Gene therapy for haploinsufficiency

Abstract

To provide methods and compositions for activating transcription in a mammalian cell.SOLUTION: Provided is a method of treating a haploinsufficiency disease in a mammalian subject, the method comprising a step of contacting a cell of the subject with a composition comprising: i) a guide RNA comprising a) a targeting region that, under conditions present in a nucleus of the cell, specifically hybridizes to a promoter region or an enhancer region operably linked to a wild-type copy of a haploinsufficient gene, and b) a CRISPR nuclease-binding region that specifically binds to a CRISPR nuclease under conditions present in a nucleus of the cell or a region that specifically binds to the CRISPR nuclease-binding region; and ii) the CRISPR nuclease.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 455,988, filed February 7, 2017, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with United States government support under Grant No. R01 DK090382 awarded by the National Institutes of Health. The United States government has certain rights in this invention.

[0003] Reference to submitting a sequence listing This application contains a Sequence Listing as a 107 kilobyte text file with filename "081906-224410PC-1072775_SequenceListing.txt", created on February 6, 2018. The data contained in this text file is incorporated herein by reference in its entirety for all purposes.

[0004] FIELD OF THE INVENTION The present disclosure relates generally to methods and compositions for activating transcription in mammalian cells. [Background technology]

[0005] Background of the Invention Genomic alterations that result in reduced transcription or activity of one or more genes or gene products are causative factors of a wide variety of mammalian diseases. One such genomic alteration is haploinsufficiency. In haploinsufficiency, there is only one functional copy of a gene, and that single copy does not produce enough gene product to result in a wild-type phenotype. Another disease is caused by genomic alterations in one or both copies of a gene that alter the gene product so that it exhibits reduced activity rather than eliminated activity. In yet another disease, genomic alterations reduce the transcription of one or both copies of a gene or reduce the stability of the transcript, resulting in an insufficient abundance of the gene product to produce a wild-type phenotype. Numerous approaches have been attempted to treat such diseases by increasing the amount or activity of one or more genes whose transcription or activity is reduced. Such approaches include delivering wild-type copies of the one or more genes into the genome. Recently, targeted introduction into the genome has been demonstrated using methods and compositions based on clustered regularly interspaced short palindromic repeats (CRISPR), zinc finger nucleases (ZFNs) (see Urnov et al., Nat. Rev. Genet., 11:636-646 (2010) (Patent Document 1)), or transcription activator-like effector nucleases (TALENs) (see Joung and Sander, Nat. Rev. Mol. Cell Biol., 1:49-55 (2013) (Patent Document 2)). Another approach to increasing the transcription of one or more target genes involves the use of antisense oligomers that promote constitutive splicing (see US 2016 / 0298121 (Non-Patent Document 1)). However, there remains a need for alternative methods and compositions that increase the transcription of target genes to treat diseases caused by reduced transcription, amount or activity of the target genes. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Urnov et al., Nat. Rev. Genet., 11:636-646 (2010) [Patent Document 2] Joung and Sander, Nat.Rev.Mol.Cell Biol.,1:49-55(2013) [Non-patent literature]

[0007] [Non-Patent Document 1] US 2016 / 0298121 Summary of the Invention

[0008] The present invention is directed to methods and compositions for increasing transcription of a target gene in a mammalian (e.g., human) subject. The inventors have discovered that such increased transcription can be achieved with a transcription-activating guide RNA (gRNA) construct (e.g., as part of a dCAS9 / gRNA complex) that targets the promoter or enhancer region of the gene. Furthermore, the inventors have discovered that transcription activation can be achieved with a non-integrating vector in sufficient amounts and for sufficient periods to treat disease. Optionally, the present methods and compositions for transcription activation do not covalently modify the genome of the host mammal by endonuclease cleavage, nicking, and / or repair. Optionally, the non-integrating vector is an episomal vector, such as an adeno-associated virus vector.

[0009] In one aspect, the present invention provides a method of treating a haploinsufficiency disease in a mammalian subject, the method comprising: (i)(a) a targeting region that specifically hybridizes to a promoter or enhancer region operably linked to a wild-type copy of a haploinsufficient gene under conditions present in the nucleus of said cell; and (b) a CRISPR nuclease binding region that specifically binds to a CRISPR nuclease under conditions present in the nucleus of the cell, or a region that specifically binds to the CRISPR nuclease binding region a guide RNA comprising (ii) CRISPR nuclease with a composition comprising The contacting step forms a complex containing the CRISPR nuclease bound to the guide RNA, and the targeting region of the guide RNA in the complex hybridizes to the promoter or enhancer; the complex comprises a catalytically inactive CRISPR nuclease and a transcription activation domain; and The complex activates transcription of a wild-type copy of the haploinsufficient gene in an amount and for a duration sufficient to treat the haploinsufficiency disease in the subject. In some embodiments, the mammalian subject is treated with a host cell obtained from the subject. In one embodiment, the mammalian subject is treated with a host cell obtained from a different (entirely separate) mammalian subject. In some embodiments, the host cell is an isolated mammalian host cell. In another embodiment, the host cell comprises an isolated mammalian host cell having one functional copy of the target gene.

[0010] In some embodiments, the contacting step comprises contacting the cell with an episomal vector encoding a guide RNA or a CRISPR nuclease. In some embodiments, the contacting step comprises contacting the cell with an episomal vector encoding a guide RNA and a CRISPR nuclease. In some embodiments, the contacting step comprises contacting the cell with an episomal vector encoding a guide RNA and a second episomal vector encoding a CRISPR nuclease. In some embodiments, the episomal vector is non-integrating. In some embodiments, the episomal vector is non-replicating. In some embodiments, the episomal vector is an adeno-associated virus (AAV) vector. In some embodiments, the episomal vector independently comprises a first end and a second end, and the first end and the second end each independently comprise an AAV inverted terminal repeat sequence.

[0011] In some embodiments, the CRISPR nuclease comprises (i) a nuclease domain modified to eliminate nuclease activity and nicking activity and (ii) a transcription activation domain. In some embodiments, the CRISPR nuclease comprises a Cas9 nuclease or a Cpf1 nuclease. In some embodiments, the modification comprises mutations at positions corresponding to D10 and H840 of S. pyogenes Cas9. In some embodiments, the CRISPR nuclease comprises D10A,H840A S. pyogenes dCas9. In some embodiments, the CRISPR nuclease comprises Staphylococcus aureus dCas9. In some embodiments, the S. aureus dCas9 comprises one or more mutations at one of the following residues: E782, K929, N968, R1015. In some embodiments, the guide RNA comprises a dead guide sequence.

[0012] In some embodiments, the guide RNA comprises a transcription activation binding domain, and the transcription activation binding domain specifically binds to a composition comprising one or more transcription activation domains.In some embodiments, the complex comprising the CRISPR nuclease bound to the guide RNA further comprises a transcription activation domain selected from the group consisting of HSF1, VP16, VP64, p65, MyoD1, RTA, SET7 / 9, VPR, histone acetyltransferase p300, the hydroxylase catalytic domain of TET family proteins (for example, TET1 hydroxylase catalytic domain), LSD1, CIB1, AD2, CR3, EKLF1, GATA4, PRVIE, p53, SP1, MEF2C, TAX and PPARγ.In some embodiments, the CRISPR nuclease is a CRISPR nuclease-VP64 fusion polypeptide.

[0013] In some embodiments, the guide RNA comprises a scaffold region. In some embodiments, the scaffold region comprises an ms2, f6, PP7, com, or L7a ligand sequence. In some embodiments, the scaffold region of the guide RNA in the complex is bound to a transcription activation domain fused to an MCP polypeptide, a COM polypeptide, a PCP polypeptide, or an L7a polypeptide. In some embodiments, the haploinsufficient gene is SIM1, leptin, leptin receptor, MC4R, SCN2A, SETD5, PAX6, PKD1, MC3R, POMC, STAT3, STAT5, SOCS3, GHR, NPY, NPY1R, NPY2R, NPY5R, PYY, AMPK (PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3), OXT, JAK2, SHP2, NOS3, NROB2, BRS3, CARTPT, FABP4 , HTR2C, IL6, NHLH2, NMU, NPB, NPBWRI, PNPLA2, UCP3, ADIPOQ, APOA5, ARNT2, ASIP, C1QTNF2, C3AR1, CCK, CPT1B, CSF2, DGAT1, DGAT2, GH RL, GHSR, HSD11B1, HTR7, INSIG1, INSIG2, LIPC, NMURI, NMUR2, NPBWR2, NTS, PPARGC1A, PPY, RETN, SIRT1, TGFBR2, WDTC1 or FOXO1.

[0014] In some embodiments, the targeting region of the guide RNA comprises: In some embodiments, the targeting region of the guide RNA is encoded by or specifically hybridizes to TIFF2025118716000002.tif18166. In some embodiments, the targeting region of the guide RNA is encoded by or specifically hybridizes to TIFF2025118716000003.tif26158. In another embodiment, the targeting region of the guide RNA is encoded by or specifically hybridizes to TIFF2025118716000004.tif33162. In some embodiments, the targeting region of the guide RNA is encoded by or specifically hybridizes to TIFF2025118716000005.tif33164. In some embodiments, the targeting region of the guide RNA is encoded by or specifically hybridizes to TIFF2025118716000006.tif33167. In some embodiments, the targeting region of the guide RNA is encoded by or specifically hybridizes to TIFF2025118716000007.tif26162. TIFF2025118716000008.tif47169 or specifically hybridizes thereto. In some embodiments, the targeting region of the guide RNA is In some embodiments, the targeting region of the guide RNA is encoded by or specifically hybridizes to TIFF2025118716000009.tif53155. In some embodiments, the targeting region of the guide RNA is encoded by or specifically hybridizes to TIFF2025118716000010.tif54166. In some embodiments, the targeting region of the guide RNA is encoded by or specifically hybridizes to TIFF2025118716000011.tif33161. In some embodiments, the targeting region of the guide RNA is encoded by or specifically hybridizes to TIFF2025118716000012.tif26128. Encoded by or specifically hybridizes to TIFF2025118716000013.tif4128.

[0015] In some embodiments, the cell is a non-dividing cell. In some embodiments, the cell is a neuron. In some embodiments, the cell is a hypothalamic cell. In some embodiments, the contacting comprises injecting a nucleic acid encoding a guide RNA and / or a CRISPR nuclease into a region of the brain comprising the hypothalamus. In some embodiments, the contacting comprises injecting an adeno-associated viral vector comprising a nucleic acid encoding a guide RNA and / or a CRISPR nuclease into a region of the brain comprising the hypothalamus. In some embodiments, the haploinsufficiency disease is selected from Table 1. In some embodiments, the haploinsufficiency disease is selected from obesity, autism, epilepsy, intellectual disability, aniridia, and polycystic kidney disease. In some embodiments, the haploinsufficiency disease is obesity.

[0016] In another aspect, the present invention provides a method for producing a composition comprising: (I.) A genome comprising at least one functional copy of a target gene, which, in the absence of transcriptional activation by a heterologous complex, does not produce sufficient corresponding gene product to result in a wild-type phenotype in the organism; and (II.) (a)(i.) A targeting region that specifically hybridizes, under conditions present in the nucleus of a cell, to a promoter or enhancer region operably linked to a functional copy of a target gene; and (ii.) a CRISPR nuclease binding domain that specifically binds to a CRISPR nuclease under conditions present in the nucleus of a cell; a guide RNA comprising (b) CRISPR nuclease and A heterologous complex comprising a mammalian host cell comprising: The guide RNA of a heterologous complex comprising a CRISPR nuclease bound to the guide RNA is hybridized to the promoter or enhancer, the CRISPR nuclease is catalytically inactive, and the complex activates transcription of a functional copy of the target gene in a sufficient amount and for a sufficient duration to produce a wild-type phenotype when the host cell is present in an organism.

