact tcgggggtcc ttcttgttca ttccaataac 2460 ctaaaacctc tcctcggaga aaataggggg cctcaaacaa acgaaattct ctagcccgct 2520 ttccccagga taaggcaggc atccaaatgg aaaaaaaggg gccggccggg ggtctcctgt 2580 cagctccttg ccctgtgaaa cccagcaggc ctgcctgtct tctgtcctct tggggctgtc 2640 caggggcgca ggcctcttgc gggggagctg gcctccccgc cccctcgcct gtggccgccc 2700 ttttcctggc aggacagagg gatcctgcag ctgtcagggg aggggcgccg gggggtgatg 2760 tcaggagggc tacaaatagt gcagacagct aaggggctcc gtcacccatc ttcacatcca 2820 ctccagccgg ctgcccgccc gctgcctcct ctgtgcgtcc gcccagccag cctcgtccac 2880 gccaagcttg cggccgcatt aaacgccacc atgtcccgct tggataagag caaggtaata 2940 aatagcgcac tcgaactcct caacggcgtg ggcatcgaag gtctgactac tcgaaagctc 3000 gcccagaaat tgggtgtgga gcaacctaca ttgtattggc atgtcaagaa caaaagagcc 3060 ctgctggacg ctcttcctat tgaaatgctt gacaggcatc acactcattc ctgccccctt 3120 gaggtcgaga gttggcaaga ttttctccga aacaatgcaa agtcctaccg ctgcgcactt 3180 ttgtcccata gggatggagc aaaagtgcac ctgggaacca ggccaacaga gaaacaatac 3240 gagactctcg agaaccagtt ggctttcttg tgccaacagg ggttctcact tgaaaatgcc 3300 ctttacgcac tgtcagccgt tggacatttt accctggggt gcgttcttga ggagcaagaa 3360 catcaggttg ctaaggagga gcgcgagact ccaaccactg attctatgcc acctttgctg 3420 aaacaggcca ttgaactttt cgatagacag ggtgctgaac ctgcctttct cttcgggttg 3480 gagctgatta tttgtggtct cgaaaaacag ctgaaatgtg aaagtggtgg ccctactgac 3540 gccctcgatg atttcgacct ggatatgctg ccagccgatg cacttgatga tttcgatttg 3600 gatatgcttc cagccgacgc actggacgac ttcgatttgg acatgcttcc cggttaaact 3660 agtctagcaa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact 3720 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg 3780 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttagtt cttgccacgg 3840 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg 3900 acaattccgt ggtgtttatt tgtgaaattt gtgatgctat tgctttattt gtaaccattc 3960 tagctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa 4020 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggagatgtg 4080 ggaggttttt taaagcgggg gatccaaatt cccgataagg atcttcctag agcatggcta 4140 cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag tgatggagtt 4200 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg 4260 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagcc ttaattaacc 4320 taattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact 4380 taatcgcctt gcagcacatc c...

Claims

1. A mutant rtTA, wherein the mutant rtTA is shown in SEQ ID NO:

13.

2. An engineered nucleic acid comprising a first nucleic acid sequence encoding the mutant rtTA according to claim 1.

3. The engineered nucleic acid of claim 2, further comprising a promoter operatively linked to the first nucleic acid sequence encoding the mutant rtTA of claim 1.

4. The engineered nucleic acid according to claim 2 or 3, wherein the engineered nucleic acid comprises RNA and / or DNA.

5. The engineered nucleic acid according to any one of claims 2-4, wherein the engineered nucleic acid is an expression vector.

6. The engineered nucleic acid according to any one of claims 2-5, wherein the first nucleic acid is codon-optimized.

7. The engineered nucleic acid according to any one of claims 2-6, wherein the first nucleic acid is as shown in SEQ ID NO:

12.

