Methods and compositions involving tret activator therapies

Abstract

The present disclosure provides methods and compositions for treating progeria or neurodegenerative diseases, particularly neurodegenerative diseases associated with amyloid deposition and neuronal death, such as Alzheimer's disease. Accordingly, aspects of the present disclosure relate to methods for treating progeria in a subject in need thereof, comprising administering to the subject a TERT-activating therapeutic agent. Other aspects relate to methods for treating a neurodegenerative disease in a subject, comprising administering to the subject a TERT-activating therapeutic agent.

Description

BACKGROUND

[0002] This application claims priority to U.S. Provisional Application No. 62 / 842323, filed May 2, 2019, the disclosure of which is incorporated by reference herein in its entirety.

[0003] This application was made with government support under Grant No. CA084628 awarded by the National Institutes of Health. The government has certain rights in the application. TECHNICAL FIELD

[0004] The present application relates to the field of medicine. In particular, the present application provides methods and compositions for treating Alzheimer’s disease.

[0005] II. BACKGROUND

[0006] Alzheimer’s disease (AD) is a progressive and degenerative disease. It is characterized by an increase in pro-inflammatory cytokine levels and accumulation of toxic beta-amyloid deposits, particularly in the hippocampus, which gradually destroys memory and learning abilities. Despite medical advances, there is no clear treatment for AD. FDA-approved drugs can only temporarily slow the worsening of symptoms, and only about half of patients take these drugs. This means that AD and other dementias cost Medicare, Medicaid, and businesses more than $148 billion in direct and indirect costs each year.

[0007] Currently approved pharmacological treatments for cognitive symptoms of AD include cholinesterase inhibitors, and “off-label” treatments for behavioral symptoms of AD include antidepressants; both of these classes of drugs cause adverse side effects and inhibit the production of tumor necrosis factor in addition to increasing the availability of neurotransmitters. Treatments for AD are needed that address the high cost, adverse side effects, and limited efficacy. Therapies that address issues related to AD, such as high cost, high incidence of adverse side effects, and the current limitations in effectively treating AD, are needed. SUMMARY

[0008] The present disclosure provides methods and compositions for treating progeria or neurodegenerative diseases, particularly those associated with amyloid deposition and neuronal death, such as Alzheimer's disease. Accordingly, aspects of the present disclosure relate to methods for treating progeria in a subject in need thereof, comprising administering to the subject a TERT-activating therapeutic. Other aspects relate to methods for treating a neurodegenerative disease in a subject, comprising administering to the subject a TERT-activating therapeutic. Other aspects relate to methods of generating new neurons in a subject in need thereof, comprising administering to the subject a TERT-activating therapeutic. Other aspects relate to methods for reducing beta-amyloid peptides in a subject in need thereof, comprising administering to the subject a TERT-activating therapeutic. Still further aspects relate to compositions comprising a nanovesicle comprising a TERT polypeptide and / or a nucleic acid encoding a TERT polypeptide. A TERT-activating therapeutic refers to a therapy that can achieve one or more of increasing expression of endogenous TERT protein, increasing concentration of TERT protein in a cell, increasing activity of TERT protein (endogenously added TERT protein or exogenously added TERT protein), and stabilizing TERT protein and / or mRNA.

[0009] In some implementations, progeria includes Hutchinson-Guilford progeria syndrome (HGPS), Nestor-Guilmore progeria syndrome, adult-onset progeria, Cockayne syndrome, Bloom syndrome, xeroderma pigmentosum, ataxia-telangiectasia, hyposulfur piloerection, congenital dyskeratosis, or mosaic aneuploidy syndrome. In some implementations, neurodegenerative diseases include Alzheimer's disease. In some implementations, progeria does not include Hutchinson-Guilford progeria syndrome (HGPS), Nestor-Guilmore progeria syndrome, adult-onset progeria, Cockayne syndrome, Bloom syndrome, xeroderma pigmentosum, ataxia-telangiectasia, hyposulfur piloerection, congenital dyskeratosis, or mosaic aneuploidy syndrome. In some implementations, neurodegenerative diseases do not include Alzheimer's disease. In some implementations, Alzheimer's disease includes early-onset Alzheimer's disease or is early-onset Alzheimer's disease. In some embodiments, Alzheimer's disease includes late-onset Alzheimer's disease or is late-onset Alzheimer's disease. In some embodiments, early-onset Alzheimer's disease or late-onset Alzheimer's disease is excluded. In some embodiments, neurodegenerative diseases include neurodegenerative diseases associated with amyloid deposition. In some embodiments, neurodegeneration is defined as a disease that includes neuronal degeneration and / or death. In some embodiments, a neurodegenerative disease is a disease that leads to neuronal cell death. In some embodiments, treatment includes increasing dendritic spine formation. In some embodiments, the increase in dendritic spine formation is in cortical neurons. In some embodiments, treatment includes increasing or enhancing neural networks. In some embodiments, treatment includes enhancing or increasing synaptic pathway activation, which promotes molecular chaperone expression and reduces the expression of AD risk genes. In some embodiments, treatment includes reducing amyloid plaques. In some embodiments, the subject has been diagnosed with the condition. In some embodiments, the subject has previously received treatment for the condition. In some embodiments, the subject has been identified as unresponsive to previous treatments. In some embodiments, the subject has not previously received treatment for the condition. In some embodiments, the subject is a person. In some implementation schemes, the target age is under 50.In some implementations, the subject is younger than or older than 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 years old (or any range that can be derived therefrom).

[0010] In some embodiments, the method further includes administering an adjunct therapy. In some embodiments, the adjunct therapy includes a cholinesterase inhibitor, such as donepezil, galantamine, or rivastigmine. In some embodiments, the adjunct therapy includes memantine. In some embodiments, the methods and compositions disclosed herein do not include one or more of donepezil, galantamine, rivastigmine, or memantine.

[0011] In some implementations, TERT activation therapy includes the delivery of nucleic acids encoding TERT peptides. In some implementations, the TERT nucleic acid comprises a nucleic acid or fragment thereof of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9, or has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9 or a fragment thereof. In some embodiments, TERT activation therapy involves administering DNA or RNA encoding a TERT peptide to the subject. In some embodiments, the TERT activation therapeutic agent comprises a TERT peptide. In some embodiments, the TERT polypeptide comprises a polypeptide or fragment thereof of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10, or has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10. In some embodiments, the TERT-activating therapeutic agent comprises a TERT polypeptide with no catalytic activity, such as a TERT polypeptide capable of transactivating genes but lacking telomerase reverse transcriptase activity. In some embodiments, the TERT polypeptide contains a D712A mutation. In some embodiments, the TERT polypeptide does not have a D712A mutation.

[0012] In some embodiments, the TERT-activating therapeutic agent comprises nanovesicles containing a TERT peptide or a nucleic acid encoding a TERT peptide. In some embodiments, the nanovesicles comprise extracellular bodies. In some embodiments, the diameter of the nanovesicles is from 10 nm to 1000 nm. In some embodiments, the diameter of the nanovesicles is at least or at most 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, or 240 nm. 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, 500nm, 5 10nm, 520nm, 530nm, 540nm, 550nm, 560nm, 570nm, 580nm, 590nm, 600nm, 610nm, 620nm, 630nm, 6 40nm, 650nm, 660nm, 670nm, 680nm, 690nm, 700nm, 710nm, 720nm, 730nm, 740nm, 750nm, 760nm, 77 0nm, 780nm, 790nm, 800nm, 810nm, 820nm, 830nm, 840nm, 850nm, 860nm, 870nm, 880nm, 890nm, 900nm, 910nm, 920nm, 930nm, 940nm, 950nm, 960nm, 970nm, 980nm, 990nm, or 1000nm (or any range thereof). In some embodiments, the nanovesicles contain CD47. In some embodiments, the nanovesicles contain CD47 expression on the surface and / or within the membrane of the nanovesicles. In some embodiments, the nanovesicles contain rabies virus glycoprotein peptides. Exemplary rabies virus glycoprotein peptides that can be used in embodiments of this disclosure include the following:

[0013] RABIES VIRUS GLYCOPOREIN PEPTIDE SEQUENCES SEQ ID NO: YTIWMPENPRPGTPCDIFTNSRGKRASNG 11 YTIWMPENPRPGTPCDIFTNSRGKRASNGC 12 YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGGC 13 YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGG9dR 14

[0014] dR = D-arginine

[0015] Rabies virus glycoprotein peptides may contain 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 (or any range thereof) or more than 42 amino acids, or may be associated with SEQ ID No: 11 to SEQ ID No: 42. IDNo:14 contains at least or at most 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 or more than 42, or any range thereof. The consecutive amino acids have at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity, identity, or homology.

[0016] Rabies virus glycoprotein peptides may contain 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 or more amino acids, or any range of these, derived from SEQ ID NO:11 to SEQ ID No:14.

[0017] In some embodiments, the rabies virus glycoprotein peptide comprises 1 to 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 amino acids (or any range thereof) from SEQ ID NO:11 to SEQ ID No:14.

[0018] Rabies virus glycoprotein peptides may contain at least, at most, or exactly 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 substitutions.

[0019] In some embodiments, the compositions and methods do not include extracellular bodies or nanovesicles as a method of TERT delivery.

[0020] The substitution can be performed at amino acid positions 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42.

[0021] The polypeptides described herein may have a fixed length of at least, at most, or exactly 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 amino acids (or any range thereof).

[0022] In some embodiments, TERT activation therapy includes regulation of histone H3K9 methyltransferase (HMT). In some embodiments, regulation includes inhibition of HMT genes or proteins. In some embodiments, inhibition includes genetic silencing of one or more HMT genes. Methods of genetic silencing are known in the art. For example, methods such as homologous directed repair and gene editing can be used to mutate one or more HMT genes in target cells, such as neuronal cells or supporting cells. In some embodiments, gene editing technologies such as CRISPR are used to reduce the expression of one or more HMTs in the target. In some embodiments, one or more HMT genes include one or more of SUV39H1 / KMT1A, SUV39H2 / KMT1B, SETDB1 / KMT1E, SETDB2 / KMT1F, PRDM2, G9A / KMT1C, GLP / KMT1D, EHMT1, and RIZ1 / KMT8. In some embodiments, the HMT formulation excludes one or more of SUV39H1 / KMT1A, SUV39H2 / KMT1B, SETDB1 / KMT1E, SETDB2 / KMT1F, PRDM2, G9A / KMT1C, GLP / KMT1D, EHMT1, and RIZ1 / KMT8. In some embodiments, the TERT activating therapeutic agent comprises an HMT inhibitor. In some embodiments, the HMT inhibitor comprises one or more of trachomatisin, BIX-01294, BIX-01338, UNC0638, and BRD4770. In some embodiments, one or more of trachomatisin, BIX-01294, BIX-01338, UNC0638, and BRD4770 are excluded. In some embodiments, the TERT activating therapeutic agent comprises trachomatisin. In some embodiments, TERT activation therapy includes administration of a histone H3K9 demethylase (HMT) peptide or a nucleic acid encoding HDM. In some embodiments, the HDM peptide comprises a peptide having demethylase activity. In some embodiments, the HDM peptide comprises one or more peptides selected from KDM1A / LSD1, KDM3A / JHDM2A, KDM3B / JHDM2B, KDM4A / JHDM3A, KDM4B / JMJD2B, KDM4C / JMJD2C, KDM4D / JMJD2D, KDM7 / JHDM1D, and PHF8. In some implementations, the HMT implementation excludes one or more of KDM1A / LSD1, KDM3A / JHDM2A, KDM3B / JHDM2B, KDM4A / JHDM3A, KDM4B / JMJD2B, KDM4C / JMJD2C, KDM4D / JMJD2D, KDM7 / JHDM1D, and PHF8.

[0023] Other implementation schemes for the rabies virus glycoprotein are further described in Oswald et al., Mol. Pharmaceutics, 2017, 14(7), pp 2177–2196, which is incorporated herein by reference.

[0024] In some embodiments, the nanovesicles are derived from fibroblasts or bone marrow dendritic cells. In some embodiments, the nanovesicles are derived from human cells. In some embodiments, the nanovesicles are derived from non-human cells.

[0025] In some embodiments, the TERT activating agent is administered intravenously. In some embodiments, the TERT activating agent is administered systemically. In some embodiments, the TERT activating agent is administered via the route of administration described herein.

[0026] In some implementations, the treatment includes one or more of the following: reduction of β-amyloid peptide, improvement of learning ability, improvement of memory, and neuron generation. The reduction or improvement can be at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, or any range derived therefrom.

[0027] In some embodiments, the TERT polypeptide comprises a polypeptide with telomerase activity. When referring to gene products, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

[0028] The terms "object," "mammal," and "patient" are used interchangeably. In some embodiments, the object is a mammal. In some embodiments, the object is a human. In some embodiments, the object is a mouse, rat, rabbit, dog, donkey, or a laboratory test animal such as a fly, zebrafish, etc.

[0029] In some implementations, the subject has previously received treatment for a disease or condition. In some implementations, the subject is resistant to the previous treatment. In some implementations, the subject is identified as someone who has had an adverse reaction to the previous treatment.

[0030] The intended methods and compositions include any embodiments excluded herein.

[0031] In this application, the term “about” is used according to its simple and common meaning in the field of cell and molecular biology to refer to a value including the error standard deviation of the apparatus or method used to determine the value.

[0032] When used with the term “including”, the absence of a quantifier can mean “one”, but it may also be consistent with the meanings of “one or more”, “at least one” and “one or more”.

[0033] As used herein, the terms “or” and “and / or” are used to describe multiple components that are combined or mutually exclusive. For example, “x, y and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y and z”, “(x and y) or z”, “x or (y and z)” or “x or y or z”. In particular, it is taken into consideration that x, y, or z can be specifically excluded from the implementation scheme.

[0034] "Contains", "has", "includes", "characterized by", or "contains" is inclusive or open-ended and does not exclude additional, unlisted elements or method steps.

[0035] The use of a composition and method may “comprise” any component or step disclosed throughout the specification, “consistently of” or “by” any component or step disclosed throughout the specification. The phrase “consistent of” excludes any unspecified element, step, or component. The phrase “consistently of” limits the scope of the subject matter to the specified materials or steps, and materials or steps that do not materially affect its essential and novel characteristics. Embodiments contemplated in the context of the term “comprise” may also be implemented in the context of the terms “consistent of” or “consistently of”.

[0036] In particular, it is worth noting that any limitations discussed with respect to one embodiment of the invention may be applied to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or utilize any composition of the invention. Aspects of the embodiments described in the examples may also be implemented in different embodiments or applications discussed elsewhere, such as the summary, detailed description, claims, and description of drawings.

[0037] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific embodiments illustrate particular implementations of the invention, they are given by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Attached Figure Description

[0038] The following figures form part of this specification and are included to further illustrate certain aspects of the invention. A better understanding of the invention can be achieved by referring to one or more of these figures in conjunction with the detailed description of the specific embodiments presented herein.

[0039] FIG. 1A-1I Tert was downregulated in two different mouse Alzheimer's disease neurons. (A) Tert mRNA levels in the cortex of 3xTg-AD and wild-type control (B6129SF2 / J) mice (n=4; 3 months old). (B) Tert mRNA levels in the hippocampus of 5xFAD and wild-type littermate control mice (n=4; 2 to 3 months old). (C) Tert mRNA levels in primary cortical and hippocampal neurons isolated from 3xTg-AD and control mice in DIV 14 (n=3). (D) Tert mRNA levels in primary cortical and hippocampal neurons isolated from 5xFAD and control mice in DIV 14 (n=3). (E) Telomerase activity in hippocampal neurons isolated from 5xFAD and control mice (n=4; 2 to 3 months old). (F) Representative view of the occupancy of the H3K9me3 inhibitory histone marker in the Tert gene of 5xFAD mice and primary neurons of control mice, with control at DIV 14. (G) mRNA levels of histone demethylases Kdm1a, Kdm4b, and Kdm4c genes in cortical and hippocampal neurons of 5xFAD and wild-type littermate control mice (n=4; 2 to 3 months old). (H) Immunostaining of KDM1A in the CA1 hippocampal subregion of 5xFAD and wild-type littermate control mice (2 to 3 months old). (I) Tert mRNA levels in the cortex and hippocampus of 5xFAD mice treated with trachomatis or BIX-01294.

[0040] FIG. 2A-2C Cre-induced generation of Tert knock-in mice (R26-CAG-LSL-mTert-IRES-eGFP-pA). (A) Construction scheme for introducing CAG-LSL-mTert-IRES-eGFP-pA into the Rosa26 locus. (B) Genotyping results of the original ES-targeted line carrying the R26-CAG-LSL-mTert-IRES-eGFP-pA allele. (C) Representative photographs of chimeric mice obtained from targeted ES cells.

[0041] FIG. 3A-3DTert activation alleviated amyloid pathology in a novel induced TERT-AD mouse model. (A) Reproductive strategies of R26-CAG-LSL-mTert mice with 3xTg-AD or 5xFAD and Camk2a-CreERT2 mice. (B) Immunostaining of Aβ in the CA1 hippocampal subregion of adult (8-month-old) mice and Tert-activated R26-CAG-LSL-mTert; 3xTg-AD; Camk2a-CreERT2 mice. (C) Quantitative comparison of Aβ-immunoreactive pyramidal neurons in the CA1 region (n = 6; 8 months old per group). (D) Immunostaining of Aβ in the hippocampus of adult (7-month-old) mice and Tert-activated R26-CAG-LSL-mTert; 5xFAD; Camk2a-CreERT2 mice.

[0042] FIG. 4A-4F Tert activation in AD neurons enhances various synaptic pathways that promote molecular chaperone expression and reduce the expression of AD risk genes. (A) mRNA levels of Tert and Terc in Tert-activated neurons of R26-CAG-LSL-mTert; 3xTg-AD; Camk2a-CreERT2 mice. (B) A Venn diagram showing the crossover of upregulated biological processes compared to control groups based on RNA-Seq results from Tert-activated cortical and hippocampal neurons of R26-CAG-LSL-mTert; 3xTg-AD; Camk2a-CreERT2 mice. (C) Top 5 overlapping pathways upregulated in Tert-activated cortical and hippocampal neurons. (D) Gene set enrichment analysis (GSEA) diagram showing the relative upregulation of synaptic signaling genes in Tert-activated cortical and hippocampal neurons compared to control neurons. (E, F) mRNA levels of the App, ApoE, Hsp70-1 and Hsp70-2 genes with or without Tert-induced induction.

[0043] FIG. 5A-5CTert activation enhanced spinal morphology and neural networks in an AD mouse model. (A) Representative images of Golgi-stained cortical neurons from aged (18-month-old) control mice and Tert-activated R26-CAG-LSL-mTert;3xTg-AD;Camk2a-CreERT2 mice. (B) High magnification of dendritic spines in impregnated pyramidal cortical neurons from aged control mice and Tert-activated R26-CAG-LSL-mTert;3xTg-AD;Camk2a-CreERT2 mice. (C) Quantification of dendritic spine density (n = 20 dendrites per group, n = 4 mice per group; 18 months old). t-tests were used for comparisons between the two groups. ****P < 0.0001; mean ± sem

[0044] FIG. 6A-6D HMT inhibitors activate the human TERT gene and cause gene silencing in human AD neurons. (A) Representative image of the occupancy of the H3K9me3 repressive histone marker in the TERT gene of neurons from APP. DP iPSCs derived from patients and non-dementia control groups (NDCs). (B, C) TERT mRNA levels (B) and TERT protein levels (C) in human AD neurons treated with chrysogenin. (D) Immunoblot of TERT protein levels in human AD neurons treated with siRNA targeting histone methyltransferase genes G9A or SETDB1.

[0045] FIG. 7A-7E TERT activation alleviates amyloid pathology in human AD neurons. (A) Clones of Flag-tagged human TERT lentiviral expression constructs. (B, C) Aβ levels in neurons transduced by EGFP or TERT, measured by sandwich ELISA. 1-40 Level (n=3), this neuron from APP DP The iPSCs are derived from the patient's own cells. (D) APP transduced with EGFP or TERT. DP Immunoblot of endogenous proteins shown in neurons. Tubulin was used as an internal control. (E) APP transduced by EGFP or TERT DP Relative gene expression (n=4) obtained by RT-PCR quantification in neurons.

[0046] FIG. 8A-8CTERT's transactivation function is independent of its catalytic activity. (A) Schematic diagram of the catalytically inactive (CI) human TERT lentiviral expression construct. White asterisks indicate the location of the single mutant D712A, which inactivates the protein's catalytic activity. (B) Immunoblot used to confirm Flag-labeled catalytically inactivating TERT expression in HEK293 cells. (C) mRNA expression levels of each gene shown. Transcriptional levels were normalized to HPRT1 mRNA.

[0047] FIG. 9A-9D Activation of neuronal TERT triggers transactivation of specific genes associated with learning processes in AD neurons. (A) A Venn diagram illustrates the crossover of upregulated biological processes based on three independent RNA-Seq results compared to each control group, derived from Tert-activated mouse cortical and hippocampal neurons (n=4 per group) from R26-CAG-LSL-mTert; 3xTg-AD; Camk2a-CreERT2 mice and TERT-activated human APP neurons. DP (a) Neurons (n=3) (all p<0.05). (b) List of 13 overlapping pathways upregulated in all Tert-activated mouse cortical and hippocampal AD neurons and Tert-activated human AD neurons. (c) GSEA plot showing the relative upregulation of learning-related genes in Tert-activated cortical and hippocampal AD neurons and Tert-activated human AD neurons compared to each control group. (d) Escape latency in aged mice (22–26 months) in the Barnes maze during training days in control and Tert-activated R26-CAG-LSL-mTert; 3xTg-AD; Camk2a-CreERT2 mice. (n=9 per group). Two-way ANOVA with Sidak multiple comparison test; t-test for two-group comparison; 1, 2, 3, or 4 signs for P<0.05, 0.01, 0.0005, 0.0001, respectively; mean ± sem

[0048] FIG. 10A-10C Neuronal TERT interacts physically with core components of the β-catenin transcription factor and RNA polymerase II complex. (A) List of TERT-interacting proteins identified by mass spectrometry in human AD neurons. (B) RNA-Seq heatmap of WNT signaling pathway genes in human AD neurons transduced by EGFP and TERT (n=3). (C) Co-immunoprecipitation of endogenous β-catenin (active), CREBBP, POLR2A, and TERT from human AD neurons.