[0017] In some embodiments, the genome comprises only one functional copy of the target gene. In some embodiments, the only functional copy of the target gene comprises a haploinsufficient gene. In some embodiments, the genome comprises fewer than two functional copies of the target gene. [The present invention 1001] 1. A method of treating a haploinsufficiency disease in a mammalian subject, comprising: (i)(a) a targeting region that specifically hybridizes to a promoter or enhancer region operably linked to a wild-type copy of a haploinsufficient gene under conditions present in the nucleus of said cell; and (b) a CRISPR nuclease binding region that specifically binds to a CRISPR nuclease under conditions present in the nucleus of the cell, or a region that specifically binds to the CRISPR nuclease binding region a guide RNA comprising (ii) CRISPR nuclease contacting the substrate with a composition comprising By the contacting step, a complex containing the CRISPR nuclease bound to the guide RNA is formed, and the targeting region of the guide RNA in the complex hybridizes to the promoter or enhancer; the complex comprises a catalytically inactive CRISPR nuclease and a transcription activation domain; and the complex activates transcription of a wild-type copy of the haploinsufficient gene in an amount and for a sufficient duration to treat the haploinsufficiency disease in the subject. The method. [The present invention 1002] 1001. The method of claim 1001, wherein the contacting step comprises contacting the cell with an episomal vector encoding a guide RNA or a CRISPR nuclease. [The present invention 1003] 1003. The method of claim 1001 or 1002, wherein the contacting step comprises contacting the cell with an episomal vector encoding a guide RNA and a CRISPR nuclease. [The present invention 1004] 1003. The method of claim 1001 or 1002, wherein the contacting step comprises contacting the cell with an episomal vector encoding a guide RNA and a second episomal vector encoding a CRISPR nuclease. [The present invention 1005] Any of the aforementioned methods of the present invention, wherein the episomal vector is non-integrating. [The present invention 1006] Any of the aforementioned methods of the present invention, wherein the episomal vector is non-replicative. [The present invention 1007] Any of the aforementioned methods of the present invention, wherein the episomal vector is an adeno-associated virus (AAV) vector. [The present invention 1008] Any of the aforementioned methods of the present invention, wherein the episomal vector independently comprises a first end and a second end, and the first end and second end each independently comprise an AAV inverted terminal repeat sequence. [The present invention 1009] Any of the aforementioned methods of the present invention, wherein the CRISPR nuclease comprises (i) a nuclease domain that has been modified to eliminate nuclease activity and nicking activity, and (ii) a transcription activation domain. [The present invention 1010] Any of the aforementioned methods of the present invention, wherein the modification comprises mutations at positions corresponding to D10 and H840 of S. pyogenes Cas9. [The present invention 1011] Any of the methods of the preceding claims, wherein the CRISPR nuclease comprises D10A,H840A Streptococcus pyogenes dCas9. [The present invention 1012] Any of the aforementioned methods of the present invention, wherein the guide RNA comprises a dead guide sequence. [The present invention 1013] Any of the aforementioned methods of the present invention, wherein the guide RNA comprises a transcription activation binding domain, and the transcription activation binding domain specifically binds to a composition comprising one or more transcription activation domains. [The present invention 1014] Any of the aforementioned methods of the present invention, wherein the complex comprising the CRISPR nuclease bound to the guide RNA further comprises a transcription activation domain selected from the group consisting of HSF1, VP16, VP64, p65, MyoD1, RTA, SET7 / 9, VPR, histone acetyltransferase p300, a hydroxylase catalytic domain of a TET family protein (e.g., a TET1 hydroxylase catalytic domain), LSD1, CIB1, AD2, CR3, EKLF1, GATA4, PRVIE, p53, SP1, MEF2C, TAX, and PPARγ. [The present invention 1015] Any of the aforementioned methods of the present invention, wherein the CRISPR nuclease is a CRISPR nuclease-VP64 fusion polypeptide. [The present invention 1016] Any of the aforementioned methods of the present invention, wherein the guide RNA comprises a scaffold region. [The present invention 1017] 1016. The method of claim 1016, wherein the scaffold region comprises an ms2, f6, PP7, com, or L7a ligand sequence. [The present invention 1018] 1017. The method of claim 1017, wherein the scaffold region of the guide RNA in the complex is bound to a transcription activation domain fused to an MCP polypeptide, a COM polypeptide, a PCP polypeptide, or an L7a polypeptide. [The present invention 1019] Haploinsufficient genes include SIM1, leptin, leptin receptor, MC4R, SCN2A, SETD5, PAX6, PKD1, MC3R, POMC, STAT3, STAT5, SOCS3, GHR, NPY, NPY1R, NPY2R, NPY5R, PYY, AMPK (PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3), OXT, JAK2, SHP2, NOS3, NROB2, BRS3, CARTPT, FABP4, HTR2C, IL6, and NH Any of the methods of the present invention, wherein the gene is LH2, NMU, NPB, NPBWRI, PNPLA2, UCP3, ADIPOQ, APOA5, ARNT2, ASIP, C1QTNF2, C3AR1, CCK, CPT1B, CSF2, DGAT1, DGAT2, GHRL, GHSR, HSD11B1, HTR7, INSIG1, INSIG2, LIPC, NMURI, NMUR2, NPBWR2, NTS, PPARGC1A, PPY, RETN, SIRT1, TGFBR2, WDTC1, or FOXO1. [The present invention 1020] The targeting region of the guide RNA is Any of the aforementioned methods of the present invention, wherein the nucleic acid is encoded by or specifically hybridizes to TIFF2025118716000014.tif37128. [The present invention 1021] The targeting region of the guide RNA is Any of the methods of 1001 to 1019, wherein the nucleic acid is encoded by or specifically hybridizes to TIFF2025118716000015.tif33128. [The present invention 1022] The targeting region of the guide RNA is Any of the methods of 1001 to 1019, wherein the nucleic acid is encoded by or specifically hybridizes to TIFF2025118716000016.tif40128. [The present invention 1023] The targeting region of the guide RNA is Any of the methods of 1001 to 1019, wherein the nucleic acid is encoded by or specifically hybridizes to TIFF2025118716000017.tif40128. [The present invention 1024] The targeting region of the guide RNA is Any of the methods of 1001 to 1019, wherein the nucleic acid is encoded by or specifically hybridizes to TIFF2025118716000018.tif40128. [The present invention 1025] The targeting region of the guide RNA is Any of the methods of 1001 to 1019, wherein the nucleic acid is encoded by or specifically hybridizes to TIFF2025118716000019.tif33128. [The present invention 1026] The targeting region of the guide RNA is Any of the methods of 1001 to 1019, wherein the nucleic acid is encoded by or specifically hybridizes to TIFF2025118716000020.tif68128. [The present invention 1027] The targeting region of the guide RNA is Any of the methods of 1001 to 1019, wherein the nucleic acid is encoded by or specifically hybridizes to TIFF2025118716000021.tif69128. [The present invention 1028] The targeting region of the guide RNA is Any of the methods of 1001 to 1019, wherein the nucleic acid is encoded by or specifically hybridizes to TIFF2025118716000022.tif68128. [The present invention 1029] The targeting region of the guide RNA is Any of the methods of 1001 to 1019, wherein the nucleic acid is encoded by or specifically hybridizes to TIFF2025118716000023.tif47128. [The present invention 1030] The targeting region of the guide RNA is Any of the methods of 1001 to 1019, wherein the nucleic acid sequence is encoded by or specifically hybridizes to TIFF2025118716000024.tif18128. [The present invention 1031] The targeting region of the guide RNA is Any of the methods of 1001 to 1019, wherein the nucleic acid is encoded by or specifically hybridizes to TIFF2025118716000025.tif4128. [The present invention 1032] Any of the aforementioned methods of the present invention, wherein the cells are non-dividing cells. [The present invention 1033] Any of the aforementioned methods of the present invention, wherein the cell is a neuron. [The present invention 1034] Any of the aforementioned methods of the present invention, wherein the cell is a hypothalamic cell. [This invention 1035] Any of the aforementioned methods of the present invention, wherein the contacting step comprises injection of a nucleic acid encoding a guide RNA and / or a CRISPR nuclease into a region of the brain that includes the hypothalamus. [The present invention 1036] Any of the aforementioned methods of the present invention, wherein the contacting step comprises injecting an adeno-associated viral vector comprising a nucleic acid encoding a guide RNA and / or a CRISPR nuclease into a region of the brain that includes the hypothalamus. [This invention 1037] Any of the aforementioned methods of the present invention, wherein the haploinsufficiency disorder is selected from the group consisting of obesity, autism, epilepsy, intellectual disability, aniridia, and polycystic kidney disease. [The present invention 1038] Any of the aforementioned methods of the present invention, wherein the haploinsufficiency disease is selected from Table 1. [This invention 1039] The method of claim 1037, wherein the haploinsufficiency disorder is obesity. [The present invention 1040] (I.) A genome comprising at least one functional copy of a target gene, which, in the absence of transcriptional activation by a heterologous complex, does not produce sufficient corresponding gene product to result in a wild-type phenotype in the organism; and (II.) (a)(i.) A targeting region that specifically hybridizes, under conditions present in the nucleus of a cell, to a promoter or enhancer region operably linked to a functional copy of a target gene; (ii.) a CRISPR nuclease binding domain that specifically binds to a CRISPR nuclease under conditions present in the nucleus of a cell; and a guide RNA, and (b) CRISPR nuclease A heterologous complex comprising 1. An isolated mammalian host cell comprising: A guide RNA of a heterologous complex comprising a CRISPR nuclease bound to the guide RNA is hybridized to the promoter or enhancer; the CRISPR nuclease is catalytically inactive, and the complex activates transcription of a functional copy of the target gene in a sufficient amount and for a sufficient duration to produce a wild-type phenotype when the host cell is present in an organism; The isolated mammalian host cell. [This invention 1041] 1040. The isolated mammalian host cell of claim 10, wherein said genome comprises only one functional copy of the target gene. [The present invention 1042] 1041. An isolated mammalian host cell of the present invention, wherein only one functional copy of the target gene comprises a haploinsufficient gene. [This invention 1043] Haploinsufficient genes include SIM1, leptin, leptin receptor, MC4R, SCN2A, SETD5, PAX6, PKD1, MC3R, POMC, STAT3, STAT5, SOCS3, GHR, NPY, NPY1R, NPY2R, NPY5R, PYY, AMPK (PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3), OXT, JAK2, SHP2, NOS3, NROB2, BRS3, CARTPT, FABP4, HTR2C, IL6, NHLH2, 1042. The isolated mammalian host cell of the present invention, wherein the host cell is NMU, NPB, NPBWRI, PNPLA2, UCP3, ADIPOQ, APOA5, ARNT2, ASIP, C1QTNF2, C3AR1, CCK, CPT1B, CSF2, DGAT1, DGAT2, GHRL, GHSR, HSD11B1, HTR7, INSIG1, INSIG2, LIPC, NMURI, NMUR2, NPBWR2, NTS, PPARGC1A, PPY, RETN, SIRT1, TGFBR2, WDTC1, or FOXO1. [This invention 1044] 1043. The isolated mammalian host cell of the present invention, wherein the haploinsufficient gene treats a haploinsufficiency disease selected from Table 1. [This invention 1045] 1044. The isolated mammalian host cell of the present invention, wherein the haploinsufficiency disorder is selected from obesity, autism, epilepsy, intellectual disability, aniridia, and polycystic kidney disease. [The present invention 1046] 1045. The isolated mammalian host cell of the present invention, wherein the haploinsufficiency disorder is obesity. [Brief explanation of the drawings]

[0018] [Figure 1A]Figure 1A-F: Transgenic CRISPRa Sim1 overexpression in vitro and in vivo. A, Schematic of the mouse Sim1 genomic locus. B, CRISPRa in Neuro-2A cells targeting the Sim1 promoter (Pr) or enhancer (Enh). Results are expressed as fold mRNA increase normalized to beta-actin using the ΔΔCT method. Mean ± SD values were obtained from three independent experiments. * = p < 0.001, *** = p < 0.0005 (ANOVA, Tukey's test). C, Schematic depicting various mouse strains and the mouse transgenic CRISPRa concept. D, Weekly weight measurements of wild-type littermates, Sim1 + / -, H11PCAG-dCas9-VP64 × ROSA26Sim1Pr-sgRNA, and H11PCAG-dCas9-VP64 × ROSA26SCE2En-sgRNA. At least 10 male and female mice were measured per genotype. Mean values ± SD are shown. E–F: Photographs of 20-week-old mice of each genotype: Sim1+ / -, H11PCAG-dCas9-VP64×ROSA26Sim1Pr-sgRNA, and wild-type littermates (E), and Sim1+ / -, H11PCAG-dCas9-VP64×ROSA26SCE2En-sgRNA, and wild-type littermates (F). Body length and weight of each mouse are shown above and below, respectively. [Figure 1B] As described in Figure 1A. [Figure 1C] As described in Figure 1A. [Figure 1D] As described in Figure 1A. [Figure 1E] As described in Figure 1A. [Figure 1F] As described in Figure 1A. [Figure 2A]Figure 2A-D: Body composition and metabolic analysis of Sim1 CRISPRa transgenic mice. A, Estimated fat percentage and their corresponding body weight measurements in wild-type littermates, Sim1 + / -, H11PCAG-dCas9-VP64×ROSA26Sim1Pr-sgRNA (PrmCRISPRa), and H11PCAG-dCas9-VP64×ROSA26SCE2En-sgRNA (EnhCRISPRa) as determined by dual-energy X-ray absorptiometry (DEXA) or Echo magnetic resonance imaging (EchoMRI). Mean values ± standard deviation (SD) were obtained from three females and three males. B, Metabolic chamber energy expenditure analysis determined over four days in three males and three females for all four genotypes. C, Food intake for all four genotypes determined over four days. Mean values ± standard deviation (SD) were obtained from three females and three males. *=p-value<0.001, ***=p-value<0.0005, ns=not significant (ANOVA, Tukey's test). D, Respiratory exchange ratio (RER; VCO2 / VO2) for all four genotypes obtained from three females and three males and plotted as mean ± sd. [Figure 2B] As described in Figure 2A. [Figure 2C] As described in Figure 2A. [Figure 2D] As described in Figure 2A. [Figure 3]Figure 3A-D: dCas9 and Sim1 mRNA expression levels in CRISPRa transgenic mice. A, Heatmap of Sim1 tissue expression. Red and gray filled boxes represent Sim1-expressing and -nonexpressing tissues, respectively, as determined by our wild-type mice. B, dCas9 mRNA expression in the hypothalamus, kidney, lung, and liver from four Sim1 + / - × H11 PCAG-dCas9-VP64 mice. Mean values ± SD were determined using the ΔΔCT method based on fold-increase in mRNA normalized to beta-actin (for hypothalamus) and Rpl38 (for kidney, lung, and liver). C-D, Sim1 mRNA expression in the hypothalamus, kidney, lung, and liver from two females (C) and two males (D) with the following genotypes: wild-type littermates, Sim1 + / -, H11PCAG-dCas9-VP64×ROSA26Sim1Pr-sgRNA (Prm-CRISPRa), and H11PCAG-dCas9-VP64×ROSA26SCE2En-sgRNA (Enh-CRISPRa). Mean values ± SD were determined based on fold mRNA increase compared to wild-type littermates using the ΔΔCT method and normalized to beta-actin or Rpl38. BDL = below the level of detection. [Figure 4]Figure 4A-E: AAV-mediated CRISPRa Sim1 overexpression in vitro and in vivo. A, AAV CRISPRa in Neuro-2A cells using viral particles containing pCMV-dCas9-VP64 (dCas9-VP64), pCMV-dCas9-VP64 and pSim1Pr-mCherry (PrmCRISPRa), and pCMV-dCas9-VP64 and pSCE2En-mCherry (EnhCRISPRa). Results are expressed as fold mRNA increase normalized to beta-actin using the ΔΔCT method. Mean values ± SD were obtained from three independent experiments. *** = p < 0.0005 (ANOVA, Tukey's test). B, Schematic showing the PVN injection area. C, Immunohistochemistry of pSim1Pr-mCherry-injected hypothalamus from a 20-week-old mouse showing mCherry expression in the PVN. D–E, Cas9 (d) and Sim1 (e) mRNA expression in mice injected with pCMV-dCas9-VP64 (dCas9-VP64), pCMV-dCas9-VP64+pSim1Pr-mCherry (PrmCRISPRa, n = 3), and pCMV-dCas9-VP64+pSCE2En-mCherry (EnhCRISPRa, n = 4). Mean values ± SD were determined based on the fold increase in mRNA compared to Sim1+ / − mice, normalized to beta-actin using the ΔΔCT method. [Figure 5] Figure 5A-C: CRISPRa-AAV injection into the PVN reduces weight gain in Sim1+ / - mice. A, Timeline of weight measurements after CRISPRa-AAV injection into the PVN. B-C, Weight gain determined over 7 weeks in Sim1+ / - mice injected with pCMV-dCas9-VP64 (dCas9-VP64), pCMV-dCas9-VP64 + pSim1Pr-mCherry (Prm-CRIPSRa), or pCMV-dCas9-VP64 + pSCE2En-mCherry (Enh-CRISPRa) compared with uninjected wild-type littermates and Sim1+ / - mice. Mean values ± SD from three females (B) and three males (C) are shown. * = p < 0.001, *** = p < 0.0005, ns = not significant; (ANOVA, Tukey's test). [Figure 6] Schematic of CRISPRa haploinsufficiency rescue experiments. The obese phenotype in Sim1+ / - mice was rescued by CRISPRa by targeting either the Sim1 promoter or enhancer using both transgenic and postnatal AAV approaches. [Figure 7A] Figures 7A-7B: CRISPRa Sim1 overexpression in vitro. Figure 7A shows an exemplary S. aureus CRISPRa system targeting the Sim1 promoter (Pr) by transfection of various sgRNAs (SEQ ID NOs: 38-43) into Neuro-2A (N2A) cells. Results are expressed as fold mRNA increase normalized to Sa-dCas9-VP64. Mean values ± SD were obtained from three independent experiments. [Figure 7B] Figures 7A-7B: CRISPRa Sim1 overexpression in vitro. Figure 7B shows exemplary S. aureus CRISPRa targeting the Sim1 promoter (Pr) in N2A cells after infection of N2A cells with AAV containing selected sgRNAs (SEQ ID NOs: 38, 40, or 42). Results are expressed as fold mRNA increase normalized to VP64 alone. Mean values ± SD were obtained from three independent experiments. [Figure 8A] Figures 8A-8B: CRISPRa Sim1 overexpression in vitro. Figure 8A shows an exemplary S. aureus CRISPRa system targeting the Sim1 SCE2 enhancer (Enh) by transfection of various sgRNAs (SEQ ID NOs: 44-49) into N2A cells. Results are expressed as fold mRNA increase normalized to Sa-dCas9-VP64. Mean values ± SD were obtained from three independent experiments. [Figure 8B]Figures 8A-8B: CRISPRa Sim1 overexpression in vitro. Figure 8B shows an exemplary S. aureus CRISPRa system targeting the Sim1 SCE2 enhancer (Enh) after infection of N2A cells with AAV containing the selected sgRNA (SEQ ID NO: 45, 46, or 47). Results are expressed as fold mRNA increase normalized to VP64 alone. Mean values ± SD were obtained from three independent experiments. [Figure 9A] Figures 9A-9B: CRISPRa Mc4r overexpression in vitro. Figure 9A shows an exemplary S. aureus CRISPRa system targeting the Mc4r promoter (Pr) by transfection of various sgRNAs (SEQ ID NOs: 50-54) into N2A cells. Results are expressed as fold mRNA increase normalized to VP64. Mean values ± SD were obtained from three independent experiments. [Figure 9B] Figures 9A-9B: CRISPRa Mc4r overexpression in vitro. Figure 9B shows an exemplary S. aureus CRISPRa system targeting the Mc4r promoter (Pr) after infection of N2A cells with AAV containing the selected sgRNA (SEQ ID NO: 51, 52, or 54). Results are expressed as fold mRNA increase normalized to VP64. Mean values ± SD were obtained from three independent experiments. [Figure 10] CRISPRa PKD1 overexpression in vitro. An exemplary S. aureus CRISPRa system targeting the PKD1 promoter (Pr) was used by transfecting human HEK293T cells with human promoter-sgRNA (SEQ ID NOs: 55-64). Results are expressed as fold mRNA increase normalized to dCas9-VP64. Mean values ± SD were obtained from three independent experiments. [Figure 11A]Figures 11A-11B: CRISPRa SETD5 overexpression in vitro. Figure 11A depicts an exemplary S. aureus CRISPRa system targeting the SETD5 promoter (Pr) or THUMPD3 by transfection of human promoter-sgRNA (SEQ ID NOs: 65-74) into human HEK293T cells. HS MIX refers to the transfection of equimolar concentrations of each of HS01-HS10 into human HEK293T cells. Results are expressed as fold mRNA increase normalized to VP64 alone. Mean values ± SD were obtained from three independent experiments. [Figure 11B] Figures 11A-11B: CRISPRa SETD5 overexpression in vitro. Figure 11B depicts an exemplary S. aureus CRISPRa system targeting the SETD5 promoter (Pr) or ROSA26 by transfection of mouse promoter-sgRNA (SEQ ID NOs: 75-84) into mouse Neuro-2A cells. MS MIX refers to the transfection of equimolar concentrations of each of MS01-MS10 into mouse Neuro-2A cells. Results are expressed as fold mRNA increase normalized to VP64 alone. Mean values ± SD were obtained from three independent experiments. [Figure 12A] Figures 12A-12B: CRISPRa Scn2A overexpression in vitro. Figure 12A depicts an exemplary Streptococcus pyogenes (Sp) Cas9 CRISPRa system targeting the Scn2a promoter (Pr) by transfection of various sgRNAs (SEQ ID NOs: 85-91) into N2A cells. Results are expressed as fold mRNA increase normalized to VP64 alone. Mean values ± SD were obtained from three independent experiments. [Figure 12B]Figures 12A-12B: CRISPRa Scn2A overexpression in vitro. Figure 12B shows an exemplary S. aureus CRISPRa system targeting the Scn2a promoter (Pr) after infection of N2A cells with AAV containing selected sgRNAs (SEQ ID NOs: 92-94). Two different multiplicities of infection (MOIs) were used: 5,000 viral genomes and 1,250 viral genomes (vg / ml). Results are expressed as fold mRNA increase normalized to VP64 alone. Mean values ± SD were obtained from three independent experiments. [Figure 13] Figure 13: CRISPRa PAX6 overexpression in vitro. This shows an exemplary Streptococcus pyogenes (Sp) Cas9 CRISPRa system targeting the PAX6 promoter (Pr) via lentiviral delivery of human promoter-sgRNA (SEQ ID NO:95) into human H1-ESC cells differentiated into neurons. Results are expressed as relative expression to HPRT. Mean values ± sd were obtained from three independent experiments. Additional neuronal markers are shown to demonstrate that PAX6 CRISPRa leads to neural induction of H1-ESCs. DETAILED DESCRIPTION OF THE INVENTION