8. The engineered nucleic acid according to any one of claims 3-7, wherein the promoter is a constitutive promoter.

9. The engineered nucleic acid according to claim 8, wherein the constitutive promoter is selected from the group consisting of: CP1, CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, Ac5, polyhedrone protein, TEF1, GDS, CaM3 5S, Ubi, H1 and U6 promoters.

10. The engineered nucleic acid according to any one of claims 3-7, wherein the promoter is a tissue-specific promoter.

11. The engineered nucleic acid of claim 10, wherein the tissue-specific promoter is specific to one or more of the following tissues: ear, nose, mouth, bone, lung, mammary gland, breast, pancreas, stomach, esophagus, muscle, liver, blood vessels, skin, hair, heart, brain, nerve, kidney, testis, prostate, penis, cloaca, fin, ovary, or intestine.

12. The engineered nucleic acid according to claim 11, wherein the oral-specific tissue-specific promoter is specific to the gingiva or tooth root.

13. The engineered nucleic acid according to claim 11, wherein the muscle-specific tissue-specific promoter is myocardial-specific.

14. The engineered nucleic acid according to any one of claims 2-13, further comprising a second nucleic acid sequence encoding a tetracycline repressor.

15. The engineered nucleic acid according to claim 14, wherein the tetracycline repressor is tetRKRAB.

16. The engineered nucleic acid according to claim 14 or 15, wherein the promoter operatively linked to the first nucleic acid sequence is also operatively linked to the second nucleic acid sequence.

17. The engineered nucleic acid according to any one of claims 2-16, further comprising a spacer sequence.

18. The engineered nucleic acid of claim 17, wherein the spacer sequence encodes an internal ribosome entry site (IRES) or a self-cleaving peptide.

19. The engineered nucleic acid according to claim 18, wherein the self-cleaving peptide is a 2A peptide.

20. The engineered nucleic acid according to claim 19, wherein the 2A peptide comprises the sequence shown in SEQ ID NO:

9.

21. The engineered nucleic acid according to any one of claims 17-20, wherein the spacer sequence is located between the first nucleic acid sequence and the second nucleic acid sequence.

22. The engineered nucleic acid according to any one of claims 2-21, wherein the engineered nucleic acid is a viral vector or a non-viral vector.

23. The engineered nucleic acid according to claim 22, wherein the non-viral vector is plasmid DNA or RNA.

24. The engineered nucleic acid according to claim 22, wherein the viral vector is selected from the group consisting of lentiviruses, adenoviruses, alphaviruses, vacciniaviruses, herpesviruses, and adeno-associated virus (AAV) vectors.

25. The engineered nucleic acid according to claim 22, wherein the viral vector is a retroviral vector.

26. The engineered nucleic acid according to claim 22 or 24, wherein the viral vector comprises an inverted terminal repeat (ITR) flanking a first nucleic acid sequence encoding the mutant rtTA.

27. The engineered nucleic acid according to claim 26, wherein the ITR consists of the sequence shown in SEQ ID NO:

22.

28. The engineered nucleic acid according to any one of claims 24, 26 and 27, wherein the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10 vector.

29. The engineered nucleic acid according to any one of claims 2-28, further comprising the marmot hepatitis posttranscriptional regulatory element 3 (WPRE3) sequence.

30. The engineered nucleic acid according to claim 29, wherein the WPRE3 sequence is shown in SEQ ID NO:

21.

31. The engineered nucleic acid according to any one of claims 2-30, further comprising a terminator sequence.

32. The engineered nucleic acid according to claim 31, wherein the terminator sequence comprises the sequence shown in SEQ ID NO:

8.

33. The engineered nucleic acid according to any one of claims 2-11, 22-24 and 26-32, wherein the engineered nucleic acid is as shown in SEQ ID NO: 17 or SEQ ID NO:

30.

34. The engineered nucleic acid according to any one of claims 2-33, wherein the engineered nucleic acid further comprises a first transgenic sequence operatively linked to a first inducible promoter containing a tetracycline response element (TRE).