[0049] FIG. 11A-11CGlobal enrichment of TERT and β-catenin / TCF7 associations at the genomic level. (A) ChIP-Seq density heatmap of TERT, β-catenin (active), and TCF7 across human AD neuronal gene promoters. (B) Chromatin state map showing β-catenin (active), TCF7, and TERT binding peaks at the WNT9B, ATP1A3, HSPA12A, HSPA6, and MYC loci, determined by ChIP-Seq. (C) Model of TERT role in transcriptional activation of AD neurons. In neurons, TERT levels decrease in the early pathological stages of AD. Activation of neuronal TERT triggers transcriptional induction of specific genes associated with synaptic signaling and learning processes in AD neurons, thereby mitigating cognitive deficits. Detailed Implementation

[0050] Telomerase reverse transcriptase (TERT), the catalytic subunit of telomerase, has been reported to have various beneficial and protective functions in multiple tissues of rodents and humans. However, the relationship between telomerase and amyloid pathology, a key hallmark of Alzheimer's disease (AD), remains largely unexplored. AD is a progressive, adult-onset neurodegenerative disease. To test the effects of TERT reactivation in the brain, the inventors established an inducible telomerase-activated AD (TERT-AD) mouse model to control the temporal regulation of TERT gene expression. As shown in Example 1, telomerase activation can alleviate AD pathology in mouse and human AD models by directly modulating the transcriptional networks of key neurons affected in AD. Increasing TERT levels and activity in the brain provides a therapeutic strategy for the prevention and treatment of Alzheimer's amyloid neuropathology.

[0051] I.TERT polypeptide

[0052] TERT, also known in humans as CMM9, DKCA2, DKCB4, EST2, PFBMFT1, TCS1, TP2, TRT, hEST2, and hTRT, and in mice as EST2, TCS1, TP2, TR, and TRT, is known in the art and is illustrated by the following mRNA and protein sequences described herein.

[0053] For example, an instance of the human TERT gene is Homo sapiens telomerase reverse transcriptase (TERT), transcript variant 2, mRNA (NCBI reference sequence: NM_001193376.1):

[0054]

[0055]

[0056]

[0057] Human telomerase reverse transcriptase isotype 2, NCBI reference sequence: P_001180305.1:

[0058]

[0059]

[0060] Human telomerase reverse transcriptase (TERT), transcript variant 1, mRNA, NCBI reference sequence: NM_198253.2:

[0061]

[0062]

[0063]

[0064] Human telomerase reverse transcriptase isoform 1, NCBI reference sequence: NP_937983.2

[0065]

[0066]

[0067] Rat telomerase reverse transcriptase (Tert), transcript variant 2, mRNA, NCBI reference sequence: NM_001362387.1:

[0068]

[0069]

[0070]

[0071] Rat telomerase reverse transcriptase isotype 2, NCBI reference sequence: NP_001349316.1:

[0072]

[0073] Mouse telomerase reverse transcriptase (Tert), transcript variant 3, mRNA, NCBI reference sequence: NM_001362388.1:

[0074]

[0075]

[0076] Rat telomerase reverse transcriptase isotype 3, NCBI reference sequence: NP_001349317.1:

[0077]

[0078] Rat telomerase reverse transcriptase (Tert), transcript variant 1, mRNA, NCBI reference sequence: NM_009354.2:

[0079]

[0080]

[0081]

[0082] Rat telomerase reverse transcriptase isotype 1, NCBI reference sequence: NP_033380.1:

[0083]

[0084]

[0085] In some embodiments, the TERT peptide or nucleic acid includes a human TERT peptide or human TERT nucleic acid. In some embodiments, the TERT peptide or TERT nucleic acid is non-human. In some embodiments, the TERT peptide or TERT nucleic acid is derived from a mouse, horse, dog, rabbit, or goat.

[0086] The polypeptides or polynucleotides disclosed herein, such as those containing or encoding TERT polypeptides, may contain 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 1, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 64 9, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682

[0087] 683, 684, 685, 686, 687, 688, 689, 690, 691

[0088] 692, 693, 694, 695, 696, 697, 698, 699, 700

[0089] 701, 702, 703, 704, 705, 706, 707, 708, 709

[0090] 710, 711, 712, 713, 714, 715, 716, 717, 718

[0091] 719, 720, 721, 722, 723, 724, 725, 726, 727

[0092] 728, 729, 730, 731, 732, 733, 734, 735, 736

[0093] 737, 738, 739, 740, 741, 742, 743, 744, 745

[0094] 746, 747, 748, 749, 750, 751, 752, 753, 754

[0095] 755, 756, 757, 758, 759, 760, 761, 762, 763

[0096] 764, 765, 766, 767, 768, 769, 770, 771, 772

[0097] 773, 774, 775, 776, 777, 778, 779, 780, 781

[0098] 782, 783, 784, 785, 786, 787, 788, 789, 790

[0099] 791, 792, 793, 794, 795, 796, 797, 798, 799

[0100] 1, 800, 801, 802, 803, 804, 805, 806, 807, 808

[0101] 809, 810, 811, 812, 813, 814, 815, 816, 817

[0102] 818, 819, 820, 821, 822, 823, 824, 825, 826

[0103] 827, 828, 829, 830, 831, 832, 833, 834, 835

[0104] 836, 837, 838, 839, 840, 841, 842, 843, 844

[0105] 1, 845, 846, 847, 848, 849, 850, 851, 852, 853

[0106] 854, 855, 856, 857, 858, 859, 860, 861, 862

[0107] 1, 863, 864, 865, 866, 867, 868, 869, 870, 871

[0108] 1, 872, 873, 874, 875, 876, 877, 878, 879, 880

[0109] 881, 882, 883, 884, 885, 886, 887, 888, 889

[0110] 890, 891, 892, 893, 894, 895, 896, 897, 898

[0111] 1, 899, 900, 901, 902, 903, 904, 905, 906, 907

[0112] 908, 909, 910, 911, 912, 913, 914, 915, 916

[0113] 917, 918, 919, 920, 921, 922, 923, 924, 925

[0114] 1, 926, 927, 928, 929, 930, 931, 932, 933, 934

[0115] 1, 935, 936, 937, 938, 939, 940, 941, 942, 943

[0116] 1, 944, 945, 946, 947, 948, 949, 950, 951, 952

[0117] 1, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980 1, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or any range thereof) or more than 1000 variations of amino acids or nucleic acids, or substitutions with SEQ ID No:1 to SEQ ID No. 10 contains at least 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 items. 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 4 02, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 44 2, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482 1, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522

[0118] 523, 524, 525, 526, 527, 528, 529, 530, 531

[0119] 532, 533, 534, 535, 536, 537, 538, 539, 540

[0120] 541, 542, 543, 544, 545, 546, 547, 548, 549

[0121] 550, 551, 552, 553, 554, 555, 556, 557, 558

[0122] 559, 560, 561, 562, 563, 564, 565, 566, 567

[0123] 568, 569, 570, 571, 572, 573, 574, 575, 576

[0124] 577, 578, 579, 580, 581, 582, 583, 584, 585

[0125] 586, 587, 588, 589, 590, 591, 592, 593, 594

[0126] 595, 596, 597, 598, 599, 600, 601, 602, 603

[0127] 604, 605, 606, 607, 608, 609, 610, 611, 612

[0128] 613, 614, 615, 616, 617, 618, 619, 620, 621

[0129] 622, 623, 624, 625, 626, 627, 628, 629, 630

[0130] 631, 632, 633, 634, 635, 636, 637, 638, 639

[0131] 640, 641, 642, 643, 644, 645, 646, 647, 648

[0132] 649, 650, 651, 652, 653, 654, 655, 656, 657

[0133] 658, 659, 660, 661, 662, 663, 664, 665, 666

[0134] 667, 668, 669, 670, 671, 672, 673, 674, 675

[0135] 676, 677, 678, 679, 680, 681, 682, 683, 684

[0136] 685, 686, 687, 688, 689, 690, 691, 692, 693

[0137] 694, 695, 696, 697, 698, 699, 700, 701, 702

[0138] 703, 704, 705, 706, 707, 708, 709, 710, 711

[0139] 712, 713, 714, 715, 716, 717, 718, 719, 720

[0140] 721, 722, 723, 724, 725, 726, 727, 728, 729

[0141] 730, 731, 732, 733, 734, 735, 736, 737, 738

[0142] 739, 740, 741, 742, 743, 744, 745, 746, 747

[0143] 748, 749, 750, 751, 752, 753, 754, 755, 756

[0144] 757, 758, 759, 760, 761, 762, 763, 764, 765

[0145] 766, 767, 768, 769, 770, 771, 772, 773, 774

[0146] 775, 776, 777, 778, 779, 780, 781, 782, 783

[0147] 784, 785, 786, 787, 788, 789, 790, 791, 792

[0148] 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 81 7, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 84 2, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 8 67, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 8 92, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991992, 993, 994, 995, 996, 997, 998, 999, or 1000 or more than 1000 or any range thereof of consecutive amino acids or nucleic acids having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity, identity, or homology. ,

[0149] The polypeptides or polynucleotides disclosed herein, such as those comprising or encoding TERT polypeptides, may comprise SEQ ID NO:1 to SEQ ID NO:1. NO: 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 6 2, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 1 17, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 45 6, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 5 45, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589

[0150] 590, 591, 592, 593, 594, 595, 596, 597, 598

[0151] 599, 600, 601, 602, 603, 604, 605, 606, 607

[0152] 608, 609, 610, 611, 612, 613, 614, 615, 616

[0153] 617, 618, 619, 620, 621, 622, 623, 624, 625

[0154] 626, 627, 628, 629, 630, 631, 632, 633, 634

[0155] 635, 636, 637, 638, 639, 640, 641, 642, 643

[0156] 644, 645, 646, 647, 648, 649, 650, 651, 652

[0157] 653, 654, 655, 656, 657, 658, 659, 660, 661

[0158] 662, 663, 664, 665, 666, 667, 668, 669, 670

[0159] 671, 672, 673, 674, 675, 676, 677, 678, 679

[0160] 680, 681, 682, 683, 684, 685, 686, 687, 688

[0161] 689, 690, 691, 692, 693, 694, 695, 696, 697

[0162] 698, 699, 700, 701, 702, 703, 704, 705, 706

[0163] 707, 708, 709, 710, 711, 712, 713, 714, 715

[0164] 716, 717, 718, 719, 720, 721, 722, 723, 724

[0165] 725, 726, 727, 728, 729, 730, 731, 732, 733

[0166] 734, 735, 736, 737, 738, 739, 740, 741, 742

[0167] 743, 744, 745, 746, 747, 748, 749, 750, 751

[0168] 752, 753, 754, 755, 756, 757, 758, 759, 760

[0169] 761, 762, 763, 764, 765, 766, 767, 768, 769

[0170] 770, 771, 772, 773, 774, 775, 776, 777, 778

[0171] 779, 780, 781, 782, 783, 784, 785, 786, 787

[0172] 788, 789, 790, 791, 792, 793, 794, 795, 796

[0173] 797, 798, 799, 800, 801, 802, 803, 804, 805

[0174] 806, 807, 808, 809, 810, 811, 812, 813, 814

[0175] 815, 816, 817, 818, 819, 820, 821, 822, 823

[0176] 824, 825, 826, 827, 828, 829, 830, 831, 832

[0177] 833, 834, 835, 836, 837, 838, 839, 840, 841

[0178] 1, 842, 843, 844, 845, 846, 847, 848, 849, 850

[0179] 851, 852, 853, 854, 855, 856, 857, 858, 859

[0180] 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 88 4, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 90 9, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 9 34, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 9 59, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 100 7, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1 028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 10481049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 109 0, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1 111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 117 3, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1 194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 12141215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 125 6, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1 277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 133 9, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1 360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 13801381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420, 1421, 142 2, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1 443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463 1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1481, 1482, 1483, 1484 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1504, 150 5, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1 526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 15461547, 1548, 1549, 1550, 1551, 1552, 1553, 1554, 1555, 1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564, 1565, 1566, 1567 1568, 1569, 1570, 1571, 1572, 1573, 1574, 1575, 1576, 1577, 1578, 1579, 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, 158 8, 1589, 1590, 1591, 1592, 1593, 1594, 1595, 1596, 1597, 1598, 1599, 1600, 1601, 1602, 1603, 1604, 1605, 1606, 1607, 1608, 1 609, 1610, 1611, 1612, 1613, 1614, 1615, 1616, 1617, 1618, 1619, 1620, 1621, 1622, 1623, 1624, 1625, 1626, 1627, 1628, 1629 1630, 1631, 1632, 1633, 1634, 1635, 1636, 1637, 1638, 1639, 1640, 1641, 1642, 1643, 1644, 1645, 1646, 1647, 1648, 1649, 1650 1651, 1652, 1653, 1654, 1655, 1656, 1657, 1658, 1659, 1660, 1661, 1662, 1663, 1664, 1665, 1666, 1667, 1668, 1669, 1670, 167 1, 1672, 1673, 1674, 1675, 1676, 1677, 1678, 1679, 1680, 1681, 1682, 1683, 1684, 1685, 1686, 1687, 1688, 1689, 1690, 1691, 1 692, 1693, 1694, 1695, 1696, 1697, 1698, 1699, 1700, 1701, 1702, 1703, 1704, 1705, 1706, 1707, 1708, 1709, 1710, 1711, 17121713, 1714, 1715, 1716, 1717, 1718, 1719, 1720, 1721, 1722, 1723, 1724, 1725, 1726, 1727, 1728, 1729, 1730, 1731, 1732, 1733 1734, 1735, 1736, 1737, 1738, 1739, 1740, 1741, 1742, 1743, 1744, 1745, 1746, 1747, 1748, 1749, 1750, 1751, 1752, 1753, 175 4, 1755, 1756, 1757, 1758, 1759, 1760, 1761, 1762, 1763, 1764, 1765, 1766, 1767, 1768, 1769, 1770, 1771, 1772, 1773, 1774, 1 775, 1776, 1777, 1778, 1779, 1780, 1781, 1782, 1783, 1784, 1785, 1786, 1787, 1788, 1789, 1790, 1791, 1792, 1793, 1794, 1795 1796, 1797, 1798, 1799, 1800, 1801, 1802, 1803, 1804, 1805, 1806, 1807, 1808, 1809, 1810, 1811, 1812, 1813, 1814, 1815, 1816 1817, 1818, 1819, 1820, 1821, 1822, 1823, 1824, 1825, 1826, 1827, 1828, 1829, 1830, 1831, 1832, 1833, 1834, 1835, 1836, 183 7, 1838, 1839, 1840, 1841, 1842, 1843, 1844, 1845, 1846, 1847, 1848, 1849, 1850, 1851, 1852, 1853, 1854, 1855, 1856, 1857, 1 858, 1859, 1860, 1861, 1862, 1863, 1864, 1865, 1866, 1867, 1868, 1869, 1870, 1871, 1872, 1873, 1874, 1875, 1876, 1877, 18781879, 1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887, 1888, 1889, 1890, 1891, 1892, 1893, 1894, 1895, 1896, 1897, 1898, 1899, 1900, 1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910 1911, 1912, 1913, 1914, 1915, 1916, 1917, 1918, 1919, 1920, 1921, 1922, 1923, 1924, 1925, 1926, 1927, 1928, 1929, 1930, 1931, 1932, 1933, 1934, 1935, 1936, 1937, 1938, 1939, 1940, 1941, 194 2, 1943, 1944, 1945, 1946, 1947, 1948, 1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959, 1960, 1961, 1962, 1963, 1964, 1965, 1966, 1967, 1968, 1969, 1970, 1971, 1972, 1973, 1 974, 1975, 1976, 1977, 1978, 1979, 1980, 1981, 1982, 1983, 1984, 1985, 1986, 1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 1999, or 2000 or more than 2000, or any range thereof, of consecutive amino acids or nucleic acids.

[0181] In some embodiments, the polypeptide comprises SEQ ID NO:1 to SEQ ID NO: 1 to 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 of 10 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 16 6, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889 1, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 93 9, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 96 4, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 98 9, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 10111012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 105 3, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1 074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 113 6, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1 157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 11771178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 121 9, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1 240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 130 2, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1 323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 13431344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 138 5, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1 406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 146 8, 1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1481, 1482, 1483, 1484, 1485, 1486, 1487, 1488, 1 489, 1490, 1491, 1492, 1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 15091510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1546, 1547, 1548, 1549, 1550, 155 1, 1552, 1553, 1554, 1555, 1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564, 1565, 1566, 1567, 1568, 1569, 1570, 1571, 1 572, 1573, 1574, 1575, 1576, 1577, 1578, 1579, 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, 1588, 1589, 1590, 1591, 1592 1593, 1594, 1595, 1596, 1597, 1598, 1599, 1600, 1601, 1602, 1603, 1604, 1605, 1606, 1607, 1608, 1609, 1610, 1611, 1612, 1613 1614, 1615, 1616, 1617, 1618, 1619, 1620, 1621, 1622, 1623, 1624, 1625, 1626, 1627, 1628, 1629, 1630, 1631, 1632, 1633, 163 4, 1635, 1636, 1637, 1638, 1639, 1640, 1641, 1642, 1643, 1644, 1645, 1646, 1647, 1648, 1649, 1650, 1651, 1652, 1653, 1654, 1 655, 1656, 1657, 1658, 1659, 1660, 1661, 1662, 1663, 1664, 1665, 1666, 1667, 1668, 1669, 1670, 1671, 1672, 1673, 1674, 16751676, 1677, 1678, 1679, 1680, 1681, 1682, 1683, 1684, 1685, 1686, 1687, 1688, 1689, 1690, 1691, 1692, 1693, 1694, 1695, 1696, 1697, 1698, 1699, 1700, 1701, 1702, 1703, 1704, 1705, 1706, 1707, 1708, 1709, 1710

[0182] 1711, 1712, 1713, 1714, 1715, 1716, 1717, 1718

[0183] 1719, 1720, 1721, 1722, 1723, 1724, 1725, 1726

[0184] 1727, 1728, 1729, 1730, 1731, 1732, 1733, 1734

[0185] 1735, 1736, 1737, 1738, 1739, 1740, 1741, 1742

[0186] 1743, 1744, 1745, 1746, 1747, 1748, 1749, 1750

[0187] 1751, 1752, 1753, 1754, 1755, 1756, 1757, 1758

[0188] 1759, 1760, 1761, 1762, 1763, 1764, 1765, 1766

[0189] 1767, 1768, 1769, 1770, 1771, 1772, 1773, 1774

[0190] 1775, 1776, 1777, 1778, 1779, 1780, 1781, 1782

[0191] 1783, 1784, 1785, 1786, 1787, 1788, 1789, 1790

[0192] 1791, 1792, 1793, 1794, 1795, 1796, 1797, 1798

[0193] 1799, 1800, 1801, 1802, 1803, 1804, 1805, 1806

[0194] 1807, 1808, 1809, 1810, 1811, 1812, 1813, 1814

[0195] 1815, 1816, 1817, 1818, 1819, 1820, 1821, 1822

[0196] 1823, 1824, 1825, 1826, 1827, 1828, 1829, 1830

[0197] 1831, 1832, 1833, 1834, 1835, 1836, 1837, 1838

[0198] 1839, 1840, 1841, 1842, 1843, 1844, 1845, 1846

[0199] 1847, 1848, 1849, 1850, 1851, 1852, 1853, 1854

[0200] 1855, 1856, 1857, 1858, 1859, 1860, 1861, 1862

[0201] 1863, 1864, 1865, 1866, 1867, 1868, 1869, 1870

[0202] 1871, 1872, 1873, 1874, 1875, 1876, 1877, 1878

[0203] 1879, 1880, 1881, 1882, 1883, 1884, 1885, 1886

[0204] 1887, 1888, 1889, 1890, 1891, 1892, 1893, 1894

[0205] 1,895, 1,896, 1,897, 1,898, 1,899, 1,900, 1,901, 1,902

[0206] 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910

[0207] 1911, 1912, 1913, 1914, 1915, 1916, 1917, 1918

[0208] 1919, 1920, 1921, 1922, 1923, 1924, 1925, 1926

[0209] 1927, 1928, 1929, 1930, 1931, 1932, 1933, 1934

[0210] 1935, 1936, 1937, 1938, 1939, 1940, 1941, 1942

[0211] 1943, 1944, 1945, 1946, 1947, 1948, 1949, 1950

[0212] 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959, 1960, 1961, 1962, 1963, 1964, 1965, 1966, 1967, 1968, 1969, 1970, 1971, 1972, 1973, 1974, 1975, 1976, 197 7, 1978, 1979, 1980, 1981, 1982, 1983, 1984, 1985, 1986, 1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 1999, or 2000 (or any range thereof) amino acids or nucleic acids.