[0019] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0020] "Treating" refers to any indication of successful treatment or amelioration or prevention of a disease, condition, or disorder, including any objective or subjective parameter, such as relief; remission; reduction in symptoms or making the disease state more tolerable to the patient; slowing the rate of degeneration or decline; or reducing the debilitating end point of degeneration. Treating or ameliorating symptoms can be based on objective or subjective parameters, including the results of a physician's examination. Thus, the term "treatment" includes the administration of a compound or agent of the present invention to prevent, delay, alleviate, or arrest or inhibit the development of symptoms or conditions associated with the diseases, conditions, or disorders described herein. The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in a subject. "Treating" using the methods of the invention includes preventing the onset of a disease or disorder in a subject who may be at increased risk for, but has not yet experienced or exhibited, a disease, condition, or disorder associated with the disease, condition, or disorder described herein; suppressing (slowing or halting) the development of, the symptoms of, the disease or disorder; providing relief from, the symptoms or side effects of, the disease (including palliative treatment); and eliminating (causing regression of) the symptoms of the disease. Treatment can be prophylactic (preventing or delaying the onset of, or preventing the manifestation of, clinical or preclinical symptoms thereof) or the therapeutic arrest or alleviation of symptoms after the manifestation of, the disease or condition. As used herein, the term "treatment" encompasses preventative (e.g., prophylactic), therapeutic, or palliative treatment.

[0021] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof, in either single- or double-stranded form. Unless otherwise specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized similarly to natural nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly indicated, but also conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0022] The term "gene" refers to the segment of DNA involved in producing a polypeptide chain, which may include intervening sequences (introns) between coding segments (exons), as well as regions preceding and following the coding region (leader and trailer).

[0023] A "promoter" is defined as a set of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as a TATA element in the case of a polymerase II type promoter. A promoter optionally includes distal enhancer or repressor elements, which may be located as far as several thousand base pairs from the start site of transcription.

[0024] An "expression cassette" is a recombinantly or synthetically produced nucleic acid construct that contains a series of designated nucleic acid elements that enable transcription of a particular polynucleotide sequence in a host cell. An expression cassette can be part of a plasmid, a viral genome, or a nucleic acid fragment. Typically, an expression cassette contains a polynucleotide to be transcribed operably linked to a promoter.

[0025] "Reporter genes" encode proteins that can be easily detected due to their biochemical characteristics, such as enzymatic activity or chemiluminescent properties. One specific example of such a reporter is green fluorescent protein. The fluorescence produced by this protein can be detected with a variety of commercially available fluorescence detection systems. Other reporters can be detected by staining. Reporters can also be enzymes that generate a detectable signal when contacted with an appropriate substrate. Reporters can be enzymes that catalyze the formation of a detectable product. Suitable enzymes include, but are not limited to, proteases, nucleases, lipases, phosphatases, and hydrolases. Reporters can encode enzymes whose substrates are substantially impermeable to the eukaryotic cell plasma membrane, thereby allowing for precise control of signal generation. Specific examples of suitable reporter genes encoding enzymes include, but are not limited to, CAT (chloramphenicol acetyltransferase; Alton and Vapnek (1979) Nature 282:864-869), luciferase (lux), β-galactosidase, LacZ, β-glucuronidase, and alkaline phosphatase (Toh, et al. (1980) Eur. J. Biochem. 182:231-238 and Hall et al. (1983) J. Mol. Appl. Gen. 2:101), each of which is incorporated by reference in its entirety. Other suitable reporters encode specific epitopes that can be detected with labeled antibodies that specifically recognize the epitopes.

[0026] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. An "amino acid mimetic" refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0027] A variety of methods are known in the art that allow for the site-specific incorporation of unnatural amino acid derivatives or analogs into polypeptide chains, see, for example, WO 02 / 086075.

[0028] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to herein by their commonly accepted single-letter codes.

[0029] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. All three terms apply not only to natural and unnatural amino acid polymers, but also to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding natural amino acids. As used herein, these terms encompass any length of amino acid chain, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds.

[0030] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to nucleic acids that encode identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because the genetic code is degenerate, any given protein can be encoded by a large number of functionally identical nucleic acids. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, wherever alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide represents every possible silent variation of that nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only methionine codon, and TGG, which is usually the only tryptophan codon) can be altered to result in a functionally identical molecule. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0031] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions in nucleic acid, peptide, polypeptide, or protein sequences that alter, add, or delete a single amino acid or a small number of amino acids in the encoded sequence are "conservatively modified variants" if the change results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the present invention. In some cases, conservatively modified variants of CRISPR nucleases such as Cas9 or guide RNAs such as small guide RNAs (sgRNAs) may have increased stability, assembly, or activity, as described in WO 2016 / 011080. The contents of WO 2016 / 011080, including but not limited to, the sgRNAs, sgRNA scaffolds, sgRNA libraries, and sgRNA binding regions described therein, are incorporated herein by reference in their entirety for all purposes.

[0032] The following eight groups each contain amino acids that are conservative substitutions for one another: (1) alanine (A), glycine (G); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamine (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); (6) phenylalanine (F), tyrosine (Y), tryptophan (W); (7) serine (S), threonine (T); and (8) Cysteine (C), Methionine (M) (See, for example, Creighton, Protein, W.H. Freeman and Co., NY (1984)).

[0033] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to herein by their commonly accepted single-letter codes.

[0034] In this application, amino acid residues are numbered according to their relative position from the leftmost residue in the unmodified wild-type polypeptide sequence, which is numbered 1.

[0035] As used herein, in the context of two or more polynucleotide or amino acid sequences, the term "identical" or "percent identity" refers to two or more sequences or subsequences that are the same, or to two or more sequences or subsequences that have a specified percentage of the same amino acid residues or nucleotides. For example, a core small guide RNA (sgRNA) sequence responsible for the assembly and activity of an sgRNA:nuclease complex, when compared across a comparison window or across a specified region and aligned for maximum identity, has at least 80% identity, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a reference sequence, as measured using one of the sequence comparison algorithms described below, or by manual alignment and visual inspection.

[0036] In sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. For sequence comparison of nucleic acids and proteins, the BLAST and BLAST 2.0 algorithms and the default parameters described below are used.

[0037] As used herein, a "comparison window" includes reference to any segment of 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, contiguous positions within which a sequence may be compared to a reference sequence of the same number of contiguous positions after optimally aligning the two sequences. Methods for aligning sequences to be compared are well known in the art. Optimal alignment of the sequences to be compared can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), or by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), or by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), or by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin)), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

[0038] Examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analysis is publicly available at the National Center for Biotechnology Information website, ncbi.nlm.nih.gov. This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in database sequences, match or meet a threshold score T with a positive value. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction is halted when the cumulative alignment score falls by an amount X from its maximum achieved value, or when the cumulative score falls below zero due to one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0039] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0040] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by a first nucleic acid immunologically cross-reacts with an antibody raised against the polypeptide encoded by a second nucleic acid, as described below. Thus, for example, where the only difference between the two polypeptides is a conservative substitution, a polypeptide is typically substantially identical to a second polypeptide. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the sequences can be amplified using the same primers. Yet another indication that two polypeptides are substantially identical is that the two polypeptides retain the same or substantially similar activity.

[0041] "Translocation sequence" or "transduction sequence" refers to a peptide or protein (or active fragment or domain thereof) sequence that directs the movement of a protein from one intracellular compartment to another or from the extracellular space through the cell or plasma membrane into a cell. A translocation sequence that directs the movement of a protein from the extracellular space through the cell or plasma membrane into a cell is a "cell penetration peptide." A translocation sequence that localizes to the nucleus of a cell is referred to as a "nuclear localization" sequence, signal, domain, peptide, etc.

[0042] Examples of translocation sequences include, but are not limited to, the TAT transduction domain (see, e.g., S. Schwarze et al., Science 285 (Sep. 3, 1999)); penetratin or penetratin peptide (D. Derossi et al., Trends in Cell Biol. 8, 84-87); herpes simplex virus type 1 VP22 (A. Phelan et al., Nature Biotech. 16, 440-443 (1998)); and polycationic (e.g., poly-arginine) peptides (Cell Mol. Life Sci. 62 (2005) 1839-1849). Other translocation sequences are known in the art. Translocation peptides can be fused, conjugated, or coupled to the compounds of the present invention (e.g., at the amino or carboxyl terminus) to produce, among other purposes, conjugated compounds that can readily enter target cells or that can readily cross the blood-brain barrier into target cells.

[0043] As used herein, the term " CRISPR " refers to the natural clustered regularly interspaced short palindromic repeat (CRISPR) system or locus or any one of their derivatives.CRISPR locus can be found in many bacterial and archaeal genomes.There are four types of CRISPR system (for example, type I, type II, type III and type U).

[0044] CRISPR loci contain polynucleotide sequences encoding CRISPR-associated (Cas) genes. Cas genes may be involved in the biogenesis and / or interference stages of crRNA function. Cas genes may be named according to the organism from which they are derived. For example, Cas genes in Staphylococcus epidermidis can be referred to as Csm-type, Cas genes in Streptococcus thermophilus can be referred to as Csn-type, and Cas genes in Pyrococcus furiosus can be referred to as Cmr-type.

[0045] As used herein, the term CRISPR nuclease refers to the nuclease polypeptide encoded by any one of the four types of CRISPR loci: type I, type II, type III and type U, or the polypeptide derived from said nuclease, wherein the native sequence of this polypeptide exhibits RNA-guided nuclease activity.CRISPR nuclease can be catalytically inactive.CRISPR nuclease that is catalytically inactive does not exhibit nuclease activity or nickase activity when it forms a complex with RNA guide and binds to a nucleic acid target that contains a target domain and, in certain embodiments, a PAM sequence.The reason why catalytically inactive CRISPR nuclease is catalytically inactive can be due to one or more mutations in the CRISPR nuclease polypeptide sequence, or due to the complex formed with guide RNA that is sufficient to provide RNA-guided targeting but insufficient to support catalytic activity (i.e., nuclease activity or nicking activity). For example, CRISPR nuclease can be wild-type CRISPR nuclease (for example, Cas9 or Cpf1 nuclease) that forms a complex with dead guide sequence.For example, Cpf1 is a class II CRISPR-Cas system, and is described in Zetsche et al., Cell, 163:759-771 (2015).Dead guide sequences and their use are further described, for example, in WO 2016 / 094872, which is incorporated herein by reference for all purposes, including dead guide sequences, complexes between CRISPR nucleases and dead guide sequences, and methods and compositions for producing and using such dead guide sequences and complexes containing them.

[0046] In certain embodiments, the CRISPR nuclease meets one or both of the following criteria: at least 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology to a reference sequence, e.g., the amino acid sequence of a naturally occurring CRISPR nuclease. or differs from a reference sequence, e.g., the amino acid sequence of a naturally occurring CRISPR nuclease, by less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, or 400 amino acid residues. Additional CRISPR nucleases include, but are not limited to, one or more CRISPR nucleases described in WO 2016 / 154579.

[0047] In certain embodiments, the CRISPR nuclease contains (i.e., is covalently or non-covalently linked to) one or more additional polypeptides or nucleic acids. For example, the CRISPR nuclease can be fused at the amino or carboxy terminus to one or more transcription activation domain polypeptides, one or more DNA-binding polypeptides, one or more affinity tags (e.g., forming a complex with one or more affinity tag ligands, such as an affinity tag ligand-transcription activation domain fusion protein), a nuclear localization sequence, or a combination thereof.

[0048] Exemplary DNA-binding polypeptides include, but are not limited to, the programmable DNA-binding domains described in Bolukbasi et al., Nature Methods 12, 1150-1156 (2015), the contents of which are incorporated herein by reference in their entirety, including, for example, the programmable DNA-binding domains (pDBDs), Cas9 mutants, and Cas9-pDBD chimeras described therein. Exemplary transcription activation domain polypeptides include, but are not limited to, activation domains of one or more of the following polypeptides, or combinations of activation domains thereof: · Heat shock transcription factor 1 (HSF1), e.g. TIFF2025118716000026.tif26148·Viral protein 16 (VP16), e.g. TIFF2025118716000027.tif4128·VP16 tetramer (VP64), e.g. TIFF2025118716000028.tif11144·p65 NF-Kβ transactivation subunit (p65), e.g. TIFF2025118716000029.tif40148·MyoD1, e.g. TIFF2025118716000030.tif47148·RTA, e.g. TIFF2025118716000031.tif33149·SET7, for example TIFF2025118716000032.tif54149·VPR, e.g. TIFF2025118716000033.tif75148·Histone acetyltransferase p300, e.g. TIFF2025118716000034.tif54148·Hydroxylase catalytic domains of TET family proteins (e.g., TET1 hydroxylase catalytic domain), e.g. TIFF2025118716000035.tif68148TIFF2025118716000036.tif210148·LSD1, for example TIFF2025118716000037.tif82149·CIB1, for example TIFF2025118716000038.tif33149·AD2, for example TIFF2025118716000039.tif96148·CR3, for example TIFF2025118716000040.tif218149·GATA4, e.g. TIFF2025118716000041.tif69148·p53, e.g. TIFF2025118716000042.tif61148·SP1, for example TIFF2025118716000043.tif110148·MEF2C, e.g. TIFF2025118716000044.tif68148·TAX, for example TIFF2025118716000045.tif54148·PPARγ, e.g. TIFF2025118716000046.tif75148 or SET9, for example TIFF2025118716000047.tif54149 or one or more of the transcription activation domains described in Chavez et al., Nat Methods. 2015 Apr;12(4):326-328, which is incorporated by reference in its entirety for all purposes, including, but not limited to, activation domain polypeptides and polynucleotides encoding same, Cas9 (e.g., dCas9) polypeptides and polynucleotides encoding same, and fusion proteins and complexes thereof (e.g., complexes with sgRNA).