35. The engineered nucleic acid of claim 34, wherein the TRE comprises at least one Tet-O sequence as shown in SEQ ID NO:

19.

36. The engineered nucleic acid according to claim 34 or 35, wherein the first transgenic sequence is a therapeutic sequence, a gene-targeting nucleic acid, or a nucleic acid encoding a protein.

37. The engineered nucleic acid according to any one of claims 34-36, wherein the inducible promoter containing TRE is the TRE3G promoter.

38. The engineered nucleic acid according to claim 37, wherein the TRE3G promoter consists of the sequence shown in SEQ ID NO:

7.

39. A recombinant virus comprising an engineered nucleic acid according to any one of claims 2-38.

40. The recombinant virus according to claim 39, wherein the engineered nucleic acid is a viral vector.

41. The recombinant virus of claim 40, wherein the viral vector is selected from the group consisting of lentiviruses, adenoviruses, alphaviruses, vaccinia virus, herpesviruses, and adeno-associated virus (AAV) vectors.

42. The recombinant virus according to claim 40, wherein the viral vector is a retroviral vector.

43. A composition comprising (i) the mutant rtTA according to claim 1; an engineered nucleic acid according to any one of claims 2-38; or a recombinant virus according to any one of claims 39-42; and (ii) a pharmaceutically acceptable excipient.

44. The composition of claim 43, further comprising a second engineered nucleic acid, wherein the second engineered nucleic acid comprises a second inducible promoter operatively linked to a second transgenic sequence, the second inducible promoter comprising a tetracycline response element (TRE).

45. The composition of claim 44, wherein the TRE of the second inducible promoter comprises at least one Tet-O sequence as shown in SEQ ID NO:

19.

46. ​​The composition according to claim 44 or 45, wherein the second engineered nucleic acid is a second expression vector.

47. The composition according to any one of claims 44-46, wherein the second inducible promoter is a TRE3G promoter composed of the nucleic acid sequence shown in SEQ ID NO:

7.

48. The composition according to any one of claims 44-47, wherein the second engineered nucleic acid is a viral vector.

49. The composition of claim 48, wherein the second engineered nucleic acid is a viral vector selected from the group consisting of lentivirus, adenovirus, alphavirus, vaccinia virus, herpesvirus, and adeno-associated virus (AAV) vector.

50. The composition according to claim 48, wherein the viral vector is a retroviral vector.

51. The composition of claim 49, wherein the AAV carrier is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 carrier.

52. The composition according to any one of claims 44-51, wherein the second engineered nucleic acid further comprises a marmot hepatitis posttranscriptional regulatory element 3 (WPRE3) sequence.

53. The composition of claim 52, wherein the WPRE3 sequence of the second engineered nucleic acid is shown in SEQ ID NO:

21.

54. The composition according to any one of claims 44-53, wherein the second engineered nucleic acid further comprises a terminator sequence.

55. The composition of claim 54, wherein the terminator sequence is as shown in SEQ ID NO:

8.

56. The composition according to any one of claims 44-55, wherein the second engineered nucleic acid further comprises an inverted terminal repeat (ITR) flanking the second transgenic sequence.

57. The composition of claim 56, wherein the ITR consists of the sequence shown in SEQ ID NO:

22.

58. The composition according to claim 56 or 57, wherein the distance between the two inverted terminal repeat sequences (ITRs) is 4.7 kb or less.

59. The composition according to any one of claims 44-49, comprising a first engineered nucleic acid, the first engineered nucleic acid being the engineered nucleic acid according to any one of claims 2-38, wherein the first or second engineered nucleic acid encodes more than one protein.

60. The composition of claim 59, wherein the first or second engineered nucleic acid further encodes a spacer sequence located between at least two nucleic acid sequences, wherein each of the at least two nucleic acid sequences encodes a protein.

61. The composition of claim 60, wherein the spacer sequence is an internal ribosome entry site (IRES) or a self-cleaving peptide.