[0213] In some embodiments, the polypeptide comprises SEQ ID NO:1 to SEQ ID NO: 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 16 6, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214215, 216, 217, 218, 219, 220, 221, 222, 223, 224

[0214] 1, 225, 226, 227, 228, 229, 230, 231, 232, 233

[0215] 1, 234, 235, 236, 237, 238, 239, 240, 241, 242

[0216] 1, 243, 244, 245, 246, 247, 248, 249, 250, 251

[0217] 1, 252, 253, 254, 255, 256, 257, 258, 259, 260

[0218] 261, 262, 263, 264, 265, 266, 267, 268, 269

[0219] 1, 270, 271, 272, 273, 274, 275, 276, 277, 278

[0220] 1, 279, 280, 281, 282, 283, 284, 285, 286, 287

[0221] 1, 288, 289, 290, 291, 292, 293, 294, 295, 296

[0222] 1, 297, 298, 299, 300, 301, 302, 303, 304, 305

[0223] 306, 307, 308, 309, 310, 311, 312, 313, 314

[0224] 315, 316, 317, 318, 319, 320, 321, 322, 323

[0225] 324, 325, 326, 327, 328, 329, 330, 331, 332

[0226] 333, 334, 335, 336, 337, 338, 339, 340, 341

[0227] 1, 342, 343, 344, 345, 346, 347, 348, 349, 350

[0228] 351, 352, 353, 354, 355, 356, 357, 358, 359

[0229] 1, 360, 361, 362, 363, 364, 365, 366, 367, 368

[0230] 369, 370, 371, 372, 373, 374, 375, 376, 377

[0231] 378, 379, 380, 381, 382, ​​383, 384, 385, 386

[0232] 387, 388, 389, 390, 391, 392, 393, 394, 395

[0233] 1, 396, 397, 398, 399, 400, 401, 402, 403, 404

[0234] 405, 406, 407, 408, 409, 410, 411, 412, 413

[0235] 414, 415, 416, 417, 418, 419, 420, 421, 422

[0236] 423, 424, 425, 426, 427, 428, 429, 430, 431

[0237] 1, 432, 433, 434, 435, 436, 437, 438, 439, 440

[0238] 441, 442, 443, 444, 445, 446, 447, 448, 449

[0239] 450, 451, 452, 453, 454, 455, 456, 457, 458

[0240] 459, 460, 461, 462, 463, 464, 465, 466, 467

[0241] 468, 469, 470, 471, 472, 473, 474, 475, 476

[0242] 477, 478, 479, 480, 481, 482, 483, 484, 485

[0243] 486, 487, 488, 489, 490, 491, 492, 493, 494

[0244] 1, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 5 57, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588 A series of amino acids, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 (or any range thereof).

[0245] In some embodiments, the polypeptide comprises SEQ ID NO:1 to SEQ ID NO: 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 16 6, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710

[0246] 711, 712, 713, 714, 715, 716, 717, 718, 719

[0247] 720, 721, 722, 723, 724, 725, 726, 727, 728

[0248] 729, 730, 731, 732, 733, 734, 735, 736, 737

[0249] 738, 739, 740, 741, 742, 743, 744, 745, 746

[0250] 747, 748, 749, 750, 751, 752, 753, 754, 755

[0251] 756, 757, 758, 759, 760, 761, 762, 763, 764

[0252] 765, 766, 767, 768, 769, 770, 771, 772, 773

[0253] 774, 775, 776, 777, 778, 779, 780, 781, 782

[0254] 783, 784, 785, 786, 787, 788, 789, 790, 791

[0255] 792, 793, 794, 795, 796, 797, 798, 799, 800

[0256] 801, 802, 803, 804, 805, 806, 807, 808, 809

[0257] 810, 811, 812, 813, 814, 815, 816, 817, 818

[0258] 819, 820, 821, 822, 823, 824, 825, 826, 827

[0259] 828, 829, 830, 831, 832, 833, 834, 835, 836

[0260] 837, 838, 839, 840, 841, 842, 843, 844, 845

[0261] 846, 847, 848, 849, 850, 851, 852, 853, 854

[0262] 855, 856, 857, 858, 859, 860, 861, 862, 863

[0263] 1, 864, 865, 866, 867, 868, 869, 870, 871, 872

[0264] 1, 873, 874, 875, 876, 877, 878, 879, 880, 881

[0265] 1, 882, 883, 884, 885, 886, 887, 888, 889, 890

[0266] 891, 892, 893, 894, 895, 896, 897, 898, 899

[0267] 1, 900, 901, 902, 903, 904, 905, 906, 907, 908

[0268] 909, 910, 911, 912, 913, 914, 915, 916, 917

[0269] 918, 919, 920, 921, 922, 923, 924, 925, 926

[0270] 927, 928, 929, 930, 931, 932, 933, 934, 935

[0271] 1, 936, 937, 938, 939, 940, 941, 942, 943, 944

[0272] 1, 945, 946, 947, 948, 949, 950, 951, 952, 953

[0273] 1, 954, 955, 956, 957, 958, 959, 960, 961, 962

[0274] 1, 963, 964, 965, 966, 967, 968, 969, 970, 971

[0275] 1, 972, 973, 974, 975, 976, 977, 978, 979, 980

[0276] 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or any range thereof) consecutive amino acids, which, together with SEQ ID NO:1 to SEQ ID One of NO:10 has at least or at most or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity, identity, or homology.

[0277] The polypeptides or polynucleotides disclosed herein may contain at least, at most, or exactly 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 1, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 1, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 1 17, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216

[0278] 1, 217, 218, 219, 220, 221, 222, 223, 224, 225

[0279] 1, 226, 227, 228, 229, 230, 231, 232, 233, 234

[0280] 1, 235, 236, 237, 238, 239, 240, 241, 242, 243

[0281] 1, 244, 245, 246, 247, 248, 249, 250, 251, 252

[0282] 1, 253, 254, 255, 256, 257, 258, 259, 260, 261

[0283] 1, 262, 263, 264, 265, 266, 267, 268, 269, 270

[0284] 1, 271, 272, 273, 274, 275, 276, 277, 278, 279

[0285] 1, 280, 281, 282, 283, 284, 285, 286, 287, 288

[0286] 1, 289, 290, 291, 292, 293, 294, 295, 296, 297

[0287] 1, 298, 299, 300, 301, 302, 303, 304, 305, 306

[0288] 307, 308, 309, 310, 311, 312, 313, 314, 315

[0289] 316, 317, 318, 319, 320, 321, 322, 323, 324

[0290] 325, 326, 327, 328, 329, 330, 331, 332, 333

[0291] 334, 335, 336, 337, 338, 339, 340, 341, 342

[0292] 343, 344, 345, 346, 347, 348, 349, 350, 351

[0293] 1, 352, 353, 354, 355, 356, 357, 358, 359, 360

[0294] 361, 362, 363, 364, 365, 366, 367, 368, 369

[0295] 370, 371, 372, 373, 374, 375, 376, 377, 378

[0296] 379, 380, 381, 382, ​​383, 384, 385, 386, 387

[0297] 388, 389, 390, 391, 392, 393, 394, 395, 396

[0298] 1, 397, 398, 399, 400, 401, 402, 403, 404, 405

[0299] 406, 407, 408, 409, 410, 411, 412, 413, 414

[0300] 415, 416, 417, 418, 419, 420, 421, 422, 423

[0301] 424, 425, 426, 427, 428, 429, 430, 431, 432

[0302] 1, 433, 434, 435, 436, 437, 438, 439, 440, 441

[0303] 442, 443, 444, 445, 446, 447, 448, 449, 450

[0304] 451, 452, 453, 454, 455, 456, 457, 458, 459

[0305] 460, 461, 462, 463, 464, 465, 466, 467, 468

[0306] 469, 470, 471, 472, 473, 474, 475, 476, 477

[0307] 478, 479, 480, 481, 482, 483, 484, 485, 486

[0308] 1, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 5 15, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572 Replacement of 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, or 600.

[0309] The substitution can be located in SEQ ID NO:1 to SEQ ID NO: One of the amino acid positions or nucleic acid positions: 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 1 45, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766、767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 82 5, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 94 2, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999 or 1000.

[0310] The polypeptides described herein can be of a fixed length of at least, at most, or exactly 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62. 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 1 20, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 or more than 500 amino acids (or any range thereof).

[0311] Substitution variants typically involve the exchange of one amino acid for another at one or more sites within a protein or polypeptide, and can be designed to modulate one or more properties of the polypeptide, with other functions or properties retained or lost. Substitutions can be conserved, meaning one amino acid is replaced by an amino acid with a similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following variations: alanine to serine; arginine to lysine; asparagine-glutamine or histidine; aspartic acid to glutamate; cysteine ​​to serine; glutamine to asparagine; glutamate to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions can be non-conservative, thus affecting the function or activity of the peptide. Non-conservative changes typically involve replacing residues with residues of different chemical properties, such as replacing non-polar or uncharged amino acids with polar or charged amino acids, and vice versa.

[0312] The protein can be recombinant or synthesized in vitro. Alternatively, non-recombinant or recombinant proteins can be isolated from bacteria. It is also anticipated that bacteria containing such variants can be incorporated into the composition and method. Therefore, protein isolation is not required.

[0313] The term “functionally equivalent codon” is used in this document to refer to a codon that encodes the same amino acid, such as the six codons for arginine or serine, and also to a codon that encodes a biologically equivalent amino acid.

[0314] It should also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, but remain substantially identical to one of the sequences disclosed herein, provided that the sequence meets the above criteria, including the maintenance of biological protein activity involved in protein expression. The addition of terminal sequences is particularly applicable to nucleic acid sequences, such as various non-coding sequences including those flanking the 5' or 3' ends of coding regions.

[0315] The following discussion focuses on creating equivalent or even improved second-generation molecules based on altering the amino acids of proteins. For example, certain amino acids can be substituted for other amino acids in a protein structure without a significant loss of their cross-linking ability. Such structures, such as enzyme catalytic domains or interacting components, can retain this function by having the substituted amino acids. Since the biological activity of a protein is determined by its interacting ability and properties, certain amino acid substitutions can be made in the protein sequence and its underlying DNA coding sequence, yet proteins with similar properties can still be produced. Therefore, the inventors anticipate that various alterations can be made to the DNA sequence of a gene without a significant loss of its biological utility or activity.

[0316] In other embodiments, peptide function is altered by introducing one or more substitutions. For example, certain amino acids can substitute for other amino acids in a protein structure to change the binding affinity of interacting components. Structures such as protein-protein interaction domains, nucleic acid interaction domains, and catalytic sites can have substituted amino acids to modify this function. Since the interacting affinity and properties of a protein determine its biological activity, certain amino acid substitutions can be made in the protein sequence and its underlying DNA coding sequence, yet still produce proteins with different properties. Therefore, the inventors anticipate that various alterations can be made in the DNA sequence of a gene, significantly changing its biological utility or activity.

[0317] When making such changes, the hydrophilicity index of amino acids can be considered. The importance of the hydrophilic amino acid index in conferring interactive biological functions of proteins is widely understood in the field (Kyte and Doolittle, 1982). It is generally accepted that the relative hydrophilicity of amino acids contributes to the formation of protein secondary structures, which in turn defines the interactions between proteins and other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0318] It is also understood in the art that similar amino acid substitutions can be efficiently performed based on hydrophilicity. U.S. Patent 4,554,101 (incorporated herein by reference) states that the maximum local average hydrophilicity of a protein (controlled by the hydrophilicity of its adjacent amino acids) is related to the protein's biological properties. It is understood that an amino acid can be substituted by another amino acid with a similar hydrophilicity value and still produce a biologically and immunologically equivalent protein.

[0319] As mentioned above, amino acid substitutions are typically based on the relative similarity of the substituents in the amino acid side chains, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account the various foregoing characteristics are well known and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0320] In specific implementations, all or part of the proteins described herein can also be synthesized in solution or on a solid support using conventional techniques. Various automated synthesizers are commercially available and can be used according to known methods. See, for example, Stewart and Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference. Alternatively, recombinant DNA techniques can be employed, in which a nucleotide sequence encoding a peptide or polypeptide is inserted into an expression vector, transformed or transfected into a suitable host cell, and cultured under suitable expression conditions. One implementation includes the application of transferring genes into cells, including microorganisms, to prepare and / or present proteins. The gene for the target protein can be transferred into a suitable host cell, and the cell can then be cultured under suitable conditions. Nucleic acids encoding virtually any polypeptide can be used. The generation of recombinant expression vectors and the elements contained therein are discussed herein. Alternatively, the protein to be prepared can be an endogenous protein synthesized by cells as commonly used in protein preparation.

[0321] II. Gene Transmission

[0322] Certain aspects of this disclosure include administering a TERT-activating therapeutic agent to a subject. This may include administering TERT nucleic acid and / or a polypeptide to the subject. TERT nucleic acid may include a TERT gene, protein, or mRNA encoded on DNA or RNA. In some embodiments, the method includes administering DNA encoding a TERT polypeptide to the subject. In some embodiments, the method includes administering RNA encoding a TERT polypeptide to the subject. Techniques related to transferring nucleic acids into cells are well known to those skilled in the art. Exemplary techniques are discussed below.

[0323] A. Viral vector

[0324] In some implementations, the transfer of the expression construct into cells is accomplished using a viral vector. The technique of using a "viral vector" is well known in the art. Viral vectors are intended to comprise constructs containing a viral sequence sufficient to (a) support the expression cassette and (b) ultimately express the recombinant gene construct cloned therein.

[0325] In a particular implementation, the viral vector is a lentiviral vector. Lentiviral vectors have been successfully used to infect stem cells and provide long-term expression.

[0326] Another nucleic acid delivery method involves the use of adenoviral vectors. Adenoviral vectors are known to have a low ability to integrate into genomic DNA. However, adenoviral vectors lead to highly efficient gene transfer.

[0327] Adenoviruses are currently the most commonly used gene transfer vectors in clinical settings. One of the advantages of these viruses is their ability to efficiently deliver genes to both non-dividing and dividing cells, and their capacity for mass production. The vector contains genetically engineered forms of adenovirus (Grunhaus et al., 1992). Unlike retroviruses, adenovirus infection of host cells does not lead to chromosomal integration because adenovirus DNA can replicate in a free manner without potential genotoxicity. Furthermore, adenoviruses are structurally stable, and no genomic rearrangements have been detected after extensive amplification.

[0328] Adenoviruses are particularly well-suited as gene transfer vectors due to their medium-sized genome, ease of manipulation, high titers, broad target cell range, and high infectivity. Experimental methods using adenoviral vectors are well-known to those skilled in the art.

[0329] The adenovirus vector may be replication-deficient, or at least conditionally replication-deficient, and the nature of the adenovirus vector is not considered critical to the successful practice of the present invention. The adenovirus can be any of the 42 different known serotypes or subgroups AF, and other serotypes or subgroups are contemplated. Subgroup C type 5 adenovirus is the starting material for obtaining the conditionally replication-deficient adenovirus vector used in the present invention. This is because type 5 adenovirus is a known human adenovirus with abundant biochemical and genetic information, and historically it has been used in most constructs using adenovirus as a vector. Adenovirus growth and manipulation are known to those skilled in the art, and it exhibits a broad host range in vitro and in vivo. Modified viruses, such as adenoviruses with altered CAR domains, may also be used. Methods for enhancing delivery or evading immune responses, such as liposome encapsulation of the virus, are also contemplated. Retroviruses are a group of single-stranded RNA viruses characterized by their ability to convert their RNA into double-stranded DNA in infected cells via a reverse transcription process (Coffin, 1990). The resulting DNA then integrates stably into the cell chromosome as a provirus and directs the synthesis of viral proteins. Integration results in the preservation of the viral gene sequence in the recipient cell and its progeny. The retroviral genome contains two long terminal repeat (LTR) sequences located at the 5′ and 3′ ends of the viral genome. These sequences contain strong promoter and enhancer sequences and are essential for integration into the host cell genome (Coffin, 1990).

[0330] To construct a retroviral vector, a nucleic acid encoding a nucleic acid or gene of interest is inserted into the viral genome, replacing certain viral sequences to generate a replication-defective virus. Well-known techniques for constructing retroviral vectors are familiar to those skilled in the art.

[0331] Adeno-associated virus (AAV) is an attractive vector system for use in this invention because of its high integration frequency and ability to infect non-dividing cells, making it suitable for gene delivery into mammalian cells in tissue culture (Muzyczka, 1992). AAV has a broad host range for infection (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988), which means it is suitable for use in this invention. Details regarding the production and use of rAAV vectors are described in U.S. Patents 5,139,941 and 4,797,368, both of which are incorporated herein by reference.

[0332] Typically, recombinant AAV (rAAV) viruses are wild-type AAV coding sequences without terminal repeats prepared by co-transfection of a plasmid containing the gene of interest and an expression plasmid, such as pIM45 (McCarty et al., 1991; incorporated herein by reference). Techniques that can be used to generate vectors using AAV viruses are familiar to those skilled in the art.

[0333] Herpes simplex virus (HSV) has generated considerable interest in treating neurological disorders due to its tropism towards neurons, but given its broad host range, the vector can also be used in other tissues. Another factor making HSV an attractive vector is its genome size and organization. Because HSV is large, there are fewer issues with incorporating multiple genes or expression cassettes compared to smaller viral systems. Furthermore, the availability of different viral control sequences with varying properties (timing, strength, etc.) allows for greater control over expression than with other systems. The relatively limited splicing information of the virus further simplifies gene manipulation, which is also an advantage.

[0334] HSV is also relatively easy to manipulate and can be grown to high titers. Therefore, delivery is not an issue in terms of obtaining the volume required for a sufficient MOI and reducing the need for repeated administrations. For a review of HSV as a gene therapy vector, see Glorioso et al., (1995). Those skilled in the art are familiar with well-known techniques for using HSV as a vector.

[0335] Vaccinia virus vectors are widely used due to their ease of construction, relatively high expression levels, broad host range, and large DNA carrying capacity. Vaccinia contains a linear double-stranded DNA genome of approximately 186 kb, exhibiting a pronounced "AT" preference. Approximately 10.5 kb of inverted terminal repeats are located on either side of the genome.

[0336] Other viral vectors can be used as constructs in this invention. For example, vectors derived from viruses such as poxviruses can be used. Molecular clones of Venezuelan equine encephalitis (VEE) virus have been genetically modified as replicative vaccine vectors expressing heterologous viral proteins (Davis et al., 1996). Studies have shown that VEE infection stimulates an effective CTL response, and VEE is considered to be a potentially very useful immune vector (Caley et al., 1997). This invention anticipates that VEE virus can be used to target dendritic cells.

[0337] Polynucleotides can be contained within viral vectors designed to express specific binding ligands. Thus, viral particles will specifically bind to homologous receptors on target cells and deliver their contents into the cells. Based on the chemical modification of retroviruses by adding lactose residues to the viral envelope, a novel approach has been developed to allow for the specific targeting of retroviral vectors. This modification allows for specific infection of hepatocytes via the sialic acid glycoprotein receptor.

[0338] Another method for targeting recombinant retroviruses was designed, which utilizes biotinylated antibodies against retroviral envelope proteins and specific cellular receptors. Streptavidin was used to conjugate antibodies via biotinylated components (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and II antigens, they demonstrated that the isotropic virus infected various human cells carrying these surface antigens in vitro (Roux et al., 1989).

[0339] B. Non-viral gene transfer

[0340] Some aspects of the invention also contemplate several non-viral methods for transferring nucleic acids into cells. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990), DEAE-glucan (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), nuclear transfection (Trompeter et al., 2003), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al., 1979) and lipid transfection of amine-DNA complexes, polyamino acids, cytosonic waves (Fechheimer et al., 1987), gene bombardment using high-speed microparticles (Yang et al., 1990), polycations (Boussif et al., 1995), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques can be successfully adapted for in vivo or in vitro use. Those skilled in the art will be familiar with techniques relating to the use of nonviral vectors and will understand that the present invention covers other types of nonviral vectors besides those disclosed herein. In another embodiment of the invention, the expression cassette may be encapsulated in liposomes or lipid formulations. A liposome is a vesicle structure characterized by a phospholipid bilayer and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. Gene constructs complexed with Lipofectamine (Gibco BRL) are also contemplated. Techniques utilizing liposomes and lipid formulations are familiar to those skilled in the art.

[0341] C. Lipid-based nanovesicles

[0342] In some embodiments, lipid-based nanovesicles, such as liposomes, extracellular bodies, lipid formulations, and lipid-based vesicles (e.g., DOTAP: cholesterol vesicles), are employed in the methods of this disclosure. In some embodiments, nanovesicles containing a TERT polypeptide or a nucleic acid encoding a TERT polypeptide are applied to a target. Lipid-based nanovesicles may be positively charged, negatively charged, or electrically neutral.

[0343] 1. Liposomes

[0344] "Liposome" is a general term encompassing various monolayer and multilayer lipid carriers formed by creating closed lipid bilayers or aggregates. Liposomes are characterized by a vesicular structure having a bilayer membrane, typically containing phospholipids, and an internal mediator that typically contains an aqueous composition. The liposomes described herein include monolayer liposomes, multilayer liposomes, and multivesicular liposomes. The liposomes described herein can be positively charged, negatively charged, or electrically neutral. In some embodiments, the liposomes are electrically neutral.

[0345] Multilayered liposomes consist of multiple lipid layers separated by an aqueous medium. These liposomes spontaneously form when phospholipid-containing lipids are suspended in excess aqueous solution. The lipid components rearrange themselves before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers. Lipophilic molecules, or molecules with lipophilic regions, can also dissolve in or bind to the lipid bilayer.

[0346] For example, in some implementations, peptides, nucleic acids, or small molecule drugs can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, linked to liposomes via linker molecules associated with liposomes and peptides / nucleic acids, encapsulated within liposomes, complexed with liposomes, etc.

[0347] The liposomes used according to this embodiment can be prepared by various methods known to those skilled in the art. For example, phospholipids (such as neutral dioleoylphosphatidylcholine (DOPC)) are dissolved in tert-butanol. The lipids are then mixed with peptides, nucleic acids, and / or other components. Tween 20 is added to the lipid mixture such that it is about 5% by mass of the composition. An excess of tert-butanol is added to the mixture such that the volume of tert-butanol is at least 95%. The mixture is vortexed, frozen in a dry ice / acetone bath, and lyophilized overnight. The lyophilized formulation can be stored at -20°C for up to three months. When needed, the lyophilized liposomes are reconstituted in 0.9% saline.