[0049] In some cases, the CRISPR nuclease is fused to one or more affinity tags. For example, the CRISPR nuclease can be a component of a SunTag. Exemplary SunTags or SunTag components include, but are not limited to, one or more of the affinity-tagged CRISPR nucleases or affinity tag ligands and fusion proteins thereof described in WO 2016 / 011070. In one embodiment, the CRISPR nuclease contains one or more affinity tags non-covalently bound to one or more ligand-transcriptional activation domain fusion proteins. In such embodiments, the transcriptional activation domain fused to the affinity tag ligand can be one or more of the transcriptional activation domains described herein, such as the transcriptional activation domains of SEQ ID NOs: 13-33, the transcriptional activation domains described in WO 2016 / 011070, or combinations or derivatives thereof.

[0050] As used herein, the terms "Cas9," "Cas9 molecule," and the like refer to a Cas9 polypeptide or a nucleic acid encoding a Cas9 polypeptide. A "Cas9 polypeptide" is a polypeptide capable of forming a complex with a guide RNA (gRNA) and binding to a nucleic acid target containing a targeting domain and, in certain embodiments, a PAM sequence. Cas9 molecules include those having a native Cas9 polypeptide sequence and engineered, altered, or modified Cas9 polypeptides that differ, e.g., by at least one amino acid residue, from a reference sequence, e.g., the most similar native Cas9 molecule. A Cas9 molecule can be a Cas9 polypeptide or a nucleic acid encoding a Cas9 polypeptide. A Cas9 molecule can be a nuclease (an enzyme that cleaves both strands of a double-stranded nucleic acid), a nickase (an enzyme that cleaves one strand of a double-stranded nucleic acid), or a catalytically inactive (or dead) Cas9 molecule. Cas9 molecules that possess nuclease or nickase activity are referred to as "catalytically active Cas9 molecules" ("caCas9" molecules). Cas9 molecules that lack the ability to cleave or nick target nucleic acids are referred to as "catalytically inactive Cas9 molecules" ("ciCas9" molecules) or "dead Cas9" ("dCas9").

[0051] In certain embodiments, the Cas9 molecule meets one or both of the following criteria: has at least 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology to a reference sequence, e.g., the amino acid sequence of a native Cas9 molecule. or differs from a reference sequence, e.g., the amino acid sequence of a naturally occurring Cas9 molecule, by less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, or 400 amino acid residues.

[0052] In some embodiments, the Cas9 molecule is Streptococcus pyogenes Cas9 (SpCas9) or a variant thereof. In some embodiments, the Cas9 molecule is Staphylococcus aureus Cas9 (SaCas9) or a variant thereof (see, e.g., Figures 7A-11B herein). In some embodiments, the Cas9 molecule is Campylobacter jejuni Cas9 (CjCas9) or a variant thereof (see, Kim et al., Nat. Comm., 8, 14500 (2017)). In some embodiments, the Cas9 molecule is Neisseria meningitides Cas9 (NmCas9) or a variant thereof (see, U.S. Patent No. 9,074,199). In some embodiments, the Cas9 molecule is Streptococcus thermophilus Cas9 (StCas9) or a variant thereof (see, e.g., Xu et al., Cell Mol Life Sci., 72:383-99 (2014)). In some embodiments, the Cas9 molecule is a dCas9 molecule.

[0053] In certain embodiments, the Cas9 molecule is a Streptococcus pyogenes Cas9 mutant. In certain embodiments, the Cas9 mutant is an EQR mutant. In certain embodiments, the Cas9 mutant is a VRER mutant. In certain embodiments, the dCas9 molecule is a Streptococcus pyogenes Cas9 mutant. In certain embodiments, the Cas9 mutant is an EQR mutant. In certain embodiments, the Cas9 mutant is a VRER mutant. In certain embodiments, the Cas9 system comprises a Cas9 molecule, such as a Cas9 molecule described herein, such as a Cas9 EQR mutant or a Cas9 VRER mutant.

[0054] In certain embodiments, the Cas9 molecule is a Staphylococcus aureus Cas9 mutant. In certain embodiments, the Cas9 mutant is a KKH (E782K / N968K / R1015H) mutant (see, e.g., Kleinstiver et al., Nature 523, 481-485 (23 July 2015) and Leenay et al. Molecular Cell, Vol. 62, Issue 1, 2016, p. 137), the contents of which are expressly incorporated herein by reference, particularly with respect to Cas (e.g., Cas9) mutants, such as those with altered PAM specificity. In certain embodiments, the Cas9 mutant is an E782K / K929R / R1015H mutant (see, e.g., Kleinstiver 2015). In certain embodiments, the Cas9 mutant is an E782K / K929R / N968K / R1015H mutant (see, e.g., Kleinstiver 2015). In certain embodiments, the Cas9 mutant comprises one or more mutations at one of the following residues: E782, K929, N968, R1015. In certain embodiments, the Cas9 mutant comprises one or more of the following mutations: E782K, K929R, N968K, R1015H, and R1015Q (see, e.g., Kleinstiver 2015). In certain embodiments, the Cas9 system comprises a Cas9 molecule, e.g., a Cas9 molecule described herein, e.g., a Cas9 KKH mutant.

[0055] As used herein, the terms "Cpf1," "Cpf1 molecule," and the like refer to a Cpf1 polypeptide or a nucleic acid encoding a Cpf1 polypeptide. A "Cpf1 polypeptide" is a polypeptide that can form a complex with a guide RNA (gRNA) and bind to a nucleic acid target containing a targeting domain and, in certain embodiments, a PAM sequence. Cpf1 molecules include those having a native Cpf1 polypeptide sequence, as well as engineered, altered, or modified Cpf1 polypeptides that differ, for example, by at least one amino acid residue, from a reference sequence, e.g., the most similar native Cpf1 molecule. A Cpf1 molecule can be a Cpf1 polypeptide or a nucleic acid encoding a Cpf1 polypeptide. Exemplary Cpf1 polypeptides include those isolated from Prevotella, Francisella novicida (FnCpf1), bacteria of the Lachnospiraceae family (LbCpf1), and Acidaminococcus sp. (AsCpf1) (see, e.g., Toth et al., Biology Direct, 11:46 (2016)).

[0056] In certain embodiments, the Cpf1 molecule meets one or both of the following criteria: has at least 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology to a reference sequence, e.g., the amino acid sequence of a native Cpf1 molecule. or differs from a reference sequence, e.g., the amino acid sequence of a naturally occurring Cpfl molecule, by less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, or 400 amino acid residues.

[0057] As used herein, the term " gRNA molecule " or " gRNA " refers to the guide RNA that has the ability to target CRISPR nuclease to target nucleic acid.In one embodiment, the term " gRNA molecule " refers to guide ribonucleic acid.In another embodiment, the term " gRNA molecule " refers to the nucleic acid that encodes gRNA.In one embodiment, the gRNA molecule is non-natural.In one embodiment, the gRNA molecule is a synthetic gRNA molecule.

[0058] Guide RNA can be scaffold RNA that binds to one or more protein or nucleic acid ligands (scaffold RNA ligands).Ligands can be fused to transcription activation domains or otherwise linked covalently or non-covalently.In an alternative embodiment, scaffold RNA is not guide RNA, in that it does not specifically associate with CRISPR nuclease. Exemplary scaffold RNAs and CRISPR nuclease / scaffold RNA complexes and methods for making and using them are described, for example, in WO 2016 / 054106 (which describes CRISPR-associating scaffold RNAs and CRISPR-independent scaffold RNAs) and Zhang et al., Scientific Reports 5, Article No. 16277 (2015); Konermann et al., 2015, Nature 517:583-8 (which describes CRISPR / gRNA-directed synergistic activation mediators (SAMs)).

[0059] As used herein, "subject" refers to either a human or a non-human animal. This term includes, but is not limited to, mammals (e.g., humans, other primates, pigs, rodents (e.g., mice and rats or hamsters), rabbits, guinea pigs, cows, horses, cats, dogs, sheep, and goats). In one embodiment, the subject is a human. In another embodiment, the subject is a poultry. In another embodiment, the subject is a fish. In certain embodiments, the subject is a human, and in some of these embodiments, the human is an infant, a toddler, a young adult, or an adult.

[0060] As used herein, the term "target nucleic acid" or "target gene" refers to a nucleic acid targeted for binding by, for example, a guide RNA, a CRISPR nuclease complexed with a guide RNA, or a scaffold RNA. In certain embodiments, the target nucleic acid comprises a gene or a promoter or enhancer operably linked to a gene. In certain embodiments, the target nucleic acid may comprise one or more genes, for example, two, three, four, or five genes, or promoters or enhancers operably linked to one or more genes. In one embodiment, the target nucleic acid may comprise a promoter region or a regulatory region of a gene. In one embodiment, the target nucleic acid may comprise an intron of a gene. In another embodiment, the target nucleic acid may comprise an exon of a gene. In one embodiment, the target nucleic acid may comprise a coding region of a gene. In one embodiment, the target nucleic acid may comprise a non-coding region of a gene. In some embodiments, the target nucleic acid is a regulatory region, promoter, enhancer, intron, exon, transcription start site, coding region, or non-coding region of a gene listed in Table 1 herein.

[0061] In some embodiments, the target nucleic acid is a regulatory region, promoter, enhancer, intron, exon, transcription start site, coding region, or non-coding region of a gene in the same pathway as a gene listed in Table 1 herein. The target nucleic acid can be, for example, a regulatory region, promoter, enhancer, intron, exon, transcription start site, coding region, or non-coding region of a gene upstream in the same pathway as a gene listed in Table 1 herein. Additionally or alternatively, when more than one gene or location is targeted, the target nucleic acid can be, for example, a regulatory region, promoter, enhancer, intron, exon, transcription start site, coding region, or non-coding region of a gene downstream in the same pathway as a gene listed in Table 1 herein. Additionally or alternatively, when more than one gene or location is targeted, the target nucleic acid can be, for example, a regulatory region, promoter, enhancer, intron, exon, transcription start site, coding region, or non-coding region of a gene in a parallel pathway to a gene listed in Table 1 herein. Exemplary genes in the same pathway or in parallel pathways as one or more of the genes listed in Table 1 are described, for example, in the KEGG pathway database (available at www.genome.jp / kegg / pathway.html).

[0062] As used herein, " target position " refers to the site on the target nucleic acid that is hybridized with guide RNA (for example, that forms a complex with CRISPR nuclease) or scaffold RNA.Optimized target position includes, but is not limited to, one or more target positions that are optimized for transcription activation as described in WO 2016 / 011080.

[0063] "Episomal vector" or "episomally propagating vector" refers to a plasmid or viral vector that persists or propagates as an episomal element in mammalian cells. The episomal vectors described herein can encode one or more components (e.g., a CRISPR nuclease, a guide RNA, a zinc finger nuclease, a TALEN, a TAL effector, a scaffold RNA, a transcriptional activator, an affinity element, or a combination thereof) for treating a disease or condition (e.g., a disease or condition in Table 1) through transcriptional activation. Episomal vectors include, but are not limited to, adeno-associated virus (AAV) vectors and Epstein-Barr virus (EBV) vectors. Suitable AAV vectors and methods for making and using such AAV vectors, for example, methods for delivering vectors into target cells, are described in Samulski Ret al. (1987), J. Virol. 61:3096-3101, Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993), Fisher KJ et al. (1996), J. Virol, 70:520-532, Samulski Ret al. (1989), J. Virol. 63:3822-3826, U.S. Patent No. 5,252,479, U.S. Patent No. 5,139,941, U.S. Patent No. 5,436,146, International Patent Application WO 94 / 13788, and International Patent Application WO 93 / 24641, the disclosures of which are all incorporated herein by reference.

[0064] As used herein, the term "zinc finger nuclease" refers to a zinc finger DNA-binding protein (or a zinc finger DNA-binding domain within a larger protein) that binds to DNA in a sequence-specific manner via one or more zinc fingers, which are regions of amino acid sequence within the zinc finger binding domain whose structure is stabilized by the coordination of zinc ions. The term zinc finger DNA-binding protein is often abbreviated as zinc finger nuclease or ZFN.

[0065] As used herein, the term " transcription activator-like effector nuclease " refers to the protein that comprises the transcription activator-like effector DNA binding domain fused with DNA cleavage domain, and binds to DNA sequence-specifically.The term transcription activator-like effector nuclease is often abbreviated as TALEN.

[0066] Detailed Description of the Invention Introduction Described herein are methods and compositions for treating a disease in a mammalian subject that is associated with, exacerbated by, or caused by reduced transcription of a gene, reduced amount of a gene product, or reduced activity of a gene product by increasing transcription of a target gene. Such methods and compositions can be useful, for example, for treating haploinsufficiency diseases in a subject. Haploinsufficiency diseases that can be treated by the methods and compositions described herein include, but are not limited to, one or more of the diseases listed in Table 1. Table 1 lists the Entrez gene ID (column 2) from the National Center for Bioinformatics (NCBI) and the corresponding gene symbol (column 1) provided by the Human Genome Nomenclature Committee (HGNC), the pubmed ID (PMID) reference number of the reference (column 4), and a brief description of the associated disorder (column 5). This table is adapted from Supplementary Table 1 of Dang et al., European Journal of Human Genetics (2008) 16, 1350-57 and from the ClinVar (https: / / www.ncbi.nlm.nih.gov / clinvar) and ClinGen (https: / / www.clinicalgenome.org) databases.

[0067] nuclease In some embodiments of the methods described herein, the host cell is contacted with one or more nucleases. In some embodiments, the nuclease is an endonuclease, a site-specific recombinase, a transposase, a topoisomerase, a zinc finger nuclease, a TALEN, including modified derivatives and mutants thereof.

[0068] In some embodiments, the nuclease has the ability to target a specified nucleotide or region within the target site. In some embodiments, the nuclease has the ability to target a region located between the 5' and 3' regions of the target site. In another embodiment, the nuclease has the ability to target a region located upstream or downstream of the 5' and 3' regions of the target site (e.g., upstream or downstream of the transcription start site (TSS)). The recognition sequence is a polynucleotide sequence that is specifically recognized and / or bound by the nuclease. The length of the recognition site sequence can vary, including, for example, a nucleotide sequence that is at least 10, 12, 14, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more nucleotides in length. In some embodiments, the recognition sequence is a batch structure. That is, a sequence on one DNA strand reads the same as in the opposite direction on the complementary DNA strand. In some embodiments, the target site for the nuclease is within the recognition sequence.

[0069] Zinc finger nuclease In some embodiments, the nuclease is a zinc finger nuclease (ZFN). ZFNs typically comprise a zinc finger DNA binding domain and a nuclease domain. Generally, ZFNs comprise two zinc finger arrays (ZFAs), each fused to a single subunit of a non-specific endonuclease that becomes active upon dimerization, such as the nuclease domain of the FokI enzyme. Typically, a single ZFA consists of three or four zinc finger domains, each designed to recognize a specific nucleotide triplet (such as GGC, GAT, etc.). Thus, a ZFN composed of two "three-finger" ZFAs has the ability to recognize an 18-base pair target site (i.e., recognition sequence). 18-base pair recognition sequences are generally unique, even within large genomes, such as human and plant genomes. By directing the colocalization and dimerization of two FokI nuclease monomers, ZFNs generate functional site-specific endonucleases that can target specific loci (e.g., genes, promoters, or enhancers).

[0070] Zinc finger nucleases useful in the methods disclosed herein include known and engineered ZFNs with specificity for one or more target sites (e.g., promoter or enhancer nucleotide sequences) described herein. The zinc finger domain can correspond to the design of a polypeptide that specifically binds to a selected polynucleotide recognition sequence within a target site in a host cell genome. ZFNs can comprise an engineered DNA-binding zinc finger domain linked to a non-specific endonuclease domain, e.g., the nuclease domain of a type IIs endonuclease such as HO or FokI. In some examples, the zinc finger DNA-binding domain can be fused to a site-specific recombinase, transposase, or derivatives thereof that retain DNA nicking and / or DNA cleavage activity.