62. The composition of claim 61, wherein the self-cleaving peptide is a 2A peptide.

63. The composition of claim 62, wherein the 2A peptide comprises the sequence shown in SEQ ID NO:

9.

64. The composition according to any one of claims 44-63, wherein the first or second transgenic sequence is a therapeutic sequence, a nucleic acid encoding a protein, or a gene-targeting nucleic acid.

65. The composition according to any one of claims 44-64, wherein the second engineered nucleic acid is in a recombinant virus.

66. The composition according to any one of claims 43-65, wherein the recombinant virus is selected from the group consisting of lentiviruses, adenoviruses, alphaviruses, vaccinia viruses, herpesviruses, and adeno-associated viruses (AAVs).

67. The composition according to any one of claims 43-66, wherein the recombinant virus is a retrovirus.

68. The composition according to any one of claims 43-67, further comprising tetracycline.

69. The composition according to claim 68, wherein the tetracycline is doxycycline.

70. A reagent kit comprising: (a) The mutant rtTA according to claim 1; (b) Engineered nucleic acids according to any one of claims 2-38; (c) The recombinant virus according to any one of claims 39-42; or (d) Any combination of the mutant rtTA of claim 1, the engineered nucleic acid of any one of claims 2-38, and the recombinant virus of any one of claims 39-42; and (e) A description of the use of (i) the mutant rtTA, (ii) the engineered nucleic acid, (iii) the recombinant virus, or (iv) any combination of the mutant rtTA, the engineered nucleic acid, and the recombinant virus.

71. The kit of claim 70, wherein the kit further comprises a pharmaceutically acceptable excipient.

72. The kit according to claim 70 or 71, further comprising a container containing: (a) the mutant rtTA, (b) the engineered nucleic acid, (c) the recombinant virus, or (d) any combination of the mutant rtTA, the engineered nucleic acid, and the recombinant virus.

73. The kit according to any one of claims 70-72, further comprising tetracycline.

74. The kit according to claim 73, wherein the tetracycline is doxycycline.

75. The composition according to any one of claims 44-69, further comprising a plurality of second engineered nucleic acids.

76. A composition comprising a recombinant virus according to any one of claims 39-42 and a second recombinant virus, the second recombinant virus comprising a second engineered nucleic acid as shown in any one of claims 44-69.

77. The composition according to claim 76, further comprising a plurality of recombinant viruses.

78. A composition comprising (a) the mutant rtTA according to claim 1, (b) an engineered nucleic acid according to any one of claims 2-38; or (c) a recombinant virus according to any one of claims 39-42; and a second engineered nucleic acid as shown in any one of claims 44-69.

79. The composition according to claim 78, further comprising a pharmaceutically acceptable excipient.

80. A kit comprising a container containing a composition according to any one of claims 76-79 and instructions for using the composition.

81. An in vitro method for promoting transgene expression in cells or tissues, comprising administering to cells or tissues: (a) The composition according to any one of claims 43-69; and (b) Tetracycline, The method described herein is for non-therapeutic or non-diagnostic purposes, wherein the cells or tissues are derived from the liver, pancreas, or kidney, and wherein the transgene is a nucleic acid encoding a protein.

82. The in vitro method according to claim 81, wherein the tetracycline is doxycycline.

83. The in vitro method according to claim 81 or 82, wherein the first and / or second engineered nucleic acid is a viral vector.

84. The in vitro method according to claim 83, wherein the viral vector is selected from the group consisting of lentiviruses, adenoviruses, alphaviruses, vacciniaviruses, herpesviruses, and adeno-associated virus (AAV) viral vectors.

85. The in vitro method according to claim 83, wherein the viral vector is a retroviral vector.

86. The in vitro method according to any one of claims 81-85, wherein the first and / or second engineered nucleic acid is present in the virus.

87. The in vitro method of claim 86, wherein the first and second engineered nucleic acids are present in separate viruses.

88. The in vitro method according to any one of claims 81-87, wherein the method further comprises removing tetracycline.

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