[0348] Alternatively, liposomes can be prepared by mixing lipids with a solvent in a container, such as a pear-shaped glass flask. The volume of the container should be ten times the volume of the desired liposome suspension. The solvent is removed using a rotary evaporator at approximately 40°C and under negative pressure. Depending on the desired liposome volume, the solvent is typically removed over approximately 5 minutes to 2 hours. The composition can be further dried in a desiccator under vacuum. The dried lipids are usually discarded after about one week, as they deteriorate over time.

[0349] The dried lipids can be hydrated in sterile, pyrogen-free water containing approximately 25 nM to 50 nM phospholipids by agitation until all lipid membranes are resuspended. The hydrated liposomes can then be aliquoted, placed in vials, lyophilized, and sealed under vacuum.

[0350] The dried lipids or lyophilized liposomes prepared as described above can be dehydrated and reconstituted in a solution of protein or peptides, and diluted to a suitable concentration with a suitable solvent such as DPBS. The mixture is then vigorously shaken in a vortex mixer. Unencapsulated additives, such as reagents including but not limited to hormones, drugs, nucleic acid constructs, etc., are removed by centrifugation at 29000×g, and the liposome pellets are washed. The washed liposomes are resuspended at an appropriate total phospholipid concentration, for example, about 50 nM to 200 nM. The amount of encapsulated additives or active agents can be determined according to standard methods. Once the amount of additives or active agents encapsulated in the liposome formulation is determined, the liposomes can be diluted to a suitable concentration and stored at 4°C until use. Pharmaceutical compositions containing liposomes typically include a sterile, pharmaceutically acceptable carrier or diluent, such as water or an aqueous saline solution.

[0351] Other liposomes that can be used in embodiments of this disclosure include cationic liposomes, such as those described in WO02 / 100435A1, U.S. Patent No. 5,962,016, U.S. Applications 2004 / 0208921, WO03 / 015757A1, WO04029213A2, U.S. Patent No. 5,030,453, and U.S. Patent No. 6,680,068, all of which are incorporated herein by reference in their entirety without disclaimer.

[0352] In preparing such liposomes, any method described herein or known to those skilled in the art may be used. Further non-limiting examples of liposome preparation are described in U.S. Patent Nos. 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706, and International Applications PCT / US85 / 01161 and PCT / US89 / 05040, all of which are incorporated herein by reference.

[0353] In some embodiments, the lipid-based nanovesicles are neutral liposomes (e.g., DOPC liposomes). As used herein, “neutral liposome” or “uncharged liposome” is defined as a liposome having one or more lipid components capable of producing a substantially neutral net charge (substantially uncharged). “Substantially neutral” or “substantially uncharged” means that a given population (e.g., a liposome population) contains very little (if any) charge that is not offset by the opposite charge of another component (i.e., less than 10% of the component contains unoffset charge, more preferably less than 5%, and most preferably less than 1%). In some embodiments, neutral liposomes may consist primarily of lipids and / or phospholipids that are themselves neutral under physiological conditions (i.e., at about pH 7).

[0354] The liposomes and / or lipid-based nanovesicles of this embodiment may contain phospholipids. In some embodiments, a single type of phospholipid may be used to prepare liposomes (e.g., neutral phospholipids such as DOPC may be used to prepare neutral liposomes). In other embodiments, more than one type of phospholipid may be used to generate liposomes. Phospholipids may be derived from natural or synthetic sources. Phospholipids include, for example, phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine; because phosphatidylethanolamine and phosphatidylcholine are uncharged under physiological conditions (i.e., approximately pH 7), these compounds may be particularly suitable for generating neutral liposomes. In some embodiments, the phospholipid DOPC is used to prepare uncharged liposomes. In some embodiments, non-phospholipid lipids (e.g., cholesterol) may be used.

[0355] Phospholipids include glycerophospholipids and certain sphingolipids. Phospholipids include, but are not limited to, dioleoylphosphatidylcholine (“DOPC”), lecithinylcholine (“EPC”), dilauroylphosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine (“DMPC”), dipalmitoylphosphatidylcholine (“DPPC”), distearylphosphatidylcholine (“DSPC”), 1-myristoyl-2-palmitoylphosphatidylcholine (“MPPC”), 1-palmitoyl-2-myristoylphosphatidylcholine (“PMPC”), 1-palmitoyl-2- Stearoyl phosphatidylcholine (“PSPC”), 1-stearoyl-2-palmitoyl phosphatidylcholine (“SPPC”), dilauroyl phosphatidylglycerol (“DLPG”), dimyristoyl phosphatidylglycerol (“DMPG”), dipalmitoyl phosphatidylglycerol (“DPPG”), distearyl phosphatidylglycerol (“DSPG”), distearyl sphingomyelin (“DSSP”), distearyl phosphatidylethanolamine (“DSPE”), dioleoyl phosphatidylglycerol (“DOPG”), dimyristoyl phosphatidylglycerol (“DOPG”), dimyristoyl phosphatidylglycerol (“DSPG”). Phosphatidic acid (“DMPA”), dipalmitoylphosphatidic acid (“DPPA”), dimyristoylphosphatidylethanolamine (“DMPE”), dipalmitoylphosphatidylethanolamine (“DPPE”), dimyristoylphosphatidylserine (“DMPS”), dipalmitoylphosphatidylserine (“DPPS”), cerebrophosphatidylserine (“BPS”), cerebrosphingomyelin (“BSP”), dipalmitoylsphingomyelin (“DPSP”), dimyristoylphosphatidylcholine (“DMPC”), 1,2- Distearatel-sn-glycerol-3-phosphate choline (“DAPC”), 1,2-disarachidoyl-sn-glycerol-3-phosphate choline (“DBPC”), 1,2-dienoenoyl-sn-glycerol-3-phosphate choline (“DEPC”), dioleoylphosphatidylethanolamine (“DOPE”), palmitoyloleoylphosphatidylcholine (“POPC”), palmitoyloleoylphosphatidylethanolamine (“POPE”), lysophosphatidylcholine, lysophosphatidylethanolamine and dilinoleoylphosphatidylcholine.

[0356] 2. Extracellular body

[0357] As used herein, the terms "nanovesicles" and "exosomes" refer to membrane-like particles having a diameter of about 10 nm to about 1000 nm, more typically between 30 nm and 1000 nm, and most typically between about 50 nm and 750 nm (or the maximum size when the particle is not spherical), wherein at least a portion of the extracellular membrane is directly derived from the cell. Most commonly, the size (average diameter) of extracellular bodies is up to 5% of the size of the donor cell. Therefore, extracellular bodies particularly considered include those that detach from the cell.

[0358] Extracellular bodies can be detected or isolated in any suitable sample type, such as body fluids. As used herein, the term "isolated" means isolated from its natural environment and is intended to include at least partial purification and may include substantial purification. As used herein, the term "sample" means any sample suitable for the methods provided by the present invention. A sample can be any sample that includes extracellular bodies suitable for detection or isolation. Sample sources include blood, bone marrow, pleural fluid, peritoneal fluid, cerebrospinal fluid, urine, saliva, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, breast milk, sweat, tears, synovial fluid, and bronchial lavage fluid. In one aspect, the sample is a blood sample, including, for example, whole blood or any part or component thereof. Blood samples suitable for the present invention can be extracted from any known source, including blood cells or components thereof, such as veins, arteries, peripheral blood, tissues, umbilical cords, etc. For example, samples can be obtained and processed using well-known routine clinical methods (e.g., procedures for drawing and processing whole blood). In another aspect, an exemplary sample may be peripheral blood drawn from a subject suffering from a disease.

[0359] Extracellular bodies can be isolated from freshly collected samples or from frozen or refrigerated samples. In some embodiments, extracellular bodies can be isolated from cell culture media. While not mandatory, higher purity extracellular bodies can be obtained if the fluid sample is clarified to remove any debris from the sample before volumetric exclusion of polymer precipitates. Clarification methods include centrifugation, ultracentrifugation, filtration, or ultrafiltration. Most typically, extracellular bodies can be isolated by a variety of methods well known in the art. A preferred method is differential centrifugation from body fluids or cell culture supernatants. Exemplary methods for isolating extracellular bodies are described in (Losche et al., 2004; Mesri and Altieri, 1998; Morel et al., 2004). Alternatively, extracellular bodies can also be isolated by flow cytometry, as described in (Combes et al., 1997).

[0360] A commonly accepted method for extracellular body isolation involves ultracentrifugation, typically combined with a sucrose density gradient or sucrose pad, to allow relatively low-density extracellular bodies to float. Sequential differential centrifugation complicates the separation of extracellular bodies because their size distribution may overlap with other microvesicles or macromolecular complexes. Furthermore, depending on the size of the microvesicles, centrifugation may not provide a sufficient method for vesicle separation. However, combining sequential centrifugation with sucrose gradient ultracentrifugation can provide a high concentration of extracellular bodies.

[0361] Separating extracellular bodies by size using ultracentrifugation is another alternative. Successful purification of extracellular bodies using ultrafiltration has been reported; this procedure is less time-consuming than ultracentrifugation and does not require specialized equipment. Similarly, commercial kits (EXOMIR) can be used. TM Bioo Scientific's method removes cells, platelets, and cell debris from a first microfilter and uses positive pressure-driven fluid to capture vesicles larger than 30 nm on a second microfilter. However, extracellular bodies are not recovered during this process; their RNA content is extracted directly from the material captured on the second microfilter and can then be used for PCR analysis. HPLC-based methods may allow for the acquisition of high-purity extracellular bodies, although these processes require specialized equipment and are difficult to scale up. A significant issue is that both blood and cell culture media contain a large number of nanoparticles (some non-vesicles) in the same size range as extracellular bodies. For example, some miRNAs may be contained within extracellular protein complexes rather than in extracellular bodies; however, protease treatment (e.g., proteinase K) can be performed to eliminate any potential "exosome" protein contamination.

[0362] a. An exemplary protocol for collecting extracellular bodies from cell cultures

[0363] On Day 1, seed sufficient cells (e.g., approximately 5 million cells) in a T225 flask containing 10% FBS to allow for approximately 70% cell confluence by Day 2. On Day 2, aspirate the culture medium from the cells, wash them twice with PBS, and then add 25 to 30 mL of basal medium (i.e., PenStrep-free or FBS-free) to the cells. Incubate the cells for 24 to 48 hours. 48 hours of incubation is preferred, but some cell lines are more sensitive to serum-free medium, so the incubation time should be reduced to 24 hours. Note that FBS contains extracellular bodies that can severely distort NanoSight results.

[0364] On days 3 / 4, collect the culture medium and centrifuge at 800×g for 5 minutes at room temperature to precipitate dead cells and large debris. Transfer the supernatant to a new conical tube and centrifuge again at 2000×g for 10 minutes to remove any remaining large debris and vesicles. Pass the medium through a 0.2 μm filter and then transfer it to ultracentrifuge tubes (e.g., 25×89 mm Beckman Ultra-Clear) in 35 mL aliquots per tube. If the medium volume per tube is less than 35 mL, fill the remaining portion of the PBS tube to reach 35 mL. Ultracentrifuge the medium at 28,000 rpm for 2 to 4 hours at 4°C using an SW 32Ti rotor (k-factor 266.7, RCF max 133907). Carefully aspirate the supernatant until approximately 1 inch of liquid remains. Tilt the tube to allow the remaining medium to slowly enter the pipette. If necessary, the extracellular particles can be resuspended in PBS and ultracentrifuged again at 28,000 rpm for 1 to 2 hours to further purify the extracellular population.

[0365] Finally, resuspend the extracellular particles in 210 μL of PBS. If multiple ultracentrifuge tubes are used for each sample, resuspend each extracellular particle consecutively in the same 210 μL of PBS. For each sample, take 10 μL and add it to 990 μL of H2O for nanoparticle tracking analysis. Use the remaining 200 μL of extracellular suspension for downstream processes or store immediately at -80°C.

[0366] b. Exemplary methods for extracting extracellular bodies from serum samples

[0367] First, thaw the serum sample on ice. Then, dilute 250 μL of cell-free serum sample in 11 mL PBS; filter through a 0.2 μm pore filter. Centrifuge the diluted sample overnight at 150,000 × g at 4 °C. The next day, carefully discard the supernatant and wash the extracellular pellet in 11 mL PBS. Perform a second round of ultracentrifugation at 150,000 × g for 2 hours at 4 °C. Finally, carefully discard the supernatant and resuspend the extracellular particles in 100 μL PBS for analysis.

[0368] c. Exemplary methods for extracellular and liposome electroporation

[0369] 1×10 8Extracellular bodies (measured by NanoSight analysis) or 100 nm liposomes (e.g., purchased from Encapsula Nano Sciences) and 1 μg siRNA (Qiagen) or shRNA were mixed in 400 μL of electroporation buffer (1.15 mM potassium phosphate, pH 7.2, 25 mM potassium chloride, 21% Optiprep). The extracellular bodies or liposomes were electroporated using 4 mm cuvettes (see, e.g., Alvarez-Erviti et al., 2011; El-Andaloussi et al., 2012). After electroporation, the extracellular bodies or liposomes were treated with protease-free RNase followed by the addition of a 10-fold concentrated RNase inhibitor. Finally, the extracellular bodies or liposomes were washed with PBS by ultracentrifugation as described above.

[0370] d. Application of therapeutic extracellular substances

[0371] Certain aspects of this disclosure provide for treating patients with extracellular bodies expressing or containing therapeutic agents (e.g., TERT peptides or nucleic acids). Since extracellular bodies are known to contain the mechanisms necessary for completing mRNA transcription and protein translation (see PCT / US2014 / 068630, which is incorporated herein by reference in its entirety), mRNA or DNA nucleic acids encoding therapeutic proteins can be transfected into extracellular bodies. Alternatively, the therapeutic protein itself can be electroporated into the extracellular body or directly incorporated into liposomes. In some embodiments, the extracellular body further contains additional therapeutic agents, such as those described herein.

[0372] This article provides methods and drugs for using engineered liposomes and extracellular bodies as delivery systems for treating diseases.

[0373] 3. Nanovesicles expressing CD47

[0374] In some embodiments, a pharmaceutical composition is provided comprising lipid-based nanovesicles containing CD47 on their surface, wherein the lipid-based nanovesicles contain a TERT polypeptide or a nucleic acid encoding a TERT polypeptide.

[0375] In some respects, lipid-based nanoparticles are liposomes or extracellular bodies. In some respects, extracellular bodies are isolated from cells that overexpress CD47. In some respects, extracellular bodies are isolated from patients requiring treatment. In some respects, extracellular bodies are isolated from fibroblasts. In some respects, liposomes are monolayer liposomes. In some respects, liposomes are multilayer liposomes.

[0376] In some respects, the composition is formulated for parenteral administration, such as intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

[0377] In some respects, the composition contains an antimicrobial agent. The antimicrobial agent may be benzalkonium chloride, benzyl chloride, benzyl alcohol, bromonitrol, bromophthalic acid trimethylammonium, cetylpyridine chloride, chlorhexidine, chlorobutanol, chlorocresol, xyloxyphenol, cresol, ethanol, glycerol, icitidine, amide, phenol, phenoxyethanol, nitrate, phenethylurea propylene glycol, or thimerosal.

[0378] In some respects, a single lipid-based nanovesicle contains more than one agent, such as a TERT peptide or nucleic acid, and one or more additional therapeutic agents as described herein.

[0379] In one embodiment, a method of administering a TERT-activating therapeutic agent to a patient is provided, wherein the TERT-activating therapeutic agent comprises an extracellular body. In some aspects, this disclosure relates to transfecting an extracellular body with a nucleic acid (e.g., DNA or RNA) encoding a TERT polypeptide, incubating the transfected extracellular body under conditions allowing TERT expression in the extracellular body, and providing the incubated extracellular body to a patient, thereby administering TERT-activating therapy to the patient.

[0380] III. Application of the therapeutic composition

[0381] The treatments provided herein may include combined administration of therapeutic agents, such as a first TERT activation therapy and a second treatment. Treatments may be administered in any suitable manner known in the art. For example, the first and second treatments may be administered sequentially (not simultaneously) or simultaneously (in the same dose). In some embodiments, the first and second treatments are administered in separate compositions. In some embodiments, the first and second treatments are in the same composition. In some embodiments, the methods and compositions of this disclosure include the administration of additional treatments. In some embodiments, the additional treatment includes a cholinesterase inhibitor, such as donepezil, galantamine, or rivastigmine. In some embodiments, the additional treatment includes memantine.

[0382] Embodiments of this disclosure relate to compositions and methods of comprising therapeutic compositions. Different treatments can be administered with one or more compositions, such as two, three, or four compositions. Various combinations of pharmaceutical agents can be used, for example, a first treatment is "A" and a second treatment is "B":

[0383] A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / BB / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / AB / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0384] The therapeutic agents disclosed herein can be administered via the same route of administration or via different routes of administration. In some embodiments, treatment is administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, intraorbitally, via implantation, via inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, antibiotics are administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, intraorbitally, via implantation, via inhalation, intrathecally, intraventricularly, or intranasally. Appropriate dosage can be determined based on the type of disease being treated, the severity and duration of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the judgment of the attending physician.

[0385] Treatment may include various “unit doses.” A unit dose is defined as containing a predetermined amount of a therapeutic composition. The dosage, specific route of administration, and formulation are within the capabilities of a person skilled in the clinical field. A unit dose does not need to be administered as a single injection, but may include continuous infusion over a set period of time. In some embodiments, a unit dose includes a single administerable dose.

[0386] The dosage, depending on the number of treatments and unit dose, depends on the desired therapeutic effect. The effective dose should be understood as the amount required to achieve a specific effect. In practice with certain implementation methods, doses in the range of 10 mg / kg to 200 mg / kg are expected to affect the protective efficacy of these agents. Therefore, the anticipated dosage includes doses of approximately 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day or mg / day or any range thereof. Furthermore, such doses may be administered multiple times a day and / or over several days, weeks or months.

[0387] In some embodiments, an effective dose of the pharmaceutical composition is a dose that provides a blood level of about 1 μM to 150 μM. In another embodiment, an effective dose provides a blood level of about 4 μM to 100 μM; or about 1 μM to 100 μM; or about 1 μM to 50 μM; or about 1 μM to 40 μM; or about 1 μM to 30 μM; or about 1 μM to 20 μM; or about 1 μM to 10 μM; or about 10 μM to 150 μM; or about 10 μM to 100 μM; or about 10 μM to 50 μM; or about 25 μM to 150 μM; or about 25 μM to 100 μM; or about 25 μM to 50 μM; or about 50 μM to 150 μM; or about 50 μM to 100 μM (or any range thereof). In other embodiments, the dose may provide blood levels of the following agents produced by administering the therapeutic agent to the subject: approximately, at least approximately, or at most approximately 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or 20 μM. , 21μM, 22μM, 23μM, 24μM, 25μM, 26μM, 27μM, 28μM, 29μM, 30μM, 31μM, 3μM, 33μM, 34μ M, 35μM, 36μM, 37μM, 38μM, 39μM, 40μM, 41μM, 42μM, 43μM, 44μM, 45μM, 46μM, 47μM, 4 8μM, 49μM, 50μM, 51μM, 52μM, 53μM, 54μM, 55μM, 56μM, 57μM, 58μM, 59μM, 60μM, 61μM , 62μM, 63μM, 64μM, 65μM, 66μM, 67μM, 68μM, 69μM, 70μM, 71μM, 72μM, 73μM, 74μM, 75 The ranges are 76 μM, 77 μM, 78 μM, 79 μM, 80 μM, 81 μM, 83 μM, 84 μM, 85 μM, 86 μM, 87 μM, 88 μM, 89 μM, 90 μM, 91 μM, 92 μM, 93 μM, 94 μM, 95 μM, 96 μM, 97 μM, 98 μM, 99 μM, or 100 μM, or any range derived therefrom. In some embodiments, the therapeutic agent administered to the subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood level may refer to the amount of the agent. Alternatively, the blood level discussed herein may refer to unmetabolized therapeutic agent in terms of the extent to which the therapeutic agent is not metabolized by the subject.

[0388] The precise amount of a therapeutic composition also depends on the practitioner's judgment and varies from person to person. Factors affecting dosage include the patient's physical condition and clinical status, route of administration, expected goals of treatment and efficacy (symptom relief versus cure), and the stability and toxicity of the particular therapeutic substance, or other therapies the subject may be receiving.

[0389] Those skilled in the art will understand and recognize that the body weight dose unit of μg / kg or mg / kg can be converted and expressed as a unit of comparable concentration such as μg / ml or mM (blood level), for example, 4 μM to 100 μM. It should also be understood that absorption depends on the disease and organ / tissue. Applicable conversion factors and physiological assumptions regarding uptake and concentration measurements are well known and allow those skilled in the art to convert one concentration measurement to another and to make reasonable comparisons and conclusions regarding the dosages, efficacy, and results described herein.

[0390] IV. Disease Treatment

[0391] The methods disclosed herein can be used to treat or prevent certain age-related diseases, conditions, or disorders. Non-limiting examples of age-related diseases, conditions, or disorders include insulin resistance (i.e., impaired glucose tolerance), benign prostatic hyperplasia, hearing loss, osteoporosis, age-related macular degeneration, neurodegenerative diseases, skin diseases, skin aging, or cancer. Non-limiting examples of neurodegenerative diseases include Alzheimer's disease; epilepsy; Huntington's disease; Parkinson's disease; stroke; spinal cord injury; traumatic brain injury; Lewy body dementia; Pick's disease; Niemann-Pick disease; amyloid angiopathy; cerebral amyloid angiopathy; systemic amyloidosis; hereditary brain hemorrhage of Dutch amyloidosis; inclusion body myositis; mild cognitive impairment; Down syndrome; and neuromuscular diseases, including amyotrophic lateral sclerosis (ALS), multiple sclerosis, and muscular dystrophys (including Dichené muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, and limb girdle muscular dystrophy (LGMD)). It also includes neurodegenerative diseases caused by stroke, head injury, spinal cord injury or other damage to the brain, peripheral nerves, central nervous system or neuromuscular system.