[0071] In a preferred embodiment, additional functionality, such as, but not limited to, a transcriptional activator domain (e.g., VP16, VP48, VP64, VP160, etc.) or a transcriptional repressor domain (e.g., KRAB, etc.), can be fused to the zinc finger binding domain. In one embodiment, the zinc finger nuclease is engineered to contain a transcriptional activator domain selected from VP16, VP48, VP64, or VP160. In one embodiment, the zinc finger nuclease is engineered to contain a transcriptional activator domain selected from HSF1, VP16, VP64, p65, RTA, MyoD1, SET7, VPR, histone acetyltransferase p300, TET1 hydroxylase catalytic domain, LSD1, CIB1, AD2, CR3, GATA4, p53, SP1, MEF2C, TAX, PPAR-gamma, and SET9. For example, an engineered zinc finger transcription activator that interacts with the promoter region of the gamma-globulin gene has been shown to enhance fetal hemoglobin production in primer adult erythroblasts (Wilber et al., Blood, 115(15):3033-3041). Other polydactyl zinc finger transcription factors are known in the art, including those disclosed by Beerli and Barbas (Nature Technology, (2002) 20:135-141).

[0072] Each zinc finger domain recognizes three consecutive base pairs in the target DNA, for example, a three-finger domain recognizes a sequence of nine consecutive nucleotides, and because the nuclease requires dimerization, it uses two sets of zinc finger triplets to bind to an 18-nucleotide recognition sequence. Useful zinc finger modules include those that recognize various GNN and ANN triplets (Dreier et al., (2001) J Biol Chem 276:29466-78; Dreier et al., (2000) J Mol Biol 303:489-502; Liu et al., (2002) J Biol Chem 277:3850-6) as well as those that recognize various CNN or TNN triplets (Dreier et al., (2005) J Biol Chem 280:35588-97; Jamieson et al., (2003) Nature Rev Drug Discovery 2:361-8).Durai et al., (2005) Nucleic Acids Res 33:5978-90, Segal, (2002) Methods 26:76-83, Porteus and Carroll, (2005) Nat Biotechnology 23:967-73, Pabo et al., (2001) Ann Rev Biochem 70:313-40, Wolfe et al. al., (2000) Ann Rev Biophys Biomol Struct 29:183-212, Segal and Barbas (2001) Curr Opin Biotechnol 12:632-7, Segal et al., (2003) Biochemistry 42:2137-48, Beerli and Barbas, (2002) Nat Biotechnol 20:135-41, Carroll et al., (2006) Nature See also Protocols 1:1329, Ordiz et al., (2002) Proc Natl Acad Sci USA 99:13290-5, Guan et al., (2002) Proc Natl Acad Sci USA 99:13296-301, WO2002099084, WO00 / 42219, WO02 / 42459, WO2003062455, US20030059767, U.S. Patent Application Publication No. 2003 / 0108880, U.S. Patent Nos. 6,140,466, 6,511,808, and 6,453,242. Useful zinc finger nucleases also include those described in WO03 / 080809, WO05 / 014791, WO05 / 084190, WO08 / 021207, WO09 / 042186, WO09 / 054985 and WO10 / 065123.

[0073] In some embodiments, the ZFN comprises a fusion protein having a cleavage domain of a type IIS restriction endonuclease fused to an engineered zinc finger binding domain, wherein the binding domain further comprises one or more transcriptional activators. In some embodiments, the type IIS restriction endonuclease is selected from HO endonuclease or FokI endonuclease. In some embodiments, the zinc finger binding domain comprises three, four, five, or six zinc fingers. In another embodiment, the zinc finger binding domain specifically binds to a recognition sequence corresponding to a promoter or enhancer disclosed herein (e.g., SIM1, MC4R, PKD1, SETD5, THUMPD3, SCN2A, and PAX6 promoters or enhancers). In one embodiment, the one or more transcriptional activators are selected from VP16, VP48, VP64, or VP160. Generally, the DNA binding domain of a ZFN contains 3-6 distinct zinc finger repeats and can recognize 9-18 consecutive nucleotides. Each ZFN can be designed to target a specific target site in the host cell genome, such as a promoter sequence, an enhancer sequence, or an exon / intron within a gene.

[0074] TALEN In some embodiments of the method, the nuclease is a TALEN. TAL effectors (TALEs) are proteins secreted by Xanthomonas bacteria that play an important role in disease or in triggering defense mechanisms by binding to host DNA and activating effector-specific host genes. See, for example, Gu et al. (2005) Nature 435:1122-5, Yang et al., (2006) Proc. Natl. Acad. Sci. USA 103:10503-8, Kay et al., (2007) Science 318:648-51, Sugio et al., (2007) Proc. Natl. Acad. Sci. USA 104:10720-5, Romer et al., (2007) Science 318:645-8, Boch et al., (2009) Science 326(5959):1509-12 and Moscou and Bogdanove, (2009) 326(5959):1501. TALENs comprise a TAL effector DNA binding domain fused to a DNA cleavage domain. DNA-binding domains interact with DNA in a sequence-specific manner through one or more tandem repeat domains. The repeats typically contain 33–34 highly conserved amino acids, differing at the 12th and 13th amino acids. These two positions, called repeat variable diresidues (RVDs), are highly variable and show a strong correlation with specific nucleotide recognition (Boch et al., (2009) Science 326(5959):1509-12 and Moscou and Bogdanove, (2009) 326(5959):1501). This relationship between amino acid sequence and DNA recognition sequence allows the engineering of specific DNA-binding domains by selecting combinations of repeat segments containing appropriate RVDs.

[0075] The TAL-effector DNA binding domain can be engineered to bind to a target DNA sequence and fused to a nuclease domain, for example, a type IIS restriction endonuclease such as FokI (see, e.g., Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93:1156-1160). In some embodiments, the nuclease domain can contain one or more mutations (e.g., FokI mutants) that improve cleavage specificity (Doyon et al., (2011) Nature Methods, 8(1):74-9) and cleavage activity (Guo et al., (2010) Journal of Molecular Biology, 400(1):96-107). Other useful endonucleases that can be used as the nuclease domain include, but are not limited to, HhaI, HindIII, Nod, BbvCI, EcoRI, BglI, and AlwI. In some embodiments, TALENs can comprise a TAL effector DNA binding domain, which comprises multiple TAL effector repeat sequences that bind to specific nucleotide sequences (i.e., recognition sequences) in target DNA. Although not intended to be limiting, TALENs useful for the methods provided herein include those described in WO10 / 079430 and US Patent Application Publication No. 2011 / 0145940.

[0076] In some embodiments, the TAL effector DNA-binding domain comprises 10 or more DNA-binding repeat sequences, preferably 15 or more DNA-binding repeat sequences. In some embodiments, each DNA-binding repeat sequence comprises an RVD that determines the recognition of base pairs in target DNA, and each DNA-binding repeat sequence is responsible for recognizing one base pair in target DNA. In some embodiments, the RVD comprises one or more of: HD for recognizing C; NG for recognizing T; NI for recognizing A; NN for recognizing G or A; NS for recognizing A or C or G or T; N* for recognizing C or T, where * represents a gap in the second position of the RVD; HG for recognizing T; H* for recognizing T, where * represents a gap in the second position of the RVD; IG for recognizing T; NK for recognizing G; HA for recognizing C; ND for recognizing C; HI for recognizing C; HN for recognizing G; NA for recognizing G; SN for recognizing G or A; and YG for recognizing T.

[0077] In a preferred embodiment, TALENs are engineered so that the TAL effector contains one or more transcription activator domains (e.g., VP16, VP48, VP64, or VP160). For example, engineered TAL effectors with transcription activator domains at the C-terminus of the TAL effector have been shown to regulate the transcription of Sox2 and Klf4 genes in human 293FT cells (Zhang et al., Nature Biotechnology, 29(2):149-153(2011)). Other TALE-TFs are also known in the art, such as those disclosed by Perez-Pinera et al. (Nature Methods, (2013)10(3):239-242), who demonstrated the regulation of IL1RN, KLK3, CEACAM5, and ERBB2 genes in human 293T cells using TAL effector transcription factors (TALE-TFs). In some embodiments, one or more transcription activator domains are located adjacent to a nuclear localization signal (NLS) present at the C-terminus of the TAL effector. In another embodiment, the TALE-TF can bind to a site upstream or downstream of the transcription start site (TSS) of the target gene. In one embodiment, the TAL effector comprises a transcription activator domain selected from VP16, VP48, VP64, or VP160. In another embodiment, the TAL effector comprises a transcription activator domain selected from HSF1, VP16, VP64, p65, RTA, MyoD1, SET7, VPR, histone acetyltransferase p300, TET1 hydroxylase catalytic domain, LSD1, CIB1, AD2, CR3, GATA4, p53, SP1, MEF2C, TAX, PPAR-gamma, and SET9.

[0078] In some embodiments, TALEN comprises a TAL effector DNA binding domain fused with a DNA cleavage domain, and the TAL effector comprises a transcription activator.In some embodiments, the DNA cleavage domain is a type IIS restriction endonuclease selected from HO endonuclease or FokI endonuclease.In some embodiments, the TAL effector DNA binding domain specifically binds to the recognition sequence corresponding to the promoter region or enhancer region disclosed herein (for example, SIM1, MC4R, PKD1, SETD5, THUMPD3, SCN2A and PAX6 promoter or enhancer).Generally, the DNA binding domain of TALEN is designed to target a specific target site in host cells, for example, a promoter sequence or an enhancer sequence.

[0079] In some embodiments, the target site of zinc finger nuclease or TALEN is endogenous to host cell, such as the native locus in the genome of host cell.In some embodiments, target site is selected according to the type of nuclease used in this method.When the nuclease used is zinc finger nuclease, the optimal target site can be selected using several publicly available online information sources.For example, see Reyon et al., BMC Genomics 12:83 (2011), which is incorporated herein by reference in its entirety. Publicly available methods for engineering zinc finger nucleases include (1) Context-dependent Assembly (CoDA), (2) Oligomerized Pool Engineering (OPEN), (3) Modular Assembly, (4) ZiFiT (internet-accessible software for designing engineered zinc finger arrays), (5) ZiFDB (an internet-accessible database of zinc fingers and engineered zinc finger arrays), and (6) ZFNGenome. OPEN, for example, is a publicly available protocol for engineering zinc finger arrays with high specificity and in vivo functionality and has been successfully used to generate ZFNs that function efficiently in plants, zebrafish, and human somatic and pluripotent stem cells. OPEN is a selection-based method that screens a pre-constructed randomized pool of candidate ZFNs to identify those with high affinity and specificity for a desired target sequence. Furthermore, ZFNGenome is a GBrowse-based tool for identifying and visualizing potential target sites for OPEN-generated ZFNs. ZFNGenome provides a list of potential ZFN target sites in the sequenced and annotated genomes of model organisms.The ZFNGenome contains over 11 million potential ZFN target sites mapped within the entire sequenced genomes of seven model organisms: Saccharomyces cerevisiae (S. cerevisiae), Chlamydomonas reinhardtii (C. reinhardtii), Arabidopsis thaliana (A. thaliana), Drosophila melanogaster (D. melanogaster), zebrafish (D. rerio), Caenorhabditis elegans (C. elegans), and human (H. sapiens). The ZFNGenome provides information about each potential ZFN target site, including its chromosomal location and relative position to the transcription start site. Users can query the ZFNGenome using several different criteria (e.g., gene ID, transcript ID, target site sequence).

[0080] In some embodiments, when the nuclease is a TALEN, optimal target sites can be selected according to the method described in Sanjana et al., Nature Protocol, 7:171-192 (2012), which is incorporated herein by reference in its entirety. TALENs function as dimers, with a pair of TALENs, referred to as left and right TALENs, targeting sequences on opposite strands of DNA. TALENs are engineered as fusions of a TALE DNA binding domain and a FokI catalytic domain monomer. To facilitate FokI dimerization, the left and right TALEN target sites are typically selected to be approximately 14-20 bases apart.

[0081] In some embodiments, one or more nucleases useful in the methods described herein are provided as purified proteins, for example, delivered into host cells. In some embodiments, one or more nucleases are provided by a polynucleotide comprising a nucleic acid encoding the nuclease. In another embodiment, one or more nucleases can be introduced into host cells as purified RNA that can be directly translated in the host cell nucleus. In a preferred embodiment, the polynucleotide comprising a nucleic acid encoding the nuclease comprises an expression vector that allows the nuclease to be expressed in the host cell. Suitable expression vectors include episomal vectors.

[0082] In some embodiments, such as zinc finger nucleases and TALENs, where nucleases function as dimers and require separate expression of each monomer, each monomer of the dimer can be expressed from the same episomal vector or from a different episomal vector. In another embodiment, where multiple nucleases are introduced into a cell to introduce double-strand breaks at different target sites, the nucleases can be encoded on a single episomal vector or on separate episomal vectors.

[0083] In one aspect, the present invention provides a method for treating haploinsufficiency disease in a mammalian subject, the method comprises contacting the cell of the subject with a composition comprising zinc finger nuclease or TALEN that specifically hybridizes to promoter or enhancer regions under conditions present in the nucleus of the cell, wherein the contacting step forms a complex comprising the DNA binding domain of zinc finger nuclease or TALEN and promoter or enhancer regions, and the complex activates the transcription of the wild-type copy of the haploinsufficient gene in an amount and for a duration sufficient to treat the haploinsufficiency disease in the subject.In some embodiments, the promoter or enhancer region corresponds to the promoter or enhancer region (i.e., the regulatory region) of any of the genes listed in Table 1.

[0084] In some embodiments, the contacting step comprises contacting the cell with an episomal vector encoding a zinc finger nuclease or TALEN. In some embodiments, the episomal vector is non-integrating. In some embodiments, the zinc finger nuclease or TALEN is modified to include one or more transcription activation domains. In one embodiment, the one or more transcription activation domains are selected from the group consisting of HSF1, VP16, VP64, p65, MyoD1, RTA, SET7 / 9, VPR, histone acetyltransferase p300, the hydroxylase catalytic domain of a TET family protein (e.g., the TET1 hydroxylase catalytic domain), LSD1, CIB1, AD2, CR3, EKLF1, GATA4, PRVIE, p53, SP1, MEF2C, TAX, and PPARγ. In some embodiments, the transcription activation domain is VP64. In some embodiments, the haploinsufficient gene is SIM1, leptin, leptin receptor, MC4R, SCN2A, SETD5, PAX6, PKD1, MC3R, POMC, STAT3, STAT5, SOCS3, GHR, NPY, NPY1R, NPY2R, NPY5R, PYY, AMPK (PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3), OXT, JAK2, SHP2, NOS3, NROB2, BRS3, CARTPT, FABP4 , HTR2C, IL6, NHLH2, NMU, NPB, NPBWRI, PNPLA2, UCP3, ADIPOQ, APOA5, ARNT2, ASIP, C1QTNF2, C3AR1, CCK, CPT1B, CSF2, DGAT1, DGAT2, GH RL, GHSR, HSD11B1, HTR7, INSIG1, INSIG2, LIPC, NMURI, NMUR2, NPBWR2, NTS, PPARGC1A, PPY, RETN, SIRT1, TGFBR2, WDTC1 or FOXO1.

[0085] Table 1. Genes associated with haploinsufficiency disorders TIFF2025118716000048.tif63160TIFF2025118716000049.tif203160TIFF2025118716000050.tif219160TIFF2025118716000051.tif214160TIFF2025118716000052.tif217160TIFF2025118716000053.tif221160TIFF2025118716000054.tif220160TIFF2025118716000055.tif206160TIFF2025118716000056.tif211160TIFF2025118716000057.tif220160TIFF2025118716000058.tif203160TIFF2025118716000059.tif221160TIFF2025118716000060.tif213160TIFF2025118716000061.tif221160TIFF2025118716000062.tif207160TIFF2025118716000063.tif222160TIFF2025118716000064.tif223160TIFF2025118716000065.tif215160TIFF2025118716000066.tif215160TIFF2025118716000067.tif222160TIFF2025118716000068.tif215160TIFF2025118716000069.tif212160TIFF2025118716000070.tif227160TIFF2025118716000071.tif217160TIFF2025118716000072.tif220160TIFF2025118716000073.tif214160TIFF2025118716000074.tif215160TIFF2025118716000075.tif180160

[0086] Composition Episomal vector The present specification describes compositions useful as components for targeting transcription activation domains to gene regulatory elements to increase the transcription of endogenous genes, thereby treating diseases or conditions associated with, exacerbated by, or caused by reduced gene transcription, reduced gene product amount, or reduced gene product activity.The components include guide RNAs, scaffold RNAs, scaffold RNA ligands, CRISPR nucleases, transcription activation domains, affinity tags, affinity tag ligands, fusion proteins of one or more of these, and combinations thereof.The components also include episomal vectors encoding one or more guide RNAs, scaffold RNAs, scaffold RNA ligands, CRISPR nucleases, transcription activation domains, affinity tags, affinity tag ligands, fusion proteins of one or more of these, and combinations thereof.The episomal vectors can be single-stranded or double-stranded DNA, single-stranded RNA, or double-stranded RNA.

[0087] In one aspect, an episomal vector is provided that encodes a CRISPR nuclease, such as a catalytically inactive CRISPR nuclease.Optionally, the episomal vector encodes the CRISPR nuclease fused to one or more transcription activation domains.Optionally, the episomal vector encodes the CRISPR nuclease fused to one or more affinity tags.Optionally, the episomal vector encodes the CRISPR nuclease fused to one or more affinity tags and one or more transcription activation domains.CRISPR nuclease fusion protein can contain a transcription activator domain and / or affinity tag fused to the amino terminus, carboxy terminus, or a combination thereof of the CRISPR nuclease.In addition to or alternatively, the CRISPR nuclease can also be modified by inserting a transcription activator domain and / or affinity tag into the surface loop. An episomal vector (e.g., an AAV vector) can contain a promoter operably linked to a CRISPR nuclease or a CRISPR nuclease fusion protein. The promoter can be a promoter endogenous to the virus from which the episomal vector is derived. For example, if the episomal vector is an AAV vector, the promoter can be an endogenous AAV promoter. Alternatively, the promoter can be a promoter heterologous to the virus from which the episomal vector is derived. For example, if the episomal vector is an AAV vector, the promoter can be a non-AAV promoter. The promoter can be the promoter of a gene (e.g., a gene selected from Table 1) targeted for transcriptional activation, or a promoter heterologous to the targeted gene.The promoter can be a constitutive promoter (e.g., a CMV promoter, a CAG promoter, a CBA promoter, an EF1a promoter, a PGK promoter, etc.), a tissue-specific promoter (e.g., a synapsin, camKIIa, GFAP, RPE, ALB, TBG, MBP, MCK, TNT or aMHC promoter, etc.), or an inducible promoter (e.g., tetracycline-inducible).

[0088] In one embodiment, an episomal vector encoding a zinc finger nuclease is provided. Optionally, the episomal vector encodes the zinc finger nuclease fused to one or more transcription activation domains. Optionally, the episomal vector encodes the zinc finger nuclease fused to one or more affinity tags. Optionally, the episomal vector encodes the zinc finger nuclease fused to one or more affinity tags and one or more transcription activation domains. The zinc finger nuclease fusion protein can contain a transcription activator domain and / or affinity tag fused to the amino terminus, carboxy terminus, or a combination thereof of the zinc finger nuclease. The episomal vector (e.g., AAV vector) can contain a promoter operably linked to the zinc finger nuclease or zinc finger nuclease fusion protein. The promoter can be an endogenous promoter for the virus from which the episomal vector is derived. For example, if the episomal vector is an AAV vector, the promoter can be an endogenous AAV promoter. Alternatively, the promoter can be a promoter heterologous to the virus from which the episomal vector is derived. For example, if the episomal vector is an AAV vector, the promoter can be a non-AAV promoter. The promoter can be the promoter of a gene targeted for transcriptional activation (e.g., a gene selected from Table 1), or a promoter heterologous to the targeted gene. The promoter can be a constitutive promoter (e.g., a CMV promoter, a CAG promoter, a CBA promoter, an EF1a promoter, a PGK promoter, etc.), a tissue-specific promoter (e.g., a synapsin, camKIIa, GFAP, RPE, ALB, TBG, MBP, MCK, TNT, or aMHC promoter, etc.), or an inducible promoter (e.g., a tetracycline-inducible promoter).

[0089] In one embodiment, an episomal vector encoding a TALEN is provided. Optionally, the episomal vector encodes a TALEN fused to one or more transcription activation domains. Optionally, the episomal vector encodes a TALEN fused to one or more affinity tags. Optionally, the episomal vector encodes a TALEN fused to one or more affinity tags and one or more transcription activation domains. The TALEN can contain a transcription activator domain and / or affinity tag fused to the amino terminus, carboxy terminus, or a combination thereof. The episomal vector (e.g., AAV vector) can contain a promoter operably linked to the TALEN. The promoter can be a promoter endogenous to the virus from which the episomal vector is derived. For example, if the episomal vector is an AAV vector, the promoter can be an endogenous AAV promoter. Alternatively, the promoter can be a promoter heterologous to the virus from which the episomal vector is derived. For example, if the episomal vector is an AAV vector, the promoter can be a non-AAV promoter. The promoter can be the promoter of a gene targeted for transcriptional activation (e.g., a gene selected from Table 1) or a promoter heterologous to the targeted gene. The promoter can be a constitutive promoter (e.g., a CMV promoter, a CAG promoter, a CBA promoter, an EF1a promoter, a PGK promoter, etc.), a tissue-specific promoter (e.g., a synapsin, camKIIa, GFAP, RPE, ALB, TBG, MBP, MCK, TNT, or aMHC promoter, etc.), or an inducible promoter (e.g., a tetracycline-inducible promoter).

[0090] In one embodiment, an episomal vector encoding a guide RNA is provided. The guide RNA can be a small guide RNA. The guide RNA can be a component of a synergistic activation mediator (e.g., those described in Zhang et al., Scientific Reports 5, Article No. 16277 (2015) and Konermann et al., 2015, Nature 517:583-8). The episomal vector (e.g., an AAV vector) can contain a promoter operably linked to the guide RNA. The promoter can be a promoter endogenous to the virus from which the episomal vector is derived. For example, if the episomal vector is an AAV vector, the promoter can be an endogenous AAV promoter. Alternatively, the promoter can be a promoter heterologous to the virus from which the episomal vector is derived. For example, if the episomal vector is an AAV vector, the promoter can be a non-AAV promoter. The promoter can be the promoter of a gene targeted for transcriptional activation (e.g., a gene selected from Table 1) or a promoter heterologous to the targeted gene. The promoter can be a constitutive promoter (e.g., a CMV promoter, a CAG promoter, a CBA promoter, an EF1a promoter, a PGK promoter, etc.), a tissue-specific promoter (e.g., a synapsin, camKIIa, GFAP, RPE, ALB, TBG, MBP, MCK, TNT, or aMHC promoter, etc.), or an inducible promoter (e.g., a tetracycline-inducible promoter).

[0091] In some embodiments, the episomal vector encodes both a CRISPR nuclease and a guide RNA. Optionally, the CRISPR nuclease is operably linked to a promoter, and the guide RNA is operably linked to a different promoter. Optionally, the two promoters are the same. Optionally, the two promoters are different. Optionally, both promoters are inducible. Optionally, both promoters are tissue-specific. Optionally, both promoters are constitutive. Optionally, one promoter is constitutive and the other promoter is tissue-specific. Optionally, one promoter is constitutive and the other promoter is inducible. Optionally, one promoter is tissue-specific and the other promoter is inducible.

[0092] In some embodiments, the episomal vector encodes scaffold RNA, for example, the scaffold RNA described in WO 2016 / 054106. In some embodiments, the episomal vector also encodes CRISPR nuclease. In addition to or instead of the above, the episomal vector can also encode one or more transcription activation domains. In some cases, the transcription activation domain is fused to a binding element that binds to the scaffold RNA (for example, binds to the ms2, f6, PP7, com or L7a sequence of the scaffold RNA).

[0093] In some embodiments, two or more different episomal vectors are provided. For example, an episomal vector encoding a CRISPR nuclease and a separate episomal vector encoding a guide RNA can be provided. Alternatively, an episomal vector encoding a CRISPR nuclease and a guide RNA can be provided, and a separate episomal vector encoding one or more transcription activation domains can be provided. Optionally, the one or more transcription activation domains are fused to a binding element that binds to the scaffold RNA (e.g., the guide RNA of a SAM). Optionally, the one or more transcription activation domains are fused to a binding element that binds to the affinity tag of the CRISPR nuclease. In some embodiments, an episomal vector encoding a scaffold RNA is provided, and a separate episomal vector encoding one or more transcription activation domains fused to a binding element that binds to the scaffold RNA is provided.

[0094] In some embodiments, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a gene listed in Table 1 or a gene in the same pathway or a parallel pathway as a gene listed in Table 1. Optionally, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a regulatory region (e.g., a promoter region or enhancer region) of a gene listed in Table 1 or a gene in the same pathway or a parallel pathway as a gene listed in Table 1.

[0095] Optionally, the episomal vector is selected from the group consisting of SIM1, leptin, leptin receptor, MC4R, SCN2A, SETD5, PAX6, PKD1, MC3R, POMC, STAT3, STAT5, SOCS3, GHR, NPY, NPY1R, NPY2R, NPY5R, PYY, AMPK (PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3), OXT, JAK2, SHP2, NOS3, NROB2, BRS3, CARTPT, FABP4, HTR2C, IL6, NHLH2, NMU, NPB, NPBWRI, PNPLA2, UCP 3, encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to ADIPOQ, APOA5, ARNT2, ASIP, C1QTNF2, C3AR1, CCK, CPT1B, CSF2, DGAT1, DGAT2, GHRL, GHSR, HSD11B1, HTR7, INSIG1, INSIG2, LIPC, NMURI, NMUR2, NPBWR2, NTS, PPARGC1A, PPY, RETN, SIRT1, TGFBR2, WDTC1, or FOXO1.

[0096] Optionally, the episomal vector is selected from the group consisting of SIM1, leptin, leptin receptor, MC4R, SCN2A, SETD5, PAX6, PKD1, MC3R, POMC, STAT3, STAT5, SOCS3, GHR, NPY, NPY1R, NPY2R, NPY5R, PYY, AMPK (PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3), OXT, JAK2, SHP2, NOS3, NROB2, BRS3, CARTPT, FABP4, HTR2C, IL6, NHLH2, NMU, NPB, NPBWRI, PNPLA2, UCP3, ADIPOQ, and APOA5. , ARNT2, ASIP, C1QTNF2, C3AR1, CCK, CPT1B, CSF2, DGAT1, DGAT2, GHRL, GHSR, HSD11B1, HTR7, INSIG1, INSIG2, LIPC, NMURI, NMUR2, NPBWR2, NTS, PPARGC1A, PPY, RETN, SIRT1, TGFBR2, WDTC1 or FOXO1.

[0097] In some cases, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a regulatory region (e.g., a promoter region or an enhancer region) of SIM1. In some cases, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a promoter region of SIM1. In some cases, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to an enhancer region of SIM1. In some cases, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a regulatory region (e.g., a promoter region or an enhancer region) of MC4R. In some cases, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a promoter region of MC4R. In some cases, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to an enhancer region of MC4R. In some cases, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a regulatory region (e.g., a promoter region or an enhancer region) of PDK1. In some cases, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a promoter region of PDK1.In some cases, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (for example, under stringent hybridization conditions) with the enhancer region of PDK1.In some cases, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (for example, under stringent hybridization conditions) with the control region (for example, promoter region or enhancer region) of SETD5.In some cases, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (for example, under stringent hybridization conditions) with the promoter region of SETD5.In some cases, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (for example, under stringent hybridization conditions) with the enhancer region of SETD5. In some cases, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a regulatory region (e.g., a promoter region or an enhancer region) of SCN2A. In some cases, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a promoter region of SCN2A. In some cases, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to an enhancer region of SCN2A. Optionally, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (eg, under stringent hybridization conditions) to a regulatory region (eg, a promoter region or enhancer region) of PAX6.In some cases, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to the promoter region of PAX6. In some cases, the episomal vector encodes a zinc finger nuclease or TALEN that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to the enhancer region of PAX6.

[0098] In some embodiments, the episomal vector encodes a guide or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a gene listed in Table 1 or a gene in the same pathway or a parallel pathway as a gene listed in Table 1. Optionally, the episomal vector encodes a guide or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a regulatory region (e.g., a promoter or enhancer region) of a gene listed in Table 1 or a gene in the same pathway or a parallel pathway as a gene listed in Table 1.

[0099] Optionally, the episomal vector is selected from the group consisting of SIM1, leptin, leptin receptor, MC4R, SCN2A, SETD5, PAX6, PKD1, MC3R, POMC, STAT3, STAT5, SOCS3, GHR, NPY, NPY1R, NPY2R, NPY5R, PYY, AMPK (PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3), OXT, JAK2, SHP2, NOS3, NROB2, BRS3, CARTPT, FABP4, HTR2C, IL6, NHLH2, NMU, NPB, NPBWRI, PNPLA2, UC Encodes a guide RNA or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to P3, ADIPOQ, APOA5, ARNT2, ASIP, C1QTNF2, C3AR1, CCK, CPT1B, CSF2, DGAT1, DGAT2, GHRL, GHSR, HSD11B1, HTR7, INSIG1, INSIG2, LIPC, NMURI, NMUR2, NPBWR2, NTS, PPARGC1A, PPY, RETN, SIRT1, TGFBR2, WDTC1, or FOXO1.

[0100] In some cases, the episomal vector is selected from the group consisting of SIM1, leptin, leptin receptor, MC4R, SCN2A, SETD5, PAX6, PKD1, MC3R, POMC, STAT3, STAT5, SOCS3, GHR, NPY, NPY1R, NPY2R, NPY5R, PYY, AMPK (PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3), OXT, JAK2, SHP2, NOS3, NROB2, BRS3, CARTPT, FABP4, HTR2C, IL6, NHLH2, NMU, NPB, NPBWRI, PNPLA2, UCP3, ADIPOQ, APOA 5, ARNT2, ASIP, C1QTNF2, C3AR1, CCK, CPT1B, CSF2, DGAT1, DGAT2, GHRL, GHSR, HSD11B1, HTR7, INSIG1, INSIG2, LIPC, NMURI, NMUR2, NPBWR2, NTS, PPARGC1A, PPY, RETN, SIRT1, TGFBR2, WDTC1 or FOXO1.

[0101] Optionally, the episomal vector encodes a guide RNA or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a regulatory region (e.g., promoter region or enhancer region) of SIM1. Optionally, the episomal vector encodes a guide RNA or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a promoter region of SIM1. Optionally, the episomal vector encodes a guide RNA or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to an enhancer region of SIM1. Optionally, the episomal vector encodes a guide RNA or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a regulatory region (e.g., promoter region or enhancer region) of MC4R. Optionally, the episomal vector encodes a guide RNA or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to the promoter region of MC4R. Optionally, the episomal vector encodes a guide RNA or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to the enhancer region of MC4R. Optionally, the episomal vector encodes a guide RNA or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a regulatory region (e.g., promoter region or enhancer region) of PDK1. Optionally, the episomal vector encodes a guide RNA or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to the promoter region of PDK1.In some cases, the episomal vector encodes a guide RNA or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to the enhancer region of PDK1. In some cases, the episomal vector encodes a guide RNA or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to the regulatory region (e.g., promoter region or enhancer region) of SETD5. In some cases, the episomal vector encodes a guide RNA or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to the promoter region of SETD5. In some cases, the episomal vector encodes a guide RNA or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to the enhancer region of SETD5. Optionally, the episomal vector encodes a guide RNA or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a regulatory region (e.g., promoter region or enhancer region) of SCN2A. Optionally, the episomal vector encodes a guide RNA or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a promoter region of SCN2A. Optionally, the episomal vector encodes a guide RNA or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to an enhancer region of SCN2A. Optionally, the episomal vector encodes a guide RNA or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to a regulatory region (e.g., promoter region or enhancer region) of PAX6.Optionally, the episomal vector encodes a guide or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to the promoter region of PAX6. Optionally, the episomal vector encodes a guide or scaffold RNA that hybridizes or specifically hybridizes (e.g., under stringent hybridization conditions) to the enhancer region of PAX6.