[0392] Some implementations of the methods described herein relate to methods for preventing disease or health-related conditions in subjects. Prevention strategies are crucial in modern medicine.

[0393] In some implementations, the treatment is for progeria or a progeria-related condition. Examples of progeria include Hutchinson-Guilford progeria syndrome (HGPS), Nestor-Guilmore progeria syndrome, adult progeria, Cockayne syndrome, Bloom syndrome, xeroderma pigmentosum, ataxia-telangiectasia, hyposulfur pilaris, congenital keratosis, or mosaic aneuploidy syndrome. In some implementations, one or more of the progeria, progeria-related conditions, age-related diseases, neurodegenerative diseases, or conditions described herein are excluded from the methods of this disclosure.

[0394] V. Reagent Kit

[0395] Some aspects involve kits that contain the compositions described herein or compositions for carrying out the methods described herein.

[0396] In various aspects, kits comprising therapeutic agents and / or other therapeutic agents and delivery agents are envisioned. In some embodiments, kits for preparing and / or administering the treatments described herein may be provided. The kit may include one or more sealed vials containing any pharmaceutical composition, therapeutic agent, and / or other therapeutic agent and delivery agent. In some embodiments, lipids are in one vial, while therapeutic agents are in separate vials. The kit may include, for example, at least one TERT-activating therapeutic agent, one or more lipid components, and reagents for preparing, formulating, and / or administering the components described herein or performing one or more steps of the method. In some embodiments, the kit may also include suitable container devices that do not react with the components of the kit, such as Ependorf tubes, assay plates, syringes, bottles, or tubes. The container may be made of a sterilizable material, such as plastic or glass.

[0397] The kit may also include instructions outlining the procedural steps of the methods described herein, and will follow substantially the same procedures as those described herein or known to a person skilled in the art. Instruction information may be in a computer-readable medium containing machine-readable instructions that, when executed using a computer, result in a real or virtual procedure for delivering a pharmaceutically effective amount of the therapeutic agent.

[0398] In some embodiments, kits can be provided to evaluate the expression of TERT or related molecules. Such kits can be prepared from readily available materials and reagents. For example, such kits may contain any one or more of the following materials: enzymes, reaction tubes, buffers, detergents, primers and probes, nucleic acid amplification and / or hybridization agents. In certain embodiments, these kits allow practitioners to obtain samples from blood, tears, semen, saliva, urine, tissue, serum, feces, colon, rectum, sputum, cerebrospinal fluid, and supernatants from cell lysates. In another embodiment, these kits include apparatus for performing RNA extraction, RT-PCR, and gel electrophoresis. Instructions for performing the assays may also be included in the kit.

[0399] The kit may include components that can be individually packaged or placed in containers, such as tubes, bottles, vials, syringes, or other suitable container devices. Components may include probes, primers, antibodies, arrays, negative and / or positive controls. Individual components may also be provided in the kit in concentrated amounts; in some embodiments, components are provided individually at the same concentration as in solutions containing other components. Component concentrations may be provided at 1x, 2x, 5x, 10x, or 20x or more.

[0400] The kit may also contain reagents for labeling TERT in samples. The kit may also include labeling reagents comprising at least one of amine-modified nucleotides, poly(A) polymerase, and poly(A) polymerase buffer. Labeling reagents may include amine-reactive dyes or any dyes known in the art.

[0401] The components of the kit can be packaged in an aqueous medium or in lyophilized form. The kit's container device typically includes at least one vial, test tube, flask, bottle, syringe, or other container device into which the components are placed and preferably appropriately aliquoted. When the kit contains more than one component (labeling reagents and labels may be packaged together), the kit typically also includes a second, third, or other additional container in which the additional components can be placed separately. However, vials can contain combinations of various components. The kit may also include a device for containing containers of nucleic acids, antibodies, or any other reagents, which are sealed for commercial sale. Such containers can include injection or blow-molded plastic containers in which the required vials are stored.

[0402] When the components of the kit are provided as one and / or more liquid solutions, the liquid solutions are aqueous solutions, and sterile aqueous solutions are particularly preferred.

[0403] Alternatively, the kit components may be provided as a dry powder. When reagents and / or components are provided in dry powder form, the powder can be reconstituted by adding a suitable solvent. It is also contemplated that the solvent may be provided in another container. In some embodiments, the labeling dye is provided as a dry powder. It is anticipated that 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 120 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1000 μg, or at least these amounts of dry dye are provided in the kit in some respects. The dye can then be resuspended in any suitable solvent, such as DMSO.

[0404] The container device typically includes at least one vial, test tube, flask, bottle, syringe, and / or other container device in which the nucleic acid preparation is placed, and preferably appropriately dispensed. The kit may also include a second container device for containing sterile, pharmaceutically acceptable buffers and / or other diluents.

[0405] The kit may include a device for containing vials in a closed space for commercial sale, such as an injection and / or blow-molded plastic container in which the desired vials are held.

[0406] The kit may also include instructions for use of the kit components and any other reagents not included in the kit. These instructions may include possible variations.

[0407] VI. Examples

[0408] The following embodiments are included to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques discovered by the inventors that work well in the practice of the invention, and therefore can be considered to constitute preferred modes of practice. However, those skilled in the art will understand from this disclosure that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention, and similar or related results can still be obtained.

[0409] Example 1 - Identifying telomerase activation as a therapeutic strategy to alleviate the pathology of Alzheimer's disease using a novel induced TERT-AD mouse model.

[0410] The inventors observed that Tert gene expression was significantly downregulated in the brain tissue of two distinct and well-established AD mouse models. 3xTg-AD showed amyloid and tau pathologies (Oddo et al., Neuron, 2003), while 5xFAD showed amyloid pathologies only in 3-month-old mice (Oakley et al., J Neurosci, 2006), exhibiting elevated Aβ levels but minimal signs of neurodegeneration. FIG. 1A , FIG. 1B With particular focus on neuronal populations, the inventors isolated and cultured primary cortical and hippocampal neurons from the brains of E18.5 to E19.5 AD mice, and examined Tert mRNA levels in vitro (DIV) for 14 days, at which point the synaptic network matured. Consistent with previous results from in vivo mouse brain tissue, Tert expression was downregulated in both 3xTg-AD and 5xFAD primary neurons compared to the wild-type control group. FIG. 1C , FIG. 1D Correspondingly, telomerase activity was also lower in freshly isolated hippocampal neurons from the 5xFAD brain compared to the wild-type control group. FIG. 1E More interestingly, the inventors observed a high occupancy of the repressive epigenetic marker H3K9me3 in the Tert gene body and promoter region of 5xFAD mouse neurons. H3K9me3 is known to accumulate primarily in the gene body and is crucial for gene repression in neurons. FIG. 1F Histone methylation is reversible, and histone demethylases mediate the removal of methyl groups from lysine residues on histones (Greerand Shi, Nat Rev Genet, 2012). Interestingly, the inventors examined the levels of histone methyltransferases and demethylases and revealed that, compared with wild-type controls, H3K9 demethylases Kdm1a, Kdm4b, and Kdm4c were significantly downregulated in cortical and hippocampal neurons of mouse AD brains. FIG. 1G , FIG. 1HTo investigate whether reversible H3K9 methylation involves Tert inhibition, the inventors evaluated the effects of the histone methyltransferase inhibitors trachomatin and BIX-01294 (i.e., a non-selective cofactor-competitive inhibitor and a selective substrate-competitive inhibitor) in an AD mouse model (Greiner et al., NatChem Biol, 2005; Kubicek et al., Mol Cell, 2007; Yuan et al., ACS Chem Biol, 2012). Peripheral administration of these compounds has been shown to reduce H3K9 methylation markers in the central nervous system (Dixit et al., CellDeath Dis, 2014; Chase et al., PLoS One, 2019). Both small-molecule histone methyltransferase inhibitors resulted in desuppression of Tert gene expression in the cortex and hippocampus of AD mice. FIG. 1I Combined with previous work showing that this epigenetic marker accumulates in the genome and is crucial for transcriptional repression of neuronal genes (Liu et al., J Neurosci, 2015), the inventors demonstrated the possibility that soluble Aβ can negatively regulate Tert gene expression by altering the expression of H3K9 demethylase or methyltransferase in AD mouse neurons.

[0411] Given the suppression of Tert gene expression in the early stages of amyloid accumulation in an AD mouse model, the inventors tested whether increased Tert gene expression in AD neurons could improve or prevent amyloid pathophysiology. To this end, the inventors generated a Cre-inducible Tert knock-in allele, consisting of a universally expressed CAG promoter followed by a loxP flanking terminator and a mouse Tert open reading frame (R26-CAG-LSL-mTert). The linearized construct was targeted to the Rosa 26 locus in C57BL / 6-derived JM8F6 embryonic stem (ES) cells via electroporation. FIG. 2A The inventors used the following primers to identify positive clones via long-range PCR (New England Biolabs): left arm 5′-GGT CGT GTG GTT CGG TGT CTCTTT-3′ and 5′-ATG GGC TAT GAACTA ATG ACC CCG-3′; right arm 5′-CAC TAC CAG CAG AAC ACCCCC ATC-3′ and 5′-GTG CCA CTA GTA CCA ACAGCC TCT-3′. FIG. 2B The inventors confirmed the correct recombination through sequencing and karyotype analysis. Ultimately, the inventors identified two independent clones and injected them into C57BL / 6 albino blastocysts to generate chimeric mice, and the chimeric mice from each clone were able to produce germline transmission. FIG. 2C).

[0412] To further investigate the role of telomerase activation in an AD mouse model, the inventors first crossed this novel Cre-inducible Tert knock-in allele with 3xTg-AD or 5xFAD. Subsequently, to selectively drive Tert expression in the neuronal population of the AD mouse model, the inventors incorporated a neuron-specific Cre allele controlled by the calcium / calmodulin-dependent protein kinase type II α promoter (Camk2a-CreERT2) (Madisen et al., Nat Neurosci, 2010). The inventors successfully established the R26-CAG-LSL-mTert;3xTg-AD;Camk2a-CreERT2 and R26-CAG-LSL-mTert;5xFAD;Camk2a-CreERT2 strains, resulting in the deletion of the termination sequence flanking the loxP site after tamoxifen administration, leading to the activation of mTert gene expression in neurons of each AD mouse strain. FIG. 3A These models enabled spatial (neuron-specific) and temporal (tamoxifen-induced) control of Tert gene expression in two independent and widely studied AD (3xTg-AD and 5xFAD) mouse models.

[0413] To examine the potential impact of telomerase activation on AD pathology in vivo, the inventors treated R26-CAG-LSL-mTert;3xTg-AD;Camk2a-CreERT2 mice with tamoxifen at 2–3 months, at which point intracellular and cytotoxic Aβ oligomers began to accumulate in the brain, and the effect of enhanced Tert expression on amyloid pathology was assessed. The inventors revealed a significant decrease in Aβ deposition in the Tert-activated R26-CAG-LSL-mTert;3xTg-AD;Camk2a-CreERT2 mouse model in the hippocampus. FIG. 3B , FIG. 3C A similar reduction in amyloid load was observed in the R26-CAG-LSL-mTert; 5xFAD; Camk2a-CreERT2 model. FIG. 3D ).

[0414] The inventors then investigated the molecular mechanisms driving the reduction of amyloid plaque burden. To gain a comprehensive understanding of the role of Tert in the neuronal population, they performed whole-genome RNA sequencing (RNA-Seq) analysis. They studied Tert expression in AD neurons of R26-CAG-LSL-mTert;3xTg-AD;Camk2a-CreERT2 mice induced in vivo by tamoxifen, and isolated cortical and hippocampal neurons from the adult mouse brains after tamoxifen treatment to probe the early transcriptional response of telomerase activation. This atlas confirmed that Tert gene expression increased in isolated neurons of the model after tamoxifen treatment, while the expression of the Terc gene, which encodes telomerase RNA components, remained unchanged. FIG. 4A Computational analysis shows that Tert induction in cortical and hippocampal AD neurons is associated with the activation of multiple signaling pathways related to the regulation of synaptic signal transduction, synaptic structure or activity, synaptic assembly, positive regulation of synaptic assembly, and regulation of synaptic organization. FIG. 4B , FIG. 4C Gene set enrichment analysis (GSEA) also showed that, following Tert induction, genes involved in synaptic signaling were upregulated in both neuronal populations. FIG. 4D The inventors also examined the expression of genes essential to the biology of Alzheimer's disease (AD). Surprisingly, they found that the expression of the App (beta-amyloid precursor protein) and ApoE (apolipoprotein E, a strong genetic risk factor for AD) genes was significantly reduced in Tert-activated AD neurons. FIG. 4E Meanwhile, Hsp70 is a molecular chaperone that can reduce Aβ-induced cytotoxicity and has been shown to effectively protect neurons in various AD animal models, significantly inducing gene expression under Tert induction. FIG. 4F By utilizing these unbiased transcriptomic analyses, the inventors determined that Tert induction in neurons can affect the expression of a large number of genes in postmitotic neurons in vivo, genes that are closely related to the pathobiology of Alzheimer's disease and are crucial for synapse formation and neuronal activity.

[0415] Synaptic loss and dysfunction are major contributing factors to cognitive decline in Alzheimer's disease (AD) (Palop and Mucke, Nat Neurosci, 2010; Hong et al., Science, 2016; Selkoe and Hardy, EMBO Mol Med, 2016). To test whether induction of neuronal Tert could lead to protection against synaptic and network dysfunction in the AD brain, the inventors examined neuronal morphology in vivo using Golgi-Cox staining. The inventors observed that, relative to the control group, Tert induction was associated with increased neuronal complexity and dendritic spine density in the aging cerebral cortex of Tert-activated R26-CAG-LSL-mTert; 3xTg-AD; Camk2a-CreERT2 mice. FIG. 5A , FIG. 5B , FIG. 5C The inventors concluded that elevated Tert expression in neurons activates synaptic signaling cascades and reduces spinal contracture and synaptic loss in AD brain neurons of mice.

[0416] Along with these observations in rodents, the inventors also sought to assess the biological effects of TERT-induced pluripotent stem cells in the context of human AD. The inventors used mature induced pluripotent stem cells (iPSCs) derived from familial AD patients carrying APP genomic duplications (APP...). DP (Israel et al., Nature, 2012). Consistent with the inventors' findings in a mouse model, the inventors found that, relative to a non-dementia control group, the APP-derived... DP The patients' AD neurons also had a high percentage of the repressive epigenetic marker H3K9me3 in the TERT gene body. FIG. 6A To further investigate the requirement of human TERT expression for H3K9 methylation, the inventors then studied the inhibitory effect of H3K9 methyltransferase in human AD neurons. This was compared with findings in mice. FIG. 1I Consistent with this, inhibiting H3K9 methylation also restored the expression of TERT mRNA and protein in human AD neurons. FIG. 6B , FIG. 6C , FIG. 6D ).

[0417] To examine whether TERT activation also affects Aβ pathology in the human environment, the inventors generated a lentiviral human TERT construct under the EF1a promoter and measured the effect of TERT induction on Aβ accumulation in differentiated human AD neurons infected with lentiviral vectors expressing TERT or EGFP. FIG. 7ASimilar to studies in rodents, the inventors found that, measured by sandwich ELISA (enzyme-linked immunosorbent assay), TERT induction resulted in a significant dose- and time-dependent reduction in intracellular Aβ accumulation in human AD neurons. FIG. 7B , FIG. 7C To further understand the potential mechanisms of TERT-mediated amyloid load decay in neurons, the inventors sought to identify possible molecular targets. In addition to reducing Aβ accumulation, TERT induction not only lowered APP protein levels but also triggered the activation of anti-aging genes (SIRT1), molecular chaperones and stress sensor genes (HSP70 and HSF1), synaptic plasticity-related genes (BDNF and PSD-95), and antioxidant genes (NRF2 and HO1). FIG. 7D , FIG. 7E These genes are well known to be crucial for reducing Aβ processing and cytotoxicity, as well as improving synaptic plasticity and memory formation in the adult brain (Evans et al., J Biol Chem, 2006; Qin et al., J Biol Chem, 2006; Herskovits and Guarente, Neuron, 2014; Lackie et al., Front Neurosci-Switz, 2017). The inventors' findings indicate that TERT activation not only reduces Aβ production but also exerts a neuroprotective effect in AD neurons through the production of neuroprotective mediators.

[0418] To further determine whether TERT-dependent gene regulation at the transcriptional level requires catalytic activity, the inventors used site-directed mutagenesis and alanine to replace aspartic acid at residue 712 to generate a catalytically inactive (CI)TERT expression construct (Weinrich et al., Nat Genet, 1997). FIG. 8A , FIG. 8B The inventors revealed that catalytically inactivated TERT mutants also lead to the upregulation of these genes. FIG. 8C This indicates that TERT's trans-activation function is independent of its catalytic activity.

[0419] To gain a deeper understanding of the functional significance of neuronal TERT activation in Alzheimer's disease (AD), the inventors conducted a cross-analysis of RNA-seq transcriptomes and pathways from mouse AD cortical neurons, mouse AD hippocampal neurons, and human AD neurons. Using this comprehensive cross-species analysis of neuronal TERT activation networks, the inventors identified several neuron-specific pathways (…). FIG. 9A The overlap between TERT activation and the learning process was the most significantly enriched pathway (all p < 0.001), followed by membrane depolarization, glutamate receptor signaling, action potentials, and synaptic signaling as downstream outcomes of TERT activation. FIG. 9BThe inventors also found that all enrichment profiles from the three groups showed a highly consistent regulation of gene sets involved in the learning process of AD neurons in mice and humans. FIG. 9C This indicates that TERT regulates key disease-related pathways in the brain of Alzheimer's disease (AD).

[0420] Because TERT induces dendritic spine formation at the cellular and tissue levels and activates genes involved in learning processes at the molecular level, the inventors next investigated whether TERT activation could improve learning deficits in an in vivo AD model. To this end, spatial learning and memory were evaluated in relation to AD control in R26-CAG-LSL-mTert; 3xTg-AD; and Camk2a-CreERT2 models. While the AD control group showed impaired spatial learning in the Barnes maze in older adults, age- and sex-matched TERT-activated AD mice exhibited significant improvements in learning ability and memory, manifested as a reduced delay in entering the escape hole. FIG. 9D Consistent with the cellular and molecular data above, the inventors' findings suggest that Tert activation attenuates age-related learning impairment in AD mice.

[0421] The inventors further investigated the mechanistic details behind the role of TERT in terminally differentiated postmitotic neurons. To determine the mechanistic basis of TERT activation and gene regulation, the inventors performed a whole-proteome analysis of potential TERT interacting partners in neurons. Characterization of TERT-containing protein complexes by mass spectrometry identified the transcriptional regulators CREB-binding protein (CREBBP) and RELA, the maximum and catalytic subunit of RNA polymerase II POLR2A, and multiple mediator complex subunits (MED1, MED4, MED12, MED15, MED16, MED23, MED24) that link transcriptional regulators to RNA polymerase II in human neurons. FIG. 10A The inventors also disclosed the use of RNA-Seq analysis ( FIG. 10B The elevation of various WNT pathway components in TERT-activated AD neurons has gained additional importance given the known neuroprotective role of WNT signaling in neurodegenerative diseases. Based on these observations, the inventors assessed whether endogenous TERT in postmitotic neurons physically interacts with a transcriptional regulatory complex containing β-catenin, a key player in WNT signaling transduction. Co-immunoprecipitation assays confirmed that neuronal TERT protein physically interacts with endogenous levels of β-catenin, as well as the activated nuclear forms of CREBBP and POLR2A, in fully differentiated human neurons. FIG. 10C ).

[0422] The inventors further evaluated the potential global enrichment of TERT and β-catenin / TCF7 associations at the genomic level. Using specific antibodies, the inventors determined the genome-wide distribution of TERT and β-catenin / TCF7 in human neurons via ChIP-Seq and found that TERT, β-catenin, and TCF7 as a transcription complex chaperone primarily occupy transcription start sites (TSS) in human neuronal gene promoters. FIG. 11A The inventors also determined that the TERT binding site is occupied by β-catenin and TCF7 in the promoter regions of highly relevant genes, including WNT9B (a member of the WNT family), ATP1A3 (a Na+ / K+-ATPase catalytic subunit, one of five overlapping genes upregulated in TERT-activated human and mouse neurons in the study), HSP70 family members HSPA12A and HSPA6, and MYC (a positive feedforward regulator of TERT). FIG. 11B The inventors' findings regarding the physical association between TERT and the β-catenin / TCF transcriptional complex in AD neurons and the TERT enhancement of β-catenin / TCF transcriptional activity in AD neurons ( FIG. 11C This study highlights the important roles of TERT and WNT signaling in the progression of Alzheimer's disease.

[0423] In this invention, the inventors discovered that mouse and human neurons from amyloid-based AD models exhibited epigenetic repression of neuronal TERT expression, prompting an exploration of the relationship between amyloid accumulation and TERT gene expression, and whether the restoration of TERT expression would affect disease trajectory. The inventors observed that TERT activation led to a significant reduction in Aβ levels in hippocampal and cortical neurons in the brains of two AD mouse models, as well as in cultured human iPSC-derived AD neurons with genomic APP repeats. Mechanistically, TERT induces gene expression and interacts with core transcriptional and β-catenin / TCF7 complex components at key neuronal gene transcription initiation sites. These genes control synaptic signaling and learning pathways and protect neuronal health in both mouse and human neurons. Neuronal TERT expression improved dendritic spine formation and cognitive function in aged AD mouse models. In conclusion, these findings support the development of somatic TERT activation therapies as a potential disease-modifying strategy for AD.

[0424] Example 2 - Extracellular-mediated TERT mRNA delivery in the Alzheimer's disease brain

[0425] Extracellular bodies are small extracellular vesicles (40 nM to 100 nM) released from cells and present in most bodily fluids, providing a useful means of delivering macromolecules such as nucleic acids and proteins to target cells. Extracellular body therapy has been explored in anticancer clinical trials and, due to their ability to easily cross the blood-brain barrier, can also be used to treat neurodegenerative diseases, whereas liposomes are preferentially degraded by enzymes, mechanical stress, and / or phagocytes before reaching their target sites. Compared to liposomes, the display of CD47 and RVG brain-targeting peptides on the surface of extracellular bodies not only increases their biostability by protecting them from degradation but also improves the overall delivery efficiency of bioactive extracellular nucleic acids to target cells in the brain. Targeted extracellular bodies exhibiting superior ability to deliver TERT mRNA to the brain could serve as an effective therapeutic strategy for AD.

[0426] A. Steps

[0427] 1. Cell preparation for generating extracellular bodies

[0428] Human fibroblasts and / or bone marrow dendritic cells (BMDCs) can be used as a source of extracellular bodies. These cells can be cultured in DMEM supplemented with 10% exosome-removed FBS and 1% penicillin-streptomycin.

[0429] 2. Generating targeted extracellular bodies by displaying RVG brain-targeting peptides and CD47 "don't eat me" signals.

[0430] Cells can be transfected with plasmids encoding CD47 and RVG (rabies virus glycoprotein)-derived peptides using X-tremeGENE transfection reagent (Roche) or Lipofectamine 2000 reagent (Invitrogen). The CD47 ligand protein interacts with signal regulatory protein α (SIRPα), initiating a "don't eat me" signal to protect the extracellular body from phagocytosis. The RVG-derived peptide on the extracellular surface target guides the extracellular body to bind to neurons expressing acetylcholine receptors, allowing the targeted extracellular body to be delivered via blood vessels to the central nervous system.

[0431] 3. Separation of target extracellular bodies via microfiltration and ultracentrifugation

[0432] Targeted extracellular bodies can be purified using differential centrifugation. The supernatant, replenished with FBS to remove the extracellular bodies, can be collected from cells, filtered through a 0.2 μm filter, and ultracentrifuged at 120,000 × g for 70 minutes at 4°C. The extracellular body particles can then be resuspended in PBS and followed by ultracentrifugation at 120,000 × g for 70 minutes at 4°C. The extracellular body particles can then be resuspended in electroporation buffer.

[0433] 4. Extracellular bodies loaded with TERT mRNA via electroporation

[0434] The isolated extracellular bodies can be mixed with TERT mRNA in electroporation buffer and electroporated at 400 mV and 125 μF. All extracellular bodies can then be resuspended in PBS and ultracentrifuged at 120,000 × g for 70 minutes at 4 °C.

[0435] 5. Systemic (iv) administration of extracellular substances to Alzheimer's disease patients.

[0436] The loaded extracellular bodies can be resuspended in PBS and then intravenously injected into Alzheimer's patients.

[0437] 6. Characterization of the therapeutic effect of extracellular-mediated TERT mRNA delivery on Alzheimer's disease pathology

[0438] Learning and memory tasks can be periodically assessed in AD subjects treated with targeted exosomes loaded with control nucleic acid or TERT mRNA. The administration of therapeutic exosomes is expected to improve learning and memory and / or increase the clearance of β-amyloid protein in the subject's brain.