[0102] In some cases, the targeting region of the guide RNA is encoded by, specifically hybridizes to, or is fully complementary to the following sequence: TIFF2025118716000076.tif26170. Optionally, the targeting region of the guide RNA is encoded by, specifically hybridizes to, or is fully complementary to the following sequence: TIFF2025118716000077.tif25160. Optionally, the targeting region of the guide RNA is encoded by, specifically hybridizes to, or is fully complementary to the following sequence: TIFF2025118716000078.tif26160. In another embodiment, the targeting region of the guide RNA is encoded by, specifically hybridizes to, or is fully complementary to the following sequence: TIFF2025118716000079.tif33164. In some embodiments, the targeting region of the guide RNA is encoded by, specifically hybridizes to, or is fully complementary to the following sequence: TIFF2025118716000080.tif26166. In some embodiments, the targeting region of the guide RNA is encoded by, specifically hybridizes to, or is fully complementary to the following sequence: TIFF2025118716000081.tif25163. In some embodiments, the targeting region of the guide RNA is encoded by, specifically hybridizes to, or is fully complementary to the following sequence: TIFF2025118716000082.tif54157. In some embodiments, the targeting region of the guide RNA is encoded by, specifically hybridizes to, or is fully complementary to the following sequence: TIFF2025118716000083.tif54155. In some embodiments, the targeting region of the guide RNA is encoded by or specifically hybridizes to the following sequence: TIFF2025118716000084.tif54166. In some embodiments, the targeting region of the guide RNA is encoded by or specifically hybridizes to the following sequence: TIFF2025118716000085.tif33159. In some embodiments, the targeting region of the guide RNA is encoded by, specifically hybridizes to, or is fully complementary to the following sequence: TIFF2025118716000086.tif26128. In some embodiments, the targeting region of the guide RNA is Encoded by, specifically hybridizes to, or is perfectly complementary to TIFF2025118716000087.tif4128.

[0103] Optionally, the targeting region of the guide RNA is encoded by, specifically hybridizes to, or is fully complementary to a sequence that is orthologous and / or homologous to a region of the mouse or human genome targeted by an sgRNA that corresponds to or includes one of SEQ ID NOs: 1-12 or 34-95. Optionally, the guide RNA is encoded by, specifically hybridizes to, or is fully complementary to a sequence that is 90%, 95%, or 99% identical to one of SEQ ID NOs: 1-12 or 34-95, or a sequence that differs by 1, 2, or 3 nucleotides from one of SEQ ID NOs: 1-12 or 34-95, or a sequence that is 1, 2, or 3 nucleotides longer or shorter at the 5' and / or 3' ends than one of SEQ ID NOs: 1-12 or 34-95.

[0104] One or more of the episomal vectors described herein can be provided as a kit for treating the disease of mammalian subjects that is associated with, aggravated by, or caused by the reduced transcription of gene or the reduced amount of gene product or the reduced activity of gene product.For example, the episomal vector that encodes CRISPR nuclease, zinc finger nuclease, TALEN, TAL effector, guide RNA, transcription activation domain, scaffold RNA, scaffold RNA ligand, affinity tag ligand, one or more fusion proteins thereof, or combinations thereof, can be provided as a component of a kit that includes episomal vector packaging plasmid, cell line or helper virus, or combinations thereof.

[0105] Optionally, the episomal vector, in which the encoded polypeptide and / or RNA is flanked by AAV inverted terminal repeat sequences, is provided as a component of a kit containing additional materials for packaging the episomal vector into functional AAV particles. Such additional materials may include one or more plasmids encoding the AAV rep and cap genes, one or more plasmids encoding adenoviral helper factors E1A, E1B, E2A, E4ORF6, and VA, adenovirus, or a combination thereof. Optionally, trans-activating and / or helper elements for AAV packaging are provided in a stable cell line as a component of the kit.

[0106] In some embodiments, the cap gene is an AAV-DJ, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 cap gene. In some embodiments, the cap gene is an AAV-DJ, AAV1, AAV2, AAV5, AAV7, AAV8, or AAV9 cap gene. In some embodiments, the cap gene is an AAV2 cap gene. In some embodiments, the cap gene is an AAV-DJ cap gene. In some embodiments, the inverted terminal repeats (ITRs) are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 ITRs. In some embodiments, the ITRs are AAV1, AAV2, AAV5, AAV7, AAV8, or AAV9 ITRs. In some embodiments, the ITRs are AAV2 ITRs. Optionally, the capsid protein encoded by the cap gene is of the same serotype as the ITRs. For example, the cap gene can be the AAV2 cap gene, and the ITR can be the AAV2 ITR. In some cases, the capsid protein encoded by the cap gene is of a different serotype from the serotype of the ITR. Thus, for example, the cap gene can be the AAV5 cap gene, and the ITR can be the AAV2 ITR. In another example, the cap gene can be the AAV-DJ gene, and the ITR can be the AAV2 ITR.

[0107] Optionally, the episomal vector can be present in target cells or cells of target tissue. Optionally, the target cells or cells of target tissue are dividing cells. Optionally, the cells are non-dividing cells. Optionally, the cells are neurons. Optionally, the cells are hypothalamic cells. Optionally, the target cells or cells of target tissue are mammalian cells that contain a genome with at least one functional copy of target gene, and in the absence of transcriptional activation by heterologous complex, this functional copy does not produce enough corresponding gene product to produce wild-type phenotype in organisms. Optionally, the mammalian cell further comprises a scaffold RNA encoded by an episomal vector described herein, a guide RNA encoded by an episomal vector described herein, a CRISPR nuclease encoded by an episomal vector described herein, a SunTag encoded by one or more episomal vectors described herein, a synergistic activation mediator (SAM) encoded by one or more episomal vectors described herein, a transcription activation domain encoded by an episomal vector described herein, an affinity tag ligand encoded by an episomal vector described herein, a fusion of one or more polypeptides described herein encoded by an episomal vector described herein, or a combination thereof.

[0108] Optionally, the episomal vector in the target cell or target tissue cells is converted into a circular form, a circular concatemer, or a linear concatemer by recombination of repeat sequence elements such as ITRs. Optionally, the episomal vector in the target cell or target tissue cells is converted from a single-stranded DNA vector into double-stranded DNA. Optionally, the double-stranded DNA is converted into a circular form, a circular concatemer, or a linear concatemer. Optionally, the episomal vector in the target cell or target tissue cells remains as an episomal element, resulting in persistent expression of a transgene (e.g., CRISPR nuclease, transcription activator, guide RNA, scaffold RNA, etc.). Optionally, the episomal element is one of the aforementioned circular forms, circular concatemers, or linear concatemers.

[0109] virus particles One or more of the above-mentioned episomal vectors can be packaged in viral particles.For example, viral particles can contain the episomal vector encoding CRISPR nuclease, guide RNA, scaffold RNA, transcription activator, affinity tag, affinity tag ligand, scaffold RNA ligand, one or more fusion proteins thereof, or one or more combinations thereof.Viral particles can be capable of delivering episomal vector to target cells or target tissues, so that episomal vector enters the nucleus of target cells or the nucleus of the cells of target tissues, and is not or substantially not integrated into the genome of cells.

[0110] In some cases, viral particles deliver episomal vectors to target cells or target tissue cells, and episomal vectors are converted into circular forms, circular concatemers or linear concatemers by the incorporation of repeat sequence elements such as ITRs.In some cases, the episomal vectors delivered by viral particles are converted from single-stranded DNA vectors into double-stranded DNA.In some cases, double-stranded DNA is converted into circular forms, circular concatemers or linear concatemers.In some cases, viral particles deliver episomal vectors to target cells or target tissue cells, and episomal vectors remain as episomal elements, resulting in persistent transgene expression.

[0111] The viral particle can be an EBV or AAV viral particle. Optionally, the viral particle is an AAV viral particle. Optionally, the viral particle is an AAV-DJ, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 viral particle. Optionally, the viral particle is an AAV-DJ, AAV1, AAV2, AAV5, AAV7, AAV8, or AAV9 viral particle. Optionally, the viral particle is an AAV2 viral particle. Optionally, the viral particle is an AAV-DJ viral particle. The genome (episomal vector) encoding one or more transgenes packaged in the viral particle can be an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 genome. Optionally, the genome is an AAV1, AAV2, AAV5, AAV7, AAV8, or AAV9 genome. Optionally, genome is AAV2 genome.Optionally, genome is the same serotype as the viral particle that it is packaged in.In other cases, genome and viral particle are different serotypes.For example, capsid can be AAV5 serotype, and episomal vector can be AAV2 serotype.In another example, capsid can be AAV-DJ serotype, and episomal vector can be AAV2 serotype.

[0112] One or more of the viral particles described herein can be provided as a kit for treating the disease of mammalian subjects that is associated with, aggravated by, or caused by the reduced transcription of gene, or the reduced amount of gene product, or the reduced activity of gene product.For example, the episomal vector that encodes CRISPR nuclease, guide RNA, transcription activation domain, scaffold RNA, scaffold RNA ligand, affinity tag ligand, one or more fusion proteins thereof, or combinations thereof can be packaged in one or more viral particles, and provided as a component of the kit with suitable pharmaceutical excipients, carriers, diluents or buffers for delivery to subjects.

[0113] In one embodiment, the viral particles are in a suitable pharmaceutical excipient, carrier, diluent, or buffer for delivery to a subject. Such excipients, carriers, diluents, and buffers may include any pharmaceutical agent that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts may also be included, such as mineral acid salts such as hydrochloride, hydrobromide, phosphate, and sulfate, and salts of organic acids such as acetate, propionate, malonate, and benzoate. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may also be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be described in detail herein. Pharmaceutically acceptable excipients are described, for example, in A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy" 20th edition, Lippincott, Williams, & Wilkins, Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Cansel et al., eds., 7 thed., Lippincott, Williams, & Wilkins and Handbook of Pharmaceutical Excipients (2000) AHKibbe et al., eds.,3 rd It has been described in detail in various publications, including ed. Amer. Pharmaceutical Assoc.

[0114] method Described herein is a method for treating a disease in a mammalian subject that is associated with, exacerbated by, or caused by reduced transcription of a gene, reduced amount of a gene product, or reduced activity of a gene product, by increasing transcription of a target gene. The method generally includes contacting a target cell or cells of a target tissue with one or more of the aforementioned episomal vectors. In some embodiments, the episomal vector is non-integrating or substantially non-integrating. In some embodiments, the episomal vector is packaged into a viral particle, and the viral particle is contacted with the target cell or cells of the target tissue. Optionally, the contacting step is performed in vivo. Optionally, the contacting step is performed in vitro (e.g., using primary cells obtained from the subject), and the contacted cells are delivered to the subject, or optionally cultured and delivered to the subject.

[0115] Episomal vectors (e.g., packaged in viral particles) can be delivered by any means known in the art. Optionally, episomal vectors are contacted with cells in vivo by systemic delivery (e.g., intravenous delivery). Optionally, episomal vectors (e.g., packaged in viral particles) are contacted with cells in vivo by site-specific delivery to diseased cells or tissues. For example, viral particles containing episomal vectors can be packaged and injected at the site of diseased cells or tissues. Optionally, two or more episomal vectors are packaged in viral particles such that each viral particle contains a single copy of one of the two or more episomal vectors or is empty (contains no genome or contains a genome lacking a functional transgene). Such viral particles can be delivered as a mixture or individually. Optionally, the particles are delivered simultaneously. Optionally, the particles are delivered sequentially. Typically, the particles are delivered such that the delivered transgenes encoded by the episomal vectors are co-expressed in the subject to treat the disease.

[0116] In one embodiment, one or more different viral particles (for example, viral particles that have the same capsid but contain vectors encoding different transgenes) are injected into the brain of a subject.Optionally, one or more viral particles are injected into the hypothalamus of a subject.The viral particles can be delivered to the anterior part of the hypothalamus, the posterior part of the hypothalamus, the ventromedial part of the hypothalamus, or a combination thereof.The viral particles can be delivered bilaterally (for example, by bilateral injection into the hypothalamus of a subject).Optionally, one or more viral particles are delivered to the neurons of a subject.Optionally, one or more viral particles are delivered by stereotactic injection.

[0117] The dose of viral particles delivered to the subject is 1 x 10 3 Virus particles / kg of target ~1 x 10 20The dose of episomal vector delivered to a subject can be 1 x 10 viral particles / kg of subject. 3 Vector molecules / kg target ~1 x 10 20 Optionally, the dose can be 1 x 10 vector molecules / kg of subject. 4~ 1×10 18 , 1×10 5 ~1×10 16 , 1×10 6 ~1×10 15 viral particles / kg subject or vector molecules / kg subject. Optionally, the dose is at least 1 x 10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 or 1×10 15 The dose is viral particles / kg subject or vector molecule / kg subject.Optionally, the vector molecule is in the form of viral genome that is delivered in viral particles.Optionally, the dose is the dose of the viral genome that is delivered (for example, packaged in viral particles) that encodes CRISPR nuclease (for example, dCas9 that is fused with activation domain) and guide RNA (for example, sgRNA).Optionally, the dose is the dose of the viral genome that is delivered (for example, packaged in viral particles) that encodes CRISPR nuclease (for example, dCas9 that is fused with activation domain), and the second dose (for example, one or more of the doses mentioned above) is the dose of the viral genome that is delivered (for example, packaged in viral particles) that encodes guide RNA (for example, sgRNA).

[0118] In some cases, systemic dose can be higher than the dose that is directly applied to the tissue or organ to be treated.For example, to treat obesity caused by the haploinsufficient sim1 gene dysregulation in hypothalamic tissue or hypothalamic cell, a lower dose can be delivered to the hypothalamus compared to the systemic dose.For example, in humans, systemic delivery can be about 6.7 x 10 13 ~2.0×10 14 viral genomes (vg) / kg (see clinicaltrials.gov / ct2 / show / NCT02122952), and neurosurgical delivery can be, for example, about 7.5 x 10 11 ~8.8×10 12 vg / kg (see clinicaltrials.gov / ct2 / show / NCT01973543).

[0119] A dose can be administered once or multiple times. Optionally, the dose is delivered at least once within a period of 30, 60, 90, 120, or 180 days. Optionally, a dose is delivered at least once every 10, 20, 30, 40, 52, 75, or 100 weeks. Optionally, a dose is delivered at least once every 6, 12, 18 months, 2, 3, 5, or 10 years. Optionally, a single dose or two, three, or four doses result in sustained and sufficient expression of the target gene that is otherwise haploinsufficient to treat at least one symptom of a disease or condition caused by haploinsufficiency for several months or years. Optionally, a dose is administered, and the sufficiency of expression of the target haploinsufficient gene (e.g., a gene in Table 1, such as sim1) is assessed (e.g., in a target tissue, such as the hypothalamus), and additional doses are delivered as needed, either by the same route or a different route. Optionally, one or more doses of viral particles described herein are delivered in an amount sufficient to increase transcription of a target gene, thereby treating at least one symptom of a disease associated with, exacerbated by, or caused by reduced transcription of the gene or reduced amount of the gene product or reduced activity of the gene product, and one or more further doses are readministered once transcription of the target gene has been reduced by at least 10%, 25%, 50%, 75%, 90% or more from its maximum expression level. [Example]

[0120] Rescue of haploinsufficiency-induced obesity I. Introduction More than 300 genes are known to cause human disease due to haploinsufficiency (1, 2), leading to a wide range of phenotypes, including cancer, neurological disorders, developmental disorders, immunological disorders, metabolic disorders, infertility, kidney disease, limb malformations, and many others (1). Large-scale exome sequencing analysis has estimated that a total of 3,230 human genes may be intolerant to heterozygous loss of function (LoF) (3). Gene therapy holds great promise for correcting haploinsufficiency disorders by inserting one or more functional recombinant copies of the mutated gene. Currently, a total of 2,300 clinical trials of gene therapy are underway, the majority of which use adeno-associated viruses (AAVs) to deliver recombinant genes (4). AAVs are the preferred gene delivery method because they can deliver DNA without integrating into the genome, do not cause pathology, and result in long-lasting transgene expression (5). However, AAV has an optimal packaging capacity of 4.7 kilobases (kb), and genes longer than 3.5 kb (considering the additional regulatory sequences required for their stable expression) limit its use in gene therapy. Analysis of 3,230 heterozygous LoF genes reveals that 715 of them (22%) have coding sequences longer than 3.5 kb and are therefore unsuitable for AAV gene therapy.