[0439] ***

[0440] Based on this disclosure, all methods disclosed and claimed herein can be made and performed without excessive experimentation. While the compositions and methods of the invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that variations may be made to the methods and the steps or order of steps thereof without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant agents can be substituted for the agents described herein while obtaining the same or similar results. All such similar substitutions and modifications that will be apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0441] The references disclosed herein are specifically incorporated herein by reference to the extent that they provide supplementation to the exemplary processes or other details set forth herein. sequence list <110> University of Texas System Board of Trustees <120> Methods and compositions involving TERT-activating therapeutic agents <130> UTSC.P1157WO <140> PCT / US2020 / 030699 <141> 2020-04-30 <150> 62 / 842,323 <151> May 2, 2019 <160> 14 <170> PatentIn Version 3.5 <210> 1 <211> 3829 <212> DNA <213> Homo sapiens <400> 1 caggcagcgc tgcgtcctgc tgcgcacgtg ggaagccctg gccccggcca cccccgcgat 60 gccgcgcgct ccccgctgcc gagccgtgcg ctccctgctg cgcagccact accgcgaggt 120 gctgccgctg gccacgttcg tgcggcgcct ggggccccag ggctggcggc tggtgcagcg 180 cggggacccg gcggctttcc gcgcgctggt ggcccagtgc ctggtgtgcg tgccctggga 240 cgcacggccg ccccccgccg ccccctcctt ccgccaggtg tcctgcctga aggagctggt 300 ggcccgagtg ctgcagaggc tgtgcgagcg cggcgcgaag aacgtgctgg ccttcggctt 360 cgcgctgctg gacggggccc gcgggggccc ccccgaggcc ttcaccacca gcgtgcgcag 420 ctacctgccc aacacggtga ccgacgcact gcgggggagc ggggcgtggg ggctgctgct 480 gcgccgcgtg ggcgacgacg tgctggttca cctgctggca cgctgcgcgc tctttgtgct 540 ggtggctccc agctgcgcct accaggtgtg cgggccgccg ctgtaccagc tcggcgctgc 600 cactcaggcc cggcccccgc cacacgctag tggaccccga aggcgtctgg gatgcgaacg 660 ggcctggaac catagcgtca gggaggccgg ggtccccctg ggcctgccag ccccgggtgc 720 gaggaggcgc gggggcagtg ccagccgaag tctgccgttg cccaagaggc ccaggcgtgg 780 cgctgcccct gagccggagc ggacgcccgt tgggcagggg tcctgggccc acccgggcag 840 gacgcgtgga ccgagtgacc gtggtttctg tgtggtgtca cctgccagac ccgccgaaga 900 agccacctct ttggagggtg cgctctctgg cacgcgccac tcccacccat ccgtgggccg 960 ccagcaccac gcgggccccc catccacatc gcggccacca cgtccctggg acacgccttg 1020 tcccccggtg tacgccgaga ccaagcactt cctctactcc tcaggcgaca aggagcagct 1080 gcggccctcc ttcctactca gctctctgag gcccagcctg actggcgctc ggaggctcgt 1140 ggagaccatc tttctgggtt ccaggccctg gatgccaggg actccccgca ggttgccccg 1200 cctgccccag cgctactggc aaatgcggcc cctgtttctg gagctgcttg ggaaccacgc 1260 gcagtgcccc tacggggtgc tcctcaagac gcactgcccg ctgcgagctg cggtcacccc 1320 agcagccggt gtctgtgccc gggagaagcc ccagggctct gtggcggccc ccgaggagga 1380 ggacacagac ccccgtcgcc tggtgcagct gctccgccag cacagcagcc cctggcaggt 1440 gtacggcttc gtgcgggcct gcctgcgccg gctggtgccc ccaggcctct ggggctccag 1500 gcacaacgaa cgccgcttcc tcaggaacac caagaagttc atctccctgg ggaagcatgc 1560 caagctctcg ctgcaggagc tgacgtggaa gatgagcgtg cgggactgcg cttggctgcg 1620 caggagccca ggggttggct gtgttccggc cgcagagcac cgtctgcgtg aggagatcct 1680 ggccaagttc ctgcactggc tgatgagtgt gtacgtcgtc gagctgctca ggtctttctt 1740 ttatgtcacg gagaccacgt ttcaaaagaa caggctcttt ttctaccgga agagtgtctg 1800 gagcaagttg caaagcattg gaatcagaca gcacttgaag agggtgcagc tgcgggagct 1860 gtcggaagca gaggtcaggc agcatcggga agccaggccc gccctgctga cgtccagact 1920 ccgcttcatc cccaagcctg acgggctgcg gccgattgtg aacatggact acgtcgtggg 1980 agccagaacg ttccgcagag aaaagagggc cgagcgtctc acctcgaggg tgaaggcact 2040 gttcagcgtg ctcaactacg agcgggcgcg gcgccccggc ctcctgggcg cctctgtgct 2100 gggcctggac gatatccaca gggcctggcg caccttcgtg ctgcgtgtgc gggcccagga 2160 cccgccgcct gagctgtact ttgtcaaggt ggatgtgacg ggcgcgtacg acaccatccc 2220 ccaggacagg ctcacggagg tcatcgccag catcatcaaa ccccagaaca cgtactgcgt 2280 gcgtcggtat gccgtggtcc agaaggccgc ccatgggcac gtccgcaagg ccttcaagag 2340 ccacgtctct accttgacag acctccagcc gtacatgcga cagttcgtgg ctcacctgca 2400 ggagaccagc ccgctgaggg atgccgtcgt catcgagcag agctcctccc tgaatgaggc 2460 cagcagtggc ctcttcgacg tcttcctacg cttcatgtgc caccacgccg tgcgcatcag 2520 gggcaagtcc tacgtccagt gccaggggat cccgcagggc tccatcctct ccacgctgct 2580 ctgcagcctg tgctacggcg acatggagaa caagctgttt gcggggattc ggcgggacgg 2640 gctgctcctg cgtttggtgg atgatttctt gttggtgaca cctcacctca cccacgcgaa 2700 aaccttcctc agctatgccc ggacctccat cagagccagt ctcaccttca accgcggctt 2760 caaggctggg aggaacatgc gtcgcaaact ctttggggtc ttgcggctga agtgtcacag 2820 cctgtttctg gatttgcagg tgaacagcct ccagacggtg tgcaccaaca tctacaagat 2880 cctcctgctg caggcgtaca ggtttcacgc atgtgtgctg cagctcccat ttcatcagca 2940 agtttggaag aaccccacat ttttcctgcg cgtcatctct gacacggcct ccctctgcta 3000 ctccatcctg aaagccaaga acgcagggat gtcgctgggg gccaagggcg ccgccggccc 3060 tctgccctcc gaggccgtgc agtggctgtg ccaccaagca ttcctgctca agctgactcg 3120 acaccgtgtc acctacgtgc cactcctggg gtcactcagg acagcccaga cgcagctgag 3180 tcggaagctc ccggggacga cgctgactgc cctggaggcc gcagccaacc cggcactgcc 3240 ctcagacttc aagaccatcc tggactgatg gccacccgcc cacagccagg ccgagagcag 3300 acaccagcag ccctgtcacg ccgggctcta cgtcccaggg agggaggggc ggcccacacc 3360 caggcccgca ccgctgggag tctgaggcct gagtgagtgt ttggccgagg cctgcatgtc 3420 cggctgaagg ctgagtgtcc ggctgaggcc tgagcgagtg tccagccaag ggctgagtgt 3480 ccagcacacc tgccgtcttc acttccccac aggctggcgc tcggctccac cccagggcca 3540 gcttttcctc accaggagcc cggcttccac tccccacata ggaatagtcc atccccagat 3600 tcgccattgt tcacccctcg ccctgccctc ctttgccttc cacccccacc atccaggtgg 3660 agaccctgag aaggaccctg ggagctctgg gaatttggag tgaccaaagg tgtgccctgt 3720 acacaggcga ggaccctgca cctggatggg ggtccctgtg ggtcaaattg gggggaggtg 3780 ctgtgggagt aaaatactga atatatgagt ttttcagttt tgaaaaaaa 3829 <210> 2 <211> 1069 <212> PRT <213> Homo sapiens <400> 2 Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser 1 5 10 15 His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly 20 25 30 Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg 35 40 45 Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro 50 55 60 Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu 65 70 75 80 Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val 85 90 95 Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro 100 105 110 Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr 115 120 125 Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 130 135 140 Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val 145 150 155 160 Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr 165 170 175 Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly 180 185 190 Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg 195 200 205 Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg 210 215 220 Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg 225 230 235 240 Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp 245 250 255 Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val 260 265 270 Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala 275 280 285 Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His 290 295 300 Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro 305 310 315 320 Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly 325 330 335 Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro 340 345 350 Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser 355 360 365 Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln 370 375 380 Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His 385 390 395 400 Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg 405 410 415 Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln 420 425 430 Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu 435 440 445 Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe 450 455 460 Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser 465 470 475 480 Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser 485 490 495 Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met 500 505 510 Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys 515 520 525 Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe 530 535 540 Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe 545 550 555 560 Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr 565 570 575 Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His 580 585 590 Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln 595 600 605 His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile 610 615 620 Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val 625 630 635 640 Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser 645 650 655 Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg 660 665 670 Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg 675 680 685 Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro 690 695 700 Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile 705 710 715 720 Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln 725 730 735 Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His 740 745 750 Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp 755 760 765 Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser 770 775 780 Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu 785 790 795 800 Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His 805 810 815 Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro 820 825 830 Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp 835 840 845 Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu 850 855 860 Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala 865 870 875 880 Lys Thr Phe Leu Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr 885 890 895 Phe Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe 900 905 910 Gly Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val 915 920 925 Asn Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu 930 935 940 Gln Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln 945 950 955 960 Gln Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr 965 970 975 Ala Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser 980 985 990 Leu Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu Ala Val Gln 995 1000 1005 Trp Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg 1010 1015 1020 Val Thr Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr 1025 1030 1035 Gln Leu Ser Arg Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu 1040 1045 1050 Ala Ala Ala Asn Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu 1055 1060 1065 Asp <210> 3 <211> 4018 <212> DNA <213> Homo sapiens <400> 3 caggcagcgc tgcgtcctgc tgcgcacgtg ggaagccctg gccccggcca cccccgcgat 60 gccgcgcgct ccccgctgcc gagccgtgcg ctccctgctg cgcagccact accgcgaggt 120 gctgccgctg gccacgttcg tgcggcgcct ggggccccag ggctggcggc tggtgcagcg 180 cggggacccg gcggctttcc gcgcgctggt ggcccagtgc ctggtgtgcg tgccctggga 240 cgcacggccg ccccccgccg ccccctcctt ccgccaggtg tcctgcctga aggagctggt 300 ggcccgagtg ctgcagaggc tgtgcgagcg cggcgcgaag aacgtgctgg ccttcggctt 360 cgcgctgctg gacggggccc gcgggggccc ccccgaggcc ttcaccacca gcgtgcgcag 420 ctacctgccc aacacggtga ccgacgcact gcgggggagc ggggcgtggg ggctgctgct 480 gcgccgcgtg ggcgacgacg tgctggttca cctgctggca cgctgcgcgc tctttgtgct 540 ggtggctccc agctgcgcct accaggtgtg cgggccgccg ctgtaccagc tcggcgctgc 600 cactcaggcc cggcccccgc cacacgctag tggaccccga aggcgtctgg gatgcgaacg 660 ggcctggaac catagcgtca gggaggccgg ggtccccctg ggcctgccag ccccgggtgc 720 gaggaggcgc gggggcagtg ccagccgaag tctgccgttg cccaagaggc ccaggcgtgg 780 cgctgcccct gagccggagc ggacgcccgt tgggcagggg tcctgggccc acccgggcag 840 gacgcgtgga ccgagtgacc gtggtttctg tgtggtgtca cctgccagac ccgccgaaga 900 agccacctct ttggagggtg cgctctctgg cacgcgccac tcccacccat ccgtgggccg 960 1020. cgcctggg acacgccttg tcccccggtg tacgccgaga ccaagcactt cctctactcc tcaggcgaca aggagcagct gcggccctcc ttcctactca gctctctgag gcccagcctg actggcgctc ggaggctcgt 1140 ggagaccatc tttctgggtt ccaggccctg gatgccaggg actccccgca ggttgccccg cctgccccag cgctactggc aaatgcggcc cctgtttctg gagctgcttg ggaaccacgc gcagtgcccc tacggggtgc tcctcaagac gcactgcccg ctgcgagctg cggtcacccc 1320 agcagccggt gtctgtgccc gggagagcc ccagggctct gtggcggccc ccgagga 1380 ggacacagac ccccgtcgcc tggtgcagct gctccgccag cacagcagcc cctggcaggt gtacggcttc gtgcggggcct gcctgcgccg gctggtgccc ccaggcctct ggggctccag gcacaacgaa cgccgcttcc tcaggaacac caagaagttc atctccctgg ggaagcatgc caagctctcg ctgcaggagc tgacgtggaa gatgagcgtg cgggactgcg cttggctgcg caggagccca ggggttggct gtgttccggc cgcagagcac cgtctgcgtg aggagatcct 1680 ggccaagttc ctgcactggc tgatgagtgt gtacgtcgtc gagctgctca ggtctttctt 1740 ttatgtcacg gagaccacgt ttcaaaagaa caggctcttt ttctaccgga agagtgtctg 1800 gagcaagttg caaagcattg gaatcagaca gcacttgaag agggtgcagc tgcgggagct 1860 gtcggaagca gaggtcaggc agcatcggga agccaggccc gccctgctga cgtccagact 1920 ccgcttcatc cccaagcctg acgggctgcg gccgattgtg aacatggact acgtcgtggg 1980 agccagaacg ttccgcagag aaaagagggc cgagcgtctc acctcgaggg tgaaggcact 2040 gttcagcgtg ctcaactacg agcgggcgcg gcgccccggc ctcctgggcg cctctgtgct 2100 gggcctggac gatatccaca gggcctggcg caccttcgtg ctgcgtgtgc gggcccagga 2160 cccgccgcct gagctgtact ttgtcaaggt ggatgtgacg ggcgcgtacg acaccatccc 2220 ccaggacagg ctcacggagg tcatcgccag catcatcaaa ccccagaaca cgtactgcgt 2280 gcgtcggtat gccgtggtcc agaaggccgc ccatgggcac gtccgcaagg ccttcaagag 2340 ccacgtctct accttgacag acctccagcc gtacatgcga cagttcgtgg ctcacctgca 2400 ggagaccagc ccgctgaggg atgccgtcgt catcgagcag agctcctccc tgaatgaggc 2460 cagcagtggc ctcttcgacg tcttcctacg cttcatgtgc caccacgccg tgcgcatcag 2520 gggcaagtcc tacgtccagt gccaggggat cccgcagggc tccatcctct ccacgctgct 2580 ctgcagcctg tgctacggcg acatggagaa caagctgttt gcggggattc ggcgggacgg 2640 gctgctcctg cgtttggtgg atgatttctt gttggtgaca cctcacctca cccacgcgaa 2700 aaccttcctc aggaccctgg tccgaggtgt ccctgagtat ggctgcgtgg tgaacttgcg 2760 gaagacagtg gtgaacttcc ctgtagaaga cgaggccctg ggtggcacgg cttttgttca 2820 gatgccggcc cacggcctat tcccctggtg cggcctgctg ctggataccc ggaccctgga 2880 ggtgcagagc gactactcca gctatgcccg gacctccatc agagccagtc tcaccttcaa 2940 ccgcggcttc aaggctggga ggaacatgcg tcgcaaactc tttggggtct tgcggctgaa 3000 gtgtcacagc ctgtttctgg atttgcaggt gaacagcctc cagacggtgt gcaccaacat 3060 ctacaagatc ctcctgctgc aggcgtacag gtttcacgca tgtgtgctgc agctcccatt 3120 tcatcagcaa gtttggaaga accccacatt tttcctgcgc gtcatctctg acacggcctc 3180 cctctgctac tccatcctga aagccaagaa cgcagggatg tcgctggggg ccaagggcgc 3240 cgccggccct ctgccctccg aggccgtgca gtggctgtgc caccaagcat tcctgctcaa 3300 gctgactcga caccgtgtca cctacgtgcc actcctgggg tcactcagga cagcccagac 3360 gcagctgagt cggaagctcc cggggacgac gctgactgcc ctggaggccg cagccaaccc 3420 ggcactgccc tcagacttca agaccatcct ggactgatgg ccacccgccc acagccaggc 3480 cgagagcaga caccagcagc cctgtcacgc cgggctctac gtcccaggga gggaggggcg 3540 gcccacaccc aggcccgcac cgctgggagt ctgaggcctg agtgagtgtt tggccgaggc 3600 ctgcatgtcc ggctgaaggc tgagtgtccg gctgaggcct gagcgagtgt ccagccaagg 3660 gctgagtgtc cagcacacct gccgtcttca cttccccaca ggctggcgct cggctccacc 3720 ccagggccag cttttcctca ccaggagccc ggcttccact ccccacatag gaatagtcca 3780 tccccagatt cgccattgtt cacccctcgc cctgccctcc tttgccttcc acccccacca 3840 tccaggtgga gaccctgaga aggaccctgg gagctctggg aatttggagt gaccaaaggt 3900 gtgccctgta cacaggcgag gaccctgcac ctggatgggg gtccctgtgg gtcaaattgg 3960 ggggaggtgc tgtgggagta aaatactgaa tatatgagtt tttcagtttt gaaaaaaa 4018 <210> 4 <211> 1132 <212> PRT <213> Homo sapiens <400> 4 Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser 1 5 10 15 His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly 20 25 30 Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg 35 40 45 Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro 50 55 60 Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu 65 70 75 80 Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val 85 90 95 Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro 100 105 110 Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr 115 120 125 Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 130 135 140 Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val 145 150 155 160 Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr 165 170 175 Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly 180 185 190 Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg 195 200 205 Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg 210 215 220 Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg 225 230 235 240 Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp 245 250 255 Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val 260 265 270 Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala 275 280 285 Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His 290 295 300 Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro 305 310 315 320 Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly 325 330 335 Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro 340 345 350 Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser 355 360 365 Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln 370 375 380 Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His 385 390 395 400 Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg 405 410 415 Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln 420 425 430 Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu 435 440 445 Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe 450 455 460 Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser 465 470 475 480 Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser 485 490 495 Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met 500 505 510 Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys 515 520 525 Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe 530 535 540 Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe 545 550 555 560 Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr 565 570 575 Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His 580 585 590 Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln 595 600 605 His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile 610 615 620 Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val 625 630 635 640 Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser 645 650 655 Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg 660 665 670 Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg 675 680 685 Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro 690 695 700 Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile 705 710 715 720 Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln 725 730 735 Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His 740 745 750 Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp 755 760 765 Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser 770 775 780 Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu 785 790 795 800 Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His 805 810 815 Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro 820 825 830 Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp 835 840 845 Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu 850 855 860 Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala 865 870 875 880 Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys 885 890 895 Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu 900 905 910 Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe 915 920 925 Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser 930 935 940 Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe 945 950 955 960 Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly 965 970 975 Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn 980 985 990 Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln 995 1000 1005 Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln 1010 1015 1020 Gln Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp 1025 1030 1035 Thr Ala Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly 1040 1045 1050 Met Ser Leu Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu 1055 1060 1065 Ala Val Gln Trp Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr 1070 1075 1080 Arg His Arg Val Thr Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr 1085 1090 1095 Ala Gln Thr Gln Leu Ser Arg Lys Leu Pro Gly Thr Thr Leu Thr 1100 1105 1110 Ala Leu Glu Ala Ala Ala Asn Pro Ala Leu Pro Ser Asp Phe Lys 1115 1120 1125 Thr Ile Leu Asp 1130[[ID=​​​​​​​​​​​​​​tgacccgcgc tcctcgttgc cccgcggtgc gctctctgct gcgcagccga taccgggagg 180 tgtggccgct ggcaaccttt gtgcggcgcc tggggcccga gggcaggcgg cttgtgcaac 240 ccggggaccc gaagatctac cgcactttgg ttgcccaatg cctagtgtgc atgcactggg 300 gctcacagcc tccacctgcc gacctttcct tccaccaggt gtcatccctg aaagagctgg 360 tggccagggt tgtgcagaga ctctgcgagc gcaacgagag aaacgtgctg gcttttggct 420 ttgagctgct taacgaggcc agaggcgggc ctcccatggc cttcactagt agcgtgcgta 480 gctacttgcc caacactgtt attgagaccc tgcgtgtcag tggtgcatgg atgctactgt 540 tgagccgagt gggcgacgac ctgctggtct acctgctggc acactgtgct ctttatcttc 600 tggtgccccc cagctgtgcc taccagggga gatggccaag agcgtctaaa cccctcattc 660 ctactcagca acctccagcc taacttgact ggggccagga gactggtgga gatcatcttt 720 ctgggctcaa ggcctaggac atcaggacca ctctgcagga cacaccgtct atcgcgtcga 780 tactggcaga tgcggcccct gttccaacag ctgctggtga accatgcaga gtgccaatat 840 gtcagactcc tcaggtcaca ttgcaggttt cgaacagcaa accaacaggt gacagatgcc 900 ttgaacacca gcccaccgca cctcatggat ttgctccgcc tgcacagcag tccctggcag 960 gtatatggtt ttcttcgggc ctgtctctgc aaggtggtgt ctgctagtct ctggggtacc 1020 aggcacaatg agcgccgctt ctttaagaac ttaaagaagt tcatctcgtt ggggaaatac 1080 ggcaagctat cactgcagga actgatgtgg aagatgaaag tagaggattg ccactggctc 1140 cgcagcagcc cggggaagga ccgtgtcccc gctgcagagc accgtctgag ggagaggatc 1200 ctggctacgt tcctgttctg gctgatggac acatacgtgg tacagctgct taggtcattc 1260 ttttacatca cagagagcac attccagaag aacaggctct tcttctaccg taagagtgtg 1320 tggagcaagc tgcagagcat tggagtcagg caacaccttg agagagtgcg gctacgggag 1380 ctgtcacaag aggaggtcag gcatcaccag gacacctggc tagccatgcc catctgcaga 1440 ctgcgcttca tccccaagcc caacggcctg cggcccattg tgaacatgag ttatagcatg 1500 ggtaccagag ctttgggcag aaggaagcag gcccagcatt tcacccagcg tctcaagact 1560 ctcttcagca tgctcaacta tgagcggaca aaacatcctc accttatggg gtcttctgta ctgggtatga atgacatcta caggacctgg cggggcctttg tgctgcgtgt gcgtgctctg 1680. gaccagacac ccaggatgta ctttgttaag gcagatgtga ccggggccta tgatgccatc ccccagggta agctggtgga ggttgttgcc aatatgatca ggcactcgga gagcacgtac tgtatccgcc agtatgcagt ggtccggaga gatagccag gccaagtcca caagtccttt 1860. aggagacagg tcaccaccct ctctgacctc cagccataca tgggccagtt ccttaagcat ctgcaggatt cagatgccag tgcactgagg aactccgttg tcatcgagca gagcatctct atgaatgaga gcagcagcag cctgtttgac ttcttcctgc acttcctgcg tcacagtgtc gtaaagattg gtgacaggtg ctatacgcag tgccagggca tcccccaggg ctccagccta tccaccctgc tctgcagtct gtgtttcgga gacatggaga acaagctgtt tgctgaggtg cagcgggatg ggttgctttt acgttttgtt gatgactttc tgttggtgac gcctcacttg 2220 2280. aaaccttcct cagcaccctg gtccatggcg ttcctgagta tgggtgcatg ataaacttgc agaagacagt ggtgaacttc cctgtggagc ctggtaccct gggtggtgca 2340 gctccatacc agctgcctgc tcactgcctg tttccctggt gtggcttgct gctggacact 2400 cagactttgg aggtgttctg tgactactca ggtatgccc agacctcaat taagacgagc 2460 ctcaccttcc agagtgtctt caaagctggg aagaccatgc ggaacaagct cctgtcggtc 2520 ttgcggttga agtgtcacgg tctatttcta gacttgcagg tgaacagcct ccagacagtc 2580 tgcatcaata tatacaagat cttcctgctt caggcctaca ggttccatgc atgtgtgatt 2640 cagcttccct ttgaccagcg tgttaggaag aacctcacat tctttctggg catcatctcc 2700 agccaagcat cctgctgcta tgctatcctg aaggtcaaga atccaggaat gacactaaag 2760 gcctctggct cctttcctcc tgaagccgca cattggctct gctaccaggc cttcctgctc 2820 aagctggctg ctcattctgt catctacaaa tgtctcctgg gacctctgag gacagcccaa 2880 aaactgctgt gccggaagct cccagaggcg acaatgacca tccttaaagc tgcagctgac 2940 ccagccctaa gcacagactt tcagaccatt ttggactaac cctgtctcct tccgctagat 3000 gaacatgggc attgtagcct cagcactcct ggatccacgt cacaagaggg actggtcagt 3060 tgtgaggcta ggtcatccctc caaacctg tgtcatgggt ggtatgggag attgtcccag 3120 tgccttgttt cctgtaacag gcttgatttc ttcctgatg ccctcaggga ggcagatcct 3180 atccttta gtggcaggga tccactagca ccagcacatg aggagtgcac ccagtgcaca 3240 tgggcactggzgagtggac aggtgtgaga ttcctgggcc ctggagtct ttcacaccta 3300 accatggagc ctgtcccagt acatcagagt gcctcggaga tgaaaaagga catcgagcca 3360 gtgacctaaa ttacagcctg atatactct gattcatgt gactgcctta gctacttctc 3420 tactgctgtg tagtaaaca ccaagccac tttaaaagc aggattttcc tactggagca 3480 gcagctgaga gtttacatct tgatccataa gcacaaagc achacaga gagagagaga 3540 gagagagagagagagagagagagagagagagagagtca gtcagtcagt 3600 ctaacaaata actaagaag gtgaagggtg atgaagtcca cagggatcac gctagggatg 3660 ttccatgcct tctctgaagc taagattcct tggcagcgtt tgacagtaac catagtgggt 3720 acctactgag atcactataa agataaaata gggggaagcg tatttgtact gaactggaaa 3780 aacatacaaa taaagagtaa atcatggaaa aaaaaaaaaa aaaaaa 3826 <210> 6 <211> 729 <212> PRT <213> Mus musculus <400> 6 Met Arg Pro Leu Phe Gln Gln Leu Leu Val Asn His Ala Glu Cys Gln 1 5 10 15 Tyr Val Arg Leu Leu Arg Ser His Cys Arg Phe Arg Thr Ala Asn Gln 20 25 30 Gln Val Thr Asp Ala Leu Asn Thr Ser Pro Pro His Leu Met Asp Leu 35 40 45 Leu Arg Leu His Ser Ser Pro Trp Gln Val Tyr Gly Phe Leu Arg Ala 50 55 60 Cys Leu Cys Lys Val Val Ser Ala Ser Leu Trp Gly Thr Arg His Asn 65 70 75 80 Glu Arg Arg Phe Phe Lys Asn Leu Lys Lys Phe Ile Ser Leu Gly Lys 85 90 95 Tyr Gly Lys Leu Ser Leu Gln Glu Leu Met Trp Lys Met Lys Val Glu 100 105 110 Asp Cys His Trp Leu Arg Ser Ser Pro Gly Lys Asp Arg Val Pro Ala 115 120 125 Ala Glu His Arg Leu Arg Glu Arg Ile Leu Ala Thr Phe Leu Phe Trp 130 135 140 Leu Met Asp Thr Tyr Val Val Gln Leu Leu Arg Ser Phe Phe Tyr Ile 145 150 155 160 Thr Glu Ser Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr Arg Lys Ser 165 170 175 Val Trp Ser Lys Leu Gln Ser Ile Gly Val Arg Gln His Leu Glu Arg 180 185 190 Val Arg Leu Arg Glu Leu Ser Gln Glu Glu Val Arg His His Gln Asp 195 200 205 Thr Trp Leu Ala Met Pro Ile Cys Arg Leu Arg Phe Ile Pro Lys Pro 210 215 220 Asn Gly Leu Arg Pro Ile Val Asn Met Ser Tyr Ser Met Gly Thr Arg 225 230 235 240 Ala Leu Gly Arg Arg Lys Gln Ala Gln His Phe Thr Gln Arg Leu Lys 245 250 255 Thr Leu Phe Ser Met Leu Asn Tyr Glu Arg Thr Lys His Pro His Leu 260 265 270 Met Gly Ser Ser Val Leu Gly Met Asn Asp Ile Tyr Arg Thr Trp Arg 275 280 285 Ala Phe Val Leu Arg Val Arg Ala Leu Asp Gln Thr Pro Arg Met Tyr 290 295 300 Phe Val Lys Ala Asp Val Thr Gly Ala Tyr Asp Ala Ile Pro Gln Gly 305 310 315 320 Lys Leu Val Glu Val Val Ala Asn Met Ile Arg His Ser Glu Ser Thr 325 330 335 Tyr Cys Ile Arg Gln Tyr Ala Val Val Arg Arg Asp Ser Gln Gly Gln 340 345 350 Val His Lys Ser Phe Arg Arg Gln Val Thr Thr Leu Ser Asp Leu Gln 355 360 365 Pro Tyr Met Gly Gln Phe Leu Lys His Leu Gln Asp Ser Asp Ala Ser 370 375 380 Ala Leu Arg Asn Ser Val Val Ile Glu Gln Ser Ile Ser Met Asn Glu 385 390 395 400 Ser Ser Ser Ser Leu Phe Asp Phe Phe Leu His Phe Leu Arg His Ser 405 410 415 Val Val Lys Ile Gly Asp Arg Cys Tyr Thr Gln Cys Gln Gly Ile Pro 420 425 430 Gln Gly Ser Ser Leu Ser Thr Leu Leu Cys Ser Leu Cys Phe Gly Asp 435 440 445 Met Glu Asn Lys Leu Phe Ala Glu Val Gln Arg Asp Gly Leu Leu Leu 450 455 460 Arg Phe Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Asp Gln Ala 465 470 475 480 Lys Thr Phe Leu Ser Thr Leu Val His Gly Val Pro Glu Tyr Gly Cys 485 490 495 Met Ile Asn Leu Gln Lys Thr Val Val Asn Phe Pro Val Glu Pro Gly 500 505 510 Thr Leu Gly Gly Ala Ala Pro Tyr Gln Leu Pro Ala His Cys Leu Phe 515 520 525 Pro Trp Cys Gly Leu Leu Leu Asp Thr Gln Thr Leu Glu Val Phe Cys 530 535 540 Asp Tyr Ser Gly Tyr Ala Gln Thr Ser Ile Lys Thr Ser Leu Thr Phe 545 550 555 560 Gln Ser Val Phe Lys Ala Gly Lys Thr Met Arg Asn Lys Leu Leu Ser 565 570 575 Val Leu Arg Leu Lys Cys His Gly Leu Phe Leu Asp Leu Gln Val Asn 580 585 590 Ser Leu Gln Thr Val Cys Ile Asn Ile Tyr Lys Ile Phe Leu Leu Gln 595 600 605 Ala Tyr Arg Phe His Ala Cys Val Ile Gln Leu Pro Phe Asp Gln Arg 610 615 620 Val Arg Lys Asn Leu Thr Phe Phe Leu Gly Ile Ile Ser Ser Gln Ala 625 630 635 640 Ser Cys Cys Tyr Ala Ile Leu Lys Val Lys Asn Pro Gly Met Thr Leu 645 650 655 Lys Ala Ser Gly Ser Phe Pro Pro Glu Ala Ala His Trp Leu Cys Tyr 66​​​​​​​​​​​​​​​​​​​​​​​​​​ gttcccagcc tcatcttttt cgtcgtggac tctcagtggc ctgggtcctg gctgttttct 60 aagcacaccc ttgcatcttg gttcccgcac gtgggaggcc catcccggcc ttgagcacaa 120 tgacccgcgc tcctcgttgc cccgcggtgc gctctctgct gcgcagccga taccgggagg 180 tgtggccgct ggcaaccttt gtgcggcgcc tggggcccga gggcaggcgg cttgtgcaac 240 ccggggaccc gaagatctac cgcactttgg ttgcccaatg cctagtgtgc atgcactggg 300 gctcacagcc tccacctgcc gacctttcct tccaccaggt gtcatccctg aaagagctgg 360 tggccagggt tgtgcagaga ctctgcgagc gcaacgagag aaacgtgctg gcttttggct 420 ttgagctgct taacgaggcc agaggcgggc ctcccatggc cttcactagt agcgtgcgta 480 gctacttgcc caacactgtt attgagaccc tgcgtgtcag tggtgcatgg atgctactgt 540 tgagccgagt gggcgacgac ctgctggtct acctgctggc acactgtgct ctttatcttc 600 tggtgccccc cagctgtgcc taccagggga gatggccaag agcgtctaaa cccctcattc 660 ctactcagca acctccagcc taacttgact ggggccagga gactggtgga gatcatcttt 720 ctgggctcaa ggcctaggac atcaggacca ctctgcagga cacaccgtct atcgcgtcga 780 tactggcaga tgcggcccct gttccaacag ctgctggtga accatgcaga gtgccaatat 840 gtcagactcc tcaggtcaca ttgcaggttt cgaacagcaa accaacaggt gacagatgcc 900 ttgaacacca gcccaccgca cctcatggat ttgctccgcc tgcacagcag tccctggcag 960 ggaaggaccg tgtccccgct gcagagcacc gtctgaggga gaggatcctg gctacgttcc 1020 tgttctggct gatggacaca tacgtggtac agctgcttag gtcattcttt tacatcacag 1080 agagcacatt ccagaagaac aggctcttct tctaccgtaa gagtgtgtgg agcaagctgc 1140 agagcattgg agtcaggcaa caccttgaga gagtgcggct acgggagctg tcacaagagg 1200 aggtcaggca tcaccaggac acctggctag ccatgcccat ctgcagactg cgcttcatcc 1260 ccaagcccaa cggcctgcgg cccattgtga acatgagtta tagcatgggt accagagctt 1320 tgggcagaag gaagcaggcc cagcatttca cccagcgtct caagactctc ttcagcatgc 1380 tcaactatga gcggacaaaa catcctcacc ttatggggtc ttctgtactg ggtatgaatg 1440 acatctacag gacctggcgg gcctttgtgc tgcgtgtgcg tgctctggac cagacaccca 1500 ggatgtactt tgttaaggca gatgtgaccg gggcctatga tgccatcccc cagggtaagc 1560 tggtggaggt tgttgccaat atgatcaggc actcggagag cacgtactgt atccgccagt 1620 atgcagtggt ccggagagat agccaaggcc aagtccacaa gtcctttagg agacaggtca 1680 ccaccctctc tgacctccag ccatacatgg gccagttcct taagcatctg caggattcag 1740 atgccagtgc actgaggaac tccgttgtca tcgagcagag catctctatg aatgagagca 1800 gcagcagcct gtttgacttc ttcctgcact tcctgcgtca cagtgtcgta aagattggtg 1860 acaggtgcta tacgcagtgc cagggcatcc cccagggctc cagcctatcc accctgctct 1920 gcagtctgtg tttcggagac atggagaaca agctgtttgc tgaggtgcag cgggatgggt 1980 tgcttttacg ttttgttgat gactttctgt tggtgacgcc tcacttggac caagcaaaaa 2040 ccttcctcag caccctggtc catggcgttc ctgagtatgg gtgcatgata aacttgcaga 2100 agacagtggt gaacttccct gtggagcctg gtaccctggg tggtgcagct ccataccagc 2160 tgcctgctca ctgcctgttt ccctggtgtg gcttgctgct ggacactcag actttggagg 2220 tgttctgtga ctactcaggt tatgcccaga cctcaattaa gacgagcctc accttccaga 2280 gtgtcttcaa agctgggaag accatgcgga acaagctcct gtcggtcttg cggttgaagt 2340 gtcacggtct atttctagac ttgcaggtga acagcctcca gacagtctgc atcaatatat 2400 acaagatctt cctgcttcag gcctacaggt tccatgcatg tgtgattcag cttccctttg 2460 accagcgtgt taggaagaac ctcacattct ttctgggcat catctccagc caagcatcct 2520 gctgctatgc tatcctgaag gtcaagaatc caggaatgac actaaaggcc tctggctcct 2580 ttcctcctga agccgcacat tggctctgct accaggcctt cctgctcaag ctggctgctc 2640 attctgtcat ctacaaatgt ctcctgggac ctctgaggac agcccaaaaa ctgctgtgcc 2700 ggaagctccc agaggcgaca atgaccatcc ttaaagctgc agctgaccca gccctaagca 2760 cagactttca gaccattttg gactaaccct gtctccttcc gctagatgaa catgggcatt 2820 gtagcctcag cactcctgga tccacgtcac aagagggact ggtcagttgt gaggctaggt 2880 catcctccaa acctctgtgt catgggtggt atgggagatt gtcccagtgc cttgtttcct 2940 gtaacaggct tgatttcttt cctgatgccc tcagggaggc agatcctatc ccttttagtg 3000 gcagggatcc actagcacca gcacatgagg agtgcaccca gtgcacatgg gcactgggac 3060 agtggacagg tgtgagattc ctgggccctg gagtcttttc acacctaacc atggagcctg 3120 tcccagtaca tcagagtgcc tcggagatga aaaaggacat cgagccagtg acctaaatta 3180 cagcctgaat atactctgaa ttcatgtgac tgccttagct acttctctac tgctgtgtag 3240 taaaacacca agccaactta taaaagcagg attttcctac tggagcagca gctgagagtt 3300 tacatcttga tccataagca caaaagcaca agacagagag agagagaga agagagaga 3360 3420 aagaaaggtg aagggtgatg aagtccacag ggatcacgct agggatgttc catgccttct 3480 ctgaagctaa gattccttgg cagcgtttga cattagaccat agtgggtacc tactgagatc 3540 actataaaga taaaataggg ggaagcgtat ttgtactgaa ctggaaaaac atacaataa 3600 agagtaaatc atggaaaaaa aaaaaaaaaa aaa 3633 <210> 8 <211> 584 <212> PRT <213> Mus musculus <400> 8 Met Asp Thr Tyr Val Val Gln Leu Leu Arg Ser Phe Phe Tyr Ile Thr 1 5 10 15 Glu Ser Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr Arg Lys Ser Val 20 25 30 Trp Ser Lys Leu Gln Ser Ile Gly Val Arg Gln His Leu Glu Arg Val 35 40 45 Arg Leu Arg Glu Leu Ser Gln Glu Glu Val Arg His His Gln Asp Thr 50 55 60 Trp Leu Ala Met Pro Ile Cys Arg Leu Arg Phe Ile Pro Lys Pro Asn 65 70 75 80 Gly Leu Arg Pro Ile Val Asn Met Ser Tyr Ser Met Gly Thr Arg Ala 85 90 95 Leu Gly Arg Arg Lys Gln Ala Gln His Phe Thr Gln Arg Leu Lys Thr 100 105 110 Leu Phe Ser Met Leu Asn Tyr Glu Arg Thr Lys His Pro His Leu Met 115 120 125 Gly Ser Ser Val Leu Gly Met Asn Asp Ile Tyr Arg Thr Trp Arg Ala 130 135 140 Phe Val Leu Arg Val Arg Ala Leu Asp Gln Thr Pro Arg Met Tyr Phe 145 150 155 160 Val Lys Ala Asp Val Thr Gly Ala Tyr Asp Ala Ile Pro Gln Gly Lys 165 170 175 Leu Val Glu Val Val Ala Asn Met Ile Arg His Ser Glu Ser Thr Tyr 180 185 190 Cys Ile Arg Gln Tyr Ala Val Val Arg Arg Asp Ser Gln Gly Gln Val 195 200 205 His Lys Ser Phe Arg Arg Gln Val Thr Thr Leu Ser Asp Leu Gln Pro 210 215 220 Tyr Met Gly Gln Phe Leu Lys His Leu Gln Asp Ser Asp Ala Ser Ala 225 230 235 240 Leu Arg Asn Ser Val Val Ile Glu Gln Ser Ile Ser Met Asn Glu Ser 245 250 255 Ser Ser Ser Leu Phe Asp Phe Phe Leu His Phe Leu Arg His Ser Val 260 265 270 Val Lys Ile Gly Asp Arg Cys Tyr Thr Gln Cys Gln Gly Ile Pro Gln 275 280 285 Gly Ser Ser Leu Ser Thr Leu Leu Cys Ser Leu Cys Phe Gly Asp Met 290 295 300 Glu Asn Lys Leu Phe Ala Glu Val Gln Arg Asp Gly Leu Leu Leu Arg 305 310 315 320 Phe Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Asp Gln Ala Lys 325 330 335 Thr Phe Leu Ser Thr Leu Val His Gly Val Pro Glu Tyr Gly Cys Met 340 345 350 Ile Asn Leu Gln Lys Thr Val Val Asn Phe Pro Val Glu Pro Gly Thr 355 360 365 Leu Gly Gly Ala Ala Pro Tyr Gln Leu Pro Ala His Cys Leu Phe Pro 370 375 380 Trp Cys Gly Leu Leu Leu Asp Thr Gln Thr Leu Glu Val Phe Cys Asp 385 390 395 400 Tyr Ser Gly Tyr Ala Gln Thr Ser Ile Lys Thr Ser Leu Thr Phe Gln 405 410 415 Ser Val Phe Lys Ala Gly Lys Thr Met Arg Asn Lys Leu Leu Ser Val 420 425 430 Leu Arg Leu Lys Cys His Gly Leu Phe Leu Asp Leu Gln Val Asn Ser 435 440 445 Leu Gln Thr Val Cys Ile Asn Ile Tyr Lys Ile Phe Leu Leu Gln Ala 450 455 460 Tyr Arg Phe His Ala Cys Val Ile Gln Leu Pro Phe Asp Gln Arg Val 465 470 475 480 Arg Lys Asn Leu Thr Phe Phe Leu Gly Ile Ile Ser Ser Gln Ala Ser 485 490 495 Cys Cys Tyr Ala Ile Leu Lys Val Lys Asn Pro Gly Met Thr Leu Lys 500 505 510 Ala Ser Gly Ser Phe Pro Pro Glu Ala Ala His Trp Leu Cys Tyr Gln 515 520 525 Ala Phe Leu Leu Lys Leu Ala Ala His Ser Val Ile Tyr Lys Cys Leu 530 535 540 Leu Gly Pro Leu Arg Thr Ala Gln Lys Leu Leu Cys Arg Lys Leu Pro 545 550 555 560 Glu Ala Thr Met Thr Ile Leu Lys Ala Ala Ala Asp Pro Ala Leu Ser 565 570 575 Thr Asp Phe Gln Thr Ile Leu Asp 580 <210> 9 <211> 4335 <212> DNA <213> Mus musculus <400> 9 gttcccagcc tcatcttttt cgtcgtggac tctcagtggc ctgggtcctg gctgttttct 60 aagcacaccc ttgcatcttg gttcccgcac gtgggaggcc catcccggcc ttgagcacaa 120 tgacccgcgc tcctcgttgc cccgcggtgc gctctctgct gcgcagccga taccgggagg 180 tgtggccgct ggcaaccttt gtgcggcgcc tggggcccga gggcaggcgg cttgtgcaac 240 ccggggaccc gaagatctac cgcactttgg ttgcccaatg cctagtgtgc atgcactggg 300 gctcacagcc tccacctgcc gacctttcct tccaccaggt gtcatccctg aaagagctgg 360 tggccagggt tgtgcagaga ctctgcgagc gcaacgagag aaacgtgctg gcttttggct 420 ttgagctgct taacgaggcc agaggcgggc ctcccatggc cttcactagt agcgtgcgta 480 gctacttgcc caacactgtt attgagaccc tgcgtgtcag tggtgcatgg atgctactgt 540 tgagccgagt gggcgacgac ctgctggtct acctgctggc acactgtgct ctttatcttc 600 tggtgccccc cagctgtgcc taccaggtgt gtgggtctcc cctgtaccaa atttgtgcca 660 ccacggatat ctggccctct gtgtccgcta gttacaggcc cacccgaccc gtgggcagga 720 780 aacccctggc cttgccatct cgaggtacaa agaggcatct gagtctcacc agtacaagtg 840 tgccttcagc taagaaggcc agatgctatc ctgtcccgag agtggaggag ggaccccaca 900 ggcaggtgct accaacccca tcaggcaaat catgggtgcc aagtcctgct cggtcccccg 960 aggtgcctac tgcagagaa gatttgtctt ctaaaggaa ggtgtctgac ctgagtctct 1020 ctgggtcggt gtgcttaaa caaagccca gctccacatc tctgctgtca ccaccccgcc 1080 aaaatgcctt tcagctcagg ccatttattg agaccagaca tttcctttac tccaggggag 1140 atggccaaga gcgtctaaac ccctcattcc tactcagcaa cctccagcct aacttgactg 1200 gggccaggag actggtggag atcatctttc tgggctcaag gcctaggaca tcaggaccac 1260 tctgcaggac acaccgtcta tcgcgtcgat actggcagat gcggcccctg ttccaacagc 1320 tgctggtgaa ccatgcagag tgccaatatg tcagactcct caggtcacat tgcaggtttc 1380 gaagacaaa ccaacaggtg acagatgcct tgaacaccag cccaccgcac ctcatggatt 1440 tgctccgcct gcacagcagt ccctggcagg tatatggttt tcttcgggcc tgtctctgca 1500 aggtggtgtc tgctagtctc tggggtacca ggcacaatga gcgccgcttc tttaagaact 1560 taaagaagtt catctcgttg gggaaatacg gcaagctatc actgcaggaa ctgatgtgga 1620 agatgaaagt agaggattgc cactggctcc gcagcagccc ggggaaggac cgtgtccccg 1680 ctgcagagca ccgtctgagg gagaggatcc tggctacgtt cctgttctgg ctgatggaca 1740 catacgtggt acagctgctt aggtcattct tttacatcac agagagcaca ttccagaaga 1800 acaggctctt cttctaccgt aagagtgtgt ggagcaagct gcagagcatt ggagtcaggc 1860 aacaccttga gagagtgcgg ctacgggagc tgtcacaaga ggaggtcagg catcaccagg 1920 acacctggct agccatgccc atctgcagac tgcgcttcat ccccaagccc aacggcctgc 1980 ggcccattgt gaacatgagt tatagcatgg gtaccagagc tttgggcaga aggaagcagg 2040 cccagcattt cacccagcgt ctcaagactc tcttcagcat gctcaactat gagcggacaa 2100 aacatcctca ccttatgggg tcttctgtac tgggtatgaa tgacatctac aggacctggc 2160 gggcctttgt gctgcgtgtg cgtgctctgg accagacacc caggatgtac tttgttaagg 2220 cagatgtgac cggggcctat gatgccatcc cccagggtaa gctggtggag gttgttgcca 2280 atatgatcag gcactcggag agcacgtact gtatccgcca gtatgcagtg gtccggagag 2340 atagccaagg ccaagtccac aagtccttta ggagacaggt caccaccctc tctgacctcc 2400 agccatacat gggccagttc cttaagcatc tgcaggattc agatgccagt gcactgagga 2460 actccgttgt catcgagcag agcatctcta tgaatgagag cagcagcagc ctgtttgact 2520 tcttcctgca cttcctgcgt cacagtgtcg taaagattgg tgacaggtgc tatacgcagt 2580 gccagggcat cccccagggc tccagcctat ccaccctgct ctgcagtctg tgtttcggag 2640 acatggagaa caagctgttt gctgaggtgc agcgggatgg gttgctttta cgttttgttg 2700 atgactttct gttggtgacg cctcacttgg accaagcaaa aaccttcctc agcaccctgg 2760 tccatggcgt tcctgagtat gggtgcatga taaacttgca gaagacagtg gtgaacttcc 2820 ctgtggagcc tggtaccctg ggtggtgcag ctccatacca gctgcctgct cactgcctgt 2880 ttccctggtg tggcttgctg ctggacactc agactttgga ggtgttctgt gactactcag 2940 gttatgccca gacctcaatt aagacgagcc tcaccttcca gagtgtcttc aaagctggga 3000 agaccatgcg gaacaagctc ctgtcggtct tgcggttgaa gtgtcacggt ctatttctag 3060 acttgcaggt gaacagcctc cagacagtct gcatcaatat atacaagatc ttcctgcttc 3120 aggcctacag gttccatgca tgtgtgattc agcttccctt tgaccagcgt gttaggaaga 3180 acctcacatt ctttctgggc atcatctcca gccaagcatc ctgctgctat gctatcctga 3240 aggtcaagaa tccaggaatg acactaaagg cctctggctc ctttcctcct gaagccgcac 3300 attggctctg ctaccaggcc ttcctgctca agctggctgc tcattctgtc atctacaaat 3360 gtctcctggg acctctgagg acagcccaaa aactgctgtg ccggaagctc ccagaggcga 3420 caatgaccat ccttaaagct gcagctgacc cagccctaag cacagacttt cagaccattt 3480 tggactaacc ctgtctcctt ccgctagatg aacatgggca ttgtagcctc agcactcctg 3540 gatccacgtc acaagaggga ctggtcagtt gtgaggctag gtcatcctcc aaacctctgt 3600 gtcatgggtg gtatgggaga ttgtcccagt gccttgtttc ctgtaacagg cttgatttct 3660 ttcctgatgc cctcagggag gcagatccta tcccttttag tggcagggat ccactagcac 3720 cagcacatga ggagtgcacc cagtgcacat gggcactggg acagtggaca ggtgtgagat 3780 tcctgggccc tggagtcttt tcacacctaa ccatggagcc tgtcccagta catcagagtg 3840 cctcggagat gaaaaaggac atcgagccag tgacctaaat tacagcctga atatactctg 3900 aattcatgtg actgccttag ctacttctct actgctgtgt agtaaaacac caagccaact 3960 tataaaagca ggattttcct actggagcag cagctgagag tttacatctt gatccataag 4020 cacaaaagca caagacagag agagagagag agagagagag agagagagag agagagagag 4080 agagagagag agagagtcag tcagtcagtc taacaaataa ctaagaaagg tgaagggtga 4140 tgaagtccac agggatcacg ctagggatgt tccatgcctt ctctgaagct aagattcctt 4200 ggcagcgttt gacagtaacc atagtgggta cctactgaga tcactataaa gataaaatag 4260 ggggaagcgt atttgtactg aactggaaaa acatacaaat aaagagtaaa tcatggaaaa 4320 aaaaaaaaaa aaaaa 4335 <210> 10 <211> 1122 <212> PRT <213> House mouse <400> 10 Met Thr Arg Ala Pro Arg Cys Pro Ala Val Arg Ser Leu Leu Arg Ser 1 5 10 15 Arg Tyr Arg Glu Val Trp Pro Leu Ala Thr Phe Val Arg Arg Leu Gly 20 25 30 Pro Glu Gly Arg Arg Leu Val Gln Pro Gly Asp Pro Lys Ile Tyr Arg 35 40 45 Thr Leu Val Ala Gln Cys Leu Val Cys Met His Trp Gly Ser Gln Pro 50 55 60 Pro Pro Ala Asp Leu Ser Phe His Gln Val Ser Ser Leu Lys Glu Leu 65 70 75 80 Val Ala Arg Val Val Gln Arg Leu Cys Glu Arg Asn Glu Arg Asn Val 85 90 95 Leu Ala Phe Gly Phe Glu Leu Leu Asn Glu Ala Arg Gly Gly Pro Pro 100 105 110 Met Ala Phe Thr Ser Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Ile 115 120 125 Glu Thr Leu Arg Val Ser Gly Ala Trp Met Leu Leu Leu Ser Arg Val 130 135 140 Gly Asp Asp Leu Leu Val Tyr Leu Leu Ala His Cys Ala Leu Tyr Leu 145 150 155 160 Leu Val Pro Pro Ser Cys Ala Tyr Gln Val Cys Gly Ser Pro Leu Tyr 165 170 175 Gln Ile Cys Ala Thr Thr Asp Ile Trp Pro Ser Val Ser Ala Ser Tyr 180 185 190 Arg Pro Thr Arg Pro Val Gly Arg Asn Phe Thr Asn Leu Arg Phe Leu 195 200 205 Gln Gln Ile Lys Ser Ser Ser Arg Gln Glu Ala Pro Lys Pro Leu Ala 210 215 220 Leu Pro Ser Arg Gly Thr Lys Arg His Leu Ser Leu Thr Ser Thr Ser 225 230 235 240 Val Pro Ser Ala Lys Lys Ala Arg Cys Tyr Pro Val Pro Arg Val Glu 245 250 255 Glu Gly Pro His Arg Gln Val Leu Pro Thr Pro Ser Gly Lys Ser Trp 260 265 270 Val Pro Ser Pro Ala Arg Ser Pro Glu Val Pro Thr Ala Glu Lys Asp 275 280 285 Leu Ser Ser Lys Gly Lys Val Ser Asp Leu Ser Leu Ser Gly Ser Val 290 295 300 Cys Cys Lys His Lys Pro Ser Ser Thr Ser Leu Leu Ser Pro Pro Arg 305 310 315 320 Gln Asn Ala Phe Gln Leu Arg Pro Phe Ile Glu Thr Arg His Phe Leu 325 330 335 Tyr Ser Arg Gly Asp Gly Gln Glu Arg Leu Asn Pro Ser Phe Leu Leu 340 345 350 Ser Asn Leu Gln Pro Asn Leu Thr Gly Ala Arg Arg Leu Val Glu Ile 355 360 365 Ile Phe Leu Gly Ser Arg Pro Arg Thr Ser Gly Pro Leu Cys Arg Thr 370 375 380 His Arg Leu Ser Arg Arg Tyr Trp Gln Met Arg Pro Leu Phe Gln Gln 385 390 395 400 Leu Leu Val Asn His Ala Glu Cys Gln Tyr Val Arg Leu Leu Arg Ser 405 410 415 His Cys Arg Phe Arg Thr Ala Asn Gln Gln Val Thr Asp Ala Leu Asn 420 425 430 Thr Ser Pro Pro His Leu Met Asp Leu Leu Arg Leu His Ser Ser Pro 435 440 445 Trp Gln Val Tyr Gly Phe Leu Arg Ala Cys Leu Cys Lys Val Val Ser 450 455 460 Ala Ser Leu Trp Gly Thr Arg His Asn Glu Arg Arg Phe Phe Lys Asn 465 470 475 480 Leu Lys Lys Phe Ile Ser Leu Gly Lys Tyr Gly Lys Leu Ser Leu Gln 485 490 495 Glu Leu Met Trp Lys Met Lys Val Glu Asp Cys His Trp Leu Arg Ser 500 505 510 Ser Pro Gly Lys Asp Arg Val Pro Ala Ala Glu His Arg Leu Arg Glu 515 520 525 Arg Ile Leu Ala Thr Phe Leu Phe Trp Leu Met Asp Thr Tyr Val Val 530 535 540 Gln Leu Leu Arg Ser Phe Phe Tyr Ile Thr Glu Ser Thr Phe Gln Lys 545 550 555 560 Asn Arg Leu Phe Phe Tyr Arg Lys Ser Val Trp Ser Lys Leu Gln Ser 565 570 575 Ile Gly Val Arg Gln His Leu Glu Arg Val Arg Leu Arg Glu Leu Ser 580 585 590 Gln Glu Glu Val Arg His His Gln Asp Thr Trp Leu Ala Met Pro Ile 595 600 605 Cys Arg Leu Arg Phe Ile Pro Lys Pro Asn Gly Leu Arg Pro Ile Val 610 615 620 Asn Met Ser Tyr Ser Met Gly Thr Arg Ala Leu Gly Arg Arg Lys Gln 625 630 635 640 Ala Gln His Phe Thr Gln Arg Leu Lys Thr Leu Phe Ser Met Leu Asn 645 650 655 Tyr Glu Arg Thr Lys His Pro His Leu Met Gly Ser Ser Val Leu Gly 660 665 670 Met Asn Asp Ile Tyr Arg Thr Trp Arg Ala Phe Val Leu Arg Val Arg 675 680 685 Ala Leu Asp Gln Thr Pro Arg Met Tyr Phe Val Lys Ala Asp Val Thr 690 695 700 Gly Ala Tyr Asp Ala Ile Pro Gln Gly Lys Leu Val Glu Val Val Ala 705 710 715 720 Asn Met Ile Arg His Ser Glu Ser Thr Tyr Cys Ile Arg Gln Tyr Ala 725 730 735 Val Val Arg Arg Asp Ser Gln Gly Gln Val His Lys Ser Phe Arg Arg 740 745 750 Gln Val Thr Thr Leu Ser Asp Leu Gln Pro Tyr Met Gly Gln Phe Leu 755 760 765 Lys His Leu Gln Asp Ser Asp Ala Ser Ala Leu Arg Asn Ser Val Val 770 775 780 Ile Glu Gln Ser Ile Ser Met Asn Glu Ser Ser Ser Ser Leu Phe Asp 785 790 795 800 Phe Phe Leu His Phe Leu Arg His Ser Val Val Lys Ile Gly Asp Arg 805 810 815 Cys Tyr Thr Gln Cys Gln Gly Ile Pro Gln Gly Ser Ser Leu Ser Thr 820 825 830 Leu Leu Cys Ser Leu Cys Phe Gly Asp Met Glu Asn Lys Leu Phe Ala 835 840 845 Glu Val Gln Arg Asp Gly Leu Leu Leu Arg Phe Val Asp Asp Phe Leu 850 855 860 Leu Val Thr Pro His Leu Asp Gln Ala Lys Thr Phe Leu Ser Thr Leu 865 870 875 880 Val His Gly Val Pro Glu Tyr Gly Cys Met Ile Asn Leu Gln Lys Thr 885 890 895 Val Val Asn Phe Pro Val Glu Pro Gly Thr Leu Gly Gly Ala Ala Pro 900 905 910 Tyr Gln Leu Pro Ala His Cys Leu Phe Pro Trp Cys Gly Leu Leu Leu 915 920 925 Asp Thr Gln Thr Leu Glu Val Phe Cys Asp Tyr Ser Gly Tyr Ala Gln 930 935 940 Thr Ser Ile Lys Thr Ser Leu Thr Phe Gln Ser Val Phe Lys Ala Gly 945 950 955 960 Lys Thr Met Arg Asn Lys Leu Leu Ser Val Leu Arg Leu Lys Cys His 965 970 975 Gly Leu Phe Leu Asp Leu Gln Val Asn Ser Leu Gln Thr Val Cys Ile 980 985 990 Asn Ile Tyr Lys Ile Phe Leu Leu Gln Ala Tyr Arg Phe His Ala Cys 995 1000 1005 Val Ile Gln Leu Pro Phe Asp Gln Arg Val Arg Lys Asn Leu Thr 1010 1015 1020 Phe Phe Leu Gly Ile Ile Ser Ser Gln Ala Ser Cys Cys Tyr Ala 1025 1030 1035 Ile Leu Lys Val Lys Asn Pro Gly Met Thr Leu Lys Ala Ser Gly 1040 1045 1050 Ser Phe Pro Pro Glu Ala Ala His Trp Leu Cys Tyr Gln Ala Phe 1055 1060 1065 Leu Leu Lys Leu Ala Ala His Ser Val Ile Tyr Lys Cys Leu Leu 1070 1075 1080 Gly Pro Leu Arg Thr Ala Gln Lys Leu Leu Cys Arg Lys Leu Pro 1085 1090 1095 Glu Ala Thr Met Thr Ile Leu Lys Ala Ala Ala Asp Pro Ala Leu 1100 1105 1110 Ser Thr Asp Phe Gln Thr Ile Leu Asp 1115 1120 <210> 11 <211> 29 <212> PRT <213> Rabies virus <400> 11 Tyr Thr Ile Trp Met Pro Glu Asn Pro Arg Pro Gly Thr Pro Cys Asp 1 5 10 15 Ile Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser Asn Gly 20 25 <210> 12 <211> 30 <212> PRT <213> House mouse <​​​​​​​​​​​​​​ <213> Rabies virus <400> 13 Tyr Thr Ile Trp Met Pro Glu Asn Pro Arg Pro Gly Thr Pro Cys Asp 1 5 10 15 Ile Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser Asn Gly Gly Gly Gly 20 25 30 Gly Cys <210> 14 <211> 42 <212> PRT <213> Rabies virus <220> <221> MISC_FEATURE <222> (34) (42) <223> Xaa is D-arginine. <400> 14 Tyr Thr Ile Trp Met Pro Glu Asn Pro Arg Pro Gly Thr Pro Cys Asp 1 5 10 15 Ile Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser Asn Gly Gly Gly Gly 20 25 30 Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40

Claims

1. Use of a TERT activating therapeutic agent in the preparation of a kit for treating a subject with Alzheimer's disease, wherein the TERT activating therapeutic agent is administered to the subject, wherein the TERT activating therapeutic agent comprises one or more nucleic acids encoding a non-catalytically active TERT polypeptide, and wherein the non-catalytically active TERT polypeptide is generated by replacing the aspartic acid residue at residue 712 of SEQ ID NO:4 with alanine.

2. The use according to claim 1, wherein the TERT activating therapeutic agent further comprises an HMT (histone methyltransferase) inhibitor, a histone H3K9 demethylase polypeptide, or a nucleic acid encoding histone H3K9 demethylase.

3. The use according to any one of claims 1 to 2, wherein the object has been previously treated for Alzheimer's disease.

4. The use according to claim 3, wherein the subject has been identified as unresponsive to prior treatment.

5. The use according to any one of claims 1 to 2, wherein the object is a person.

6. The use according to claim 5, wherein the subject is older than 50 years old.

7. The use according to any one of claims 1 to 2, wherein an additional therapy is further administered.

8. The use according to claim 2, wherein the HMT inhibitor comprises one or more of trachomatis, BIX-01294, BIX-01338, UNC0638 and BRD4770.

9. The use according to claim 2, wherein the histone H3K9 demethylase polypeptide comprises one or more polypeptides selected from KDM1A / LSD1, KDM3A / JHDM2A, KDM3B / JHDM2B, KDM4A / JHDM3A, KDM4B / JMJD2B, KDM4C / JMJD2C, KDM4D / JMJD2D, KDM7 / JHDM1D, and PHF8.

10. The use according to any one of claims 1 to 2, wherein the TERT activating therapeutic agent is for administration by intravenous injection.

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