[0121] CRISPR gene editing could potentially repair haploinsufficient mutations, but this would require customizing editing strategies for each mutation. Furthermore, this is not a viable treatment for heterozygous LoF microdeletions. To address these challenges, we devised a novel haploinsufficient treatment strategy using CRISPR activation (CRISPRa). CRISPRa utilizes the RNA-guided targeting ability of CRISPR to target nuclease-deficient Cas9 (dCas9) along with transcriptional activators to regulatory elements of specific genes, thereby increasing their expression (6-10). Here, we investigated whether this system could be used to increase transcription of unaltered endogenous genes to rescue disease phenotypes in haploinsufficient diseases.

[0122] SIM1 is a transcription factor expressed in the developing kidney and central nervous system and is essential for the formation of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus (11). It is also thought to play a major role in the leptin pathway (12). In humans, haploinsufficiency of SIM1 due to chromosomal abnormalities (12, 13) leads to hyperphagia-induced obesity (13), and SIM1 coding mutations, many of which are loss-of-function, are thought to be the primary cause of severe obesity in humans (14-16). Sim1 homozygous null mice die perinatally, whereas Sim1 heterozygous mice (Sim1 + / - ) survive, are hyperphagic, and develop early-onset obesity accompanied by increased linear growth, hyperinsulinemia, and hyperleptinemia (17). Postnatal conditional knockout of hypothalamic Sim1 leads to a similar phenotype in heterozygous mice (18), suggesting that Sim1 is a key regulator of energy homeostasis. Overexpression of SIM1 in mice using human bacterial artificial chromosomes rescued diet-induced obesity and reduced food intake (19), suggesting a potential role for Sim1 as a general therapeutic target for obesity. Here, we used Sim1 as our proof-of-concept model for our CRISPRa therapeutic strategy. We demonstrate that Sim1 + / - The ability of CRISPRa to rescue the obesity phenotype in mice was tested using both transgenic and AAV-based approaches targeting the Sim1 promoter or its hypothalamus-specific enhancer. Our results suggest a novel therapeutic approach for treating haploinsufficiency disorders or other diseases caused by altered gene dosage.

[0123] II. Results A. Upregulation of Sim1 in vitro We first set out to optimize our CRISPRa conditions in vitro. SIM1 has a well-characterized promoter (20) and a distal hypothalamic enhancer (approximately 270 kb from the transcription start site), Sim1 candidate enhancer 2 (SCE2) (21), both of which were selected as targets for CRISPRa (Figure 1A). We designed sgRNAs against the Sim1 promoter or Sim1 enhancer (SCE2). Using these guides, we tested whether dCas9 fused to VP64 (dCas9-VP64), a transcriptional activator containing four tandem copies of VP16 (herpes simplex virus type 1 transcription factor) (22), could overexpress Sim1 in mouse neuroblastoma cells (Neuro-2a). We chose this activator because its activation level is lower than that of other known activators (23). We aimed to achieve therapeutic Sim1 levels similar to wild-type levels in vivo. Cells were transfected with dCas9-VP64 and various guides, and Sim1 mRNA levels were measured 48 hours later using quantitative PCR (qPCR). We identified one sgRNA for the promoter and one for SCE2 that could overexpress endogenous Sim1 by 13-fold and 4-fold, respectively (Figure 1B). Additionally, we identified four sgRNAs for the Sim1 promoter that could overexpress endogenous Sim1 by more than fourfold (Figure 7A) and at least one sgRNA for SCE2 that could overexpress endogenous Sim1 by more than twofold (Figure 8A).

[0124] B. Transgenic CRISPRa rescues obesity To test the ability of our CRISPRa system to activate Sim1 in vivo, we used the TARGATT technique (24) to transfect dCas9-VP64 (H11P) inserted into the mouse Hipp11 locus. CAG-dCas9-VP64 ) and sgRNA targeting the Sim1 promoter at the Rosa26 locus (ROSA26 Sim1Pr-sgRNA ) or sgRNA targeting SCE2 (ROSA26SCE2En-sgRNA ) or Sim1 ), which causes severe obesity (Figure 1C). + / - The mice were crossed with Sim1 mice (17). + / - ×H11P CAG-dCas9-VP64 and ROSA26 Sim1Pr-sgRNA or ROSA26 SCE2En-sgRNA ) were fed breeder's chow (picodiet-5058) and bred to produce Sim1 mice, both of which became severely obese, along with wild-type littermates. + / - ×H11P CAG-dCas9-VP64 Mouse and Sim1 + / - The mice were weighed weekly for up to 16 weeks, along with a negative control. At least seven females and seven males per condition were analyzed. Sim1 mice carrying both dCas9-VP64 and either the Sim1 promoter sgRNA or the Sim1 enhancer sgRNA were identified. + / - Mouse is Sim1 + / - ×H11P CAG-dCas9-VP64 and Sim1 + / - There was a significant reduction in body weight compared to littermates (Fig. 1D-F).

[0125] C.CRISPRa is Sim1 + / - Correcting your metabolic profile To correlate weight loss with body composition and metabolic parameters, we next investigated Sim1 + / - ×H11P CAG-dCas9-VP64 ×ROSA26 Sim1Pr-sgRNA (Prm CRISPRa), Sim1 + / - ×H11P CAG-dCas9-VP64 ×ROSA26 SCE2En-sgRNA Metabolic profiling was performed on the Enh-CRISPRa and other mouse strains of the present inventors. Three mice of each genotype were analyzed for body composition and metabolic profiling at 6-8 weeks of age, just at the onset of the obesity phase. Both Prm-CRISPRa and Enh-CRISPRa mice were Sim1-positive. + / -Compared with Sim1, both males and females showed a significant reduction in body fat content (Fig. 2A). + / - Metabolic chamber analysis of other distinguishing features of obese mice showed a shift toward wild-type metabolic parameters in Prm-CRISPRa and Enh-CRISPRa mice (Figure 2B-C). In addition, their respiratory exchange ratio (RER; VCO2 / VO2), an indirect way of defining basal metabolic rate, also showed parameters similar to those of wild-type littermates (Figure 2D). However, we did not observe any significant differences in their physical activity in the individual chambers. Taken together, these results indicate that both Prm-CRISPRa and Enh-CRISPRa mice have less body fat and exhibit improved metabolic parameters that contribute to their reduced overall body weight.

[0126] D. Sim1 activation is tissue-specific To test the level and tissue specificity of Sim1 activation in our mice, we measured its mRNA expression levels in various tissues. We selected two tissues known to express Sim1, the hypothalamus and kidney, and two tissues that do not express Sim1, the lung and liver (25) (Figure 3A). We first measured dCas9 expression and found that it was expressed in all four tissues, as expected, because we used the ubiquitous CMV enhancer chicken beta-actin (CAG) promoter to drive its expression (Figure 3B). In contrast, for Sim1, we found that Sim1 + / - We observed significantly higher mRNA levels in the hypothalamus and kidney of Prm-CRISPRa mice compared with Enh-CRISPRa mice, and only in the hypothalamus of Enh-CRISPRa mice (Figure 3C-D). Since we did not observe any significant differences in the obesity phenotype between Prm-CRISPRa and Enh-CRISPRa mice, we hypothesized that Sim1 + / -We speculated that activation of Sim1 in the hypothalamus was sufficient to rescue the obesity phenotype. Interestingly, we could not detect Sim1 expression in tissues that do not express Sim1 (i.e., liver and lung) in either Prm-CRISPRa or Enh-CRISPRa mice, despite observing Cas9 expression. These results imply that, under the in vivo conditions of our study, dCas9-VP64 was only able to upregulate expression in tissues where the cis-regulatory elements of its target genes were active. This suggests that cis-regulatory elements may be used to determine the tissue specificity of CRISPRa.

[0127] E.CRISPRa AAV is Sim1 + / - Reduces weight gain To further bridge this approach to a therapeutic strategy for haploinsufficiency, we utilized AAV to transfect Sim1 + / -We delivered CRISPRa to the hypothalamus of mice. We generated three AAV vectors: (1) dCas9-VP64 driven by the cytomegalovirus (CMV) promoter (pCMV-dCas9-VP64), (2) Sim1 promoter-sgRNA and mCherry (pU6-Sim1Pr-CMV-mCherry), and (3) SCE2 sgRNA and mCherry (pU6-SCE2-CMV-mCherry). For the pCMV-dCas9-VP64 vector, we obtained a 5.4 kb insert due to the size of the dCas9-VP64 expression cassette. While this insert size exceeds the 4.7 kb limit, inserts larger than 5 kb have been shown to still be usable for delivery, albeit at reduced transgene expression levels (26). These vectors were individually packaged into AAV-DJ serotypes, chimeras of types 2, 8, and 9 (27), which have been shown to achieve high expression levels in multiple tissues (Figure 4A). We observed low but usable viral titers with pCMV-dCas9-VP64 AAV (see Methods). We first tested whether our AAV CRISPRa vectors could overexpress Sim1 in vitro in Neuro-2a cells. We observed a 4-fold and 5-fold upregulation of Sim1 mRNA expression when targeting the promoter or enhancer, respectively (Figure 4A). We observed that our AAV CRISPRa vectors could overexpress Sim1 in vitro using Neuro-2a cells with additional sgRNAs (SEQ ID NOs: 38, 40, or 42). We observed a 2- to 6-fold upregulation of Sim1 mRNA expression when targeting the promoter (Figure 7B) and a 2- to 4.5-fold upregulation of Sim1 mRNA expression when targeting the enhancer (SCE2) (Figure 8B).

[0128] Next, the inventors transfected Sim1Pr-CMV-mCherry (Prm-CRISPRa-AAV) or pU6-SCE2-CMV-mCherry (Enh-CRISPRa-AAV) viruses carrying pCMV-dCas9-VP64 and either pU6-Sim1Pr-CMV-mCherry (Prm-CRISPRa-AAV) or pU6-SCE2-CMV-mCherry (Enh-CRISPRa-AAV) viruses. + / - To deliver Sim1 to the PVN of the hypothalamus, we performed stereotaxic injections at 4 weeks of age, before the mice began to develop obesity. + / - Mice were also injected with the pCMV-dCas9-VP64 virus alone. We tested the expression of our sgRNA-CMV-mCherry cassette by immunostaining in the hypothalamus of injected mice and found that it was expressed in the PVN (Figure 4B-C). + / - To test whether delivery of CRISPRa-AAV to the hypothalamus of mice increases Sim1 expression levels, we measured mRNA expression of both dCas9 and Sim1 in 11-week-old AAV-injected mice. We found that dCas9 was expressed in all hypothalamuses of our pCMV-dCas9-VP64 AAV-injected mice (Figure 4D). Upregulation of Sim1 was observed in both Prm-CRISPRa-AAV-injected and Enh-CRISPRa-AAV-injected hypothalamus, but not in mice injected with pCMV-dCas9-VP64-AAV alone (Figure 4E). We measured the body weight of injected mice up to 11 weeks of age (Figure 5A). We found that Sim1 expression was significantly elevated in Prm-CRISPRa-AAV-injected or Enh-CRISPRa-AAV-injected mice. + / - Sim1 or pCMV-dCas9-VP64-AAV injected mice + / - We observed significant weight reduction compared to control mice (Figure 5B-C). These results demonstrate that CRISPRa-AAV-mediated upregulation can be used as a viable gene therapy for treating haploinsufficiency.

[0129] F. Upregulation of Mc4r in vitro More than 70% of obesity cases with a genetic basis are caused by defects in the leptin pathway. MC4R is part of the leptin pathway, and mutations therein are the most common mutations in obese individuals (approximately 5% of the 1st percentile obese population). Because it is a downstream factor, upregulation of MC4R and SIM1 could potentially rescue obesity caused by mutations in these other leptin pathway genes. Here, we have shown that MC4R can be upregulated by targeting the MC4R promoter, and that upregulation of SIM1 can increase MC4R expression. We were also able to rescue the obese phenotype in Mc4r heterozygous mice (essentially as described for in vitro Sim1 upregulation above). Therefore, upregulation of MC4R could be used as a treatment for obesity.

[0130] We designed sgRNAs for the Mc4r promoter (see SEQ ID NOs: 50-54). Using these guides, we tested whether dCas9 fused to VP64 (dCas9-VP64) could overexpress Mc4r in mouse neuroblastoma cells (Neuro-2a). Cells were transfected with dCas9-VP64 and various guides, and Mc4r mRNA levels were measured 48 hours later using quantitative PCR (qPCR). We identified one sgRNA for the Mc4r promoter that could overexpress endogenous Mc4r by 7-fold (Figure 9A).

[0131] G. CRISPRa AAV induces upregulation of Mc4r We next tested whether our AAV CRISPRa vectors (prepared essentially as described above for Sim1 CRISPRa AAV) containing sgRNA SEQ ID NOs:51, 52, or 54 could overexpress Mc4r in Neuro-2a cells in vitro. We observed a 3.4- to 6.6-fold upregulation of Mc4r mRNA expression when the promoter was targeted (Figure 9B).

[0132] H. Upregulation of SCN2A in vitro Mutations in SCN2A are the most common mutations in individuals with autism spectrum disorder (ASD) and epilepsy. The majority of mutations are loss-of-function mutations leading to ASD due to haploinsufficiency. Here, we demonstrate that SCN2A can be upregulated by targeting its promoter. Therefore, upregulation of SCN2A could be used as a treatment for ASD and epilepsy.

[0133] We designed sgRNAs for the Scn2a promoter (see SEQ ID NOs: 85-91). Using these guides, we tested whether dCas9 fused to VP64 (dCas9-VP64) could overexpress Scn2a in mouse neuroblastoma cells (Neuro-2a). Cells were transfected with dCas9-VP64 and various guides, and 48 hours later, Scn2a mRNA levels were measured using quantitative PCR (qPCR). We identified four sgRNAs for the Scn2a promoter that could overexpress endogenous Scn2a by more than twofold (Figure 12A).

[0134] I. CRISPRa AAV induces upregulation of Scn2A We next tested whether our AAV CRISPRa vectors containing sgRNAs SEQ ID NOs:92-94 (prepared essentially as described above for Sim1 CRISPRa AAV) could overexpress Scn2a in Neuro-2a cells in vitro. Two different multiplicities of infection (MOIs) were used: 5,000 viral genomes and 1,750 viral genomes (vg / ml). We observed a slight upregulation of Scn2a mRNA expression when the promoter was targeted at an MOI of 5,000 viral genomes per ml (Figure 12B).

[0135] J. Upregulation of SETD5 in vitro Mutations in SETD5 lead to mental retardation, including intellectual disability and dysmorphic features (OMIM number 615761). Here, we demonstrated that SETD5 can be upregulated by targeting the SETD5 promoter. Therefore, upregulation of SETD5 could be used as a treatment for intellectual disability.

[0136] We designed sgRNAs for the Setd5 promoter (see SEQ ID NOs: 75-84). Using these guides, we tested whether dCas9 fused to VP64 (dCas9-VP64) could overexpress Setd5 in mouse neuroblastoma cells (Neuro-2a). Cells were transfected with dCas9-VP64 and various guides, and Setd5 mRNA levels were measured 48 hours later using quantitative PCR (qPCR). We identified two sgRNAs for the Setd5 promoter that could overexpress endogenous Setd5 by 1.5-fold or more (Figure 11B).

[0137] Next, we designed sgRNAs for the human SETD5 promoter (see SEQ ID NOs: 65-74). Using these guides, we tested whether dCas9 fused to VP64 (dCas9-VP64) could overexpress SETD5 in human HEK293T cells. Cells were transfected with dCas9-VP64 and various guides, and SETD5 mRNA levels were measured 48 hours later using quantitative PCR (qPCR). We identified at least one sgRNA for the SETD5 promoter that could overexpress endogenous SETD5 by more than 2.5-fold (Figure 11A).

[0138] K. Upregulation of PKD1 in vitro Mutations in PKD1 lead to autosomal dominant polycystic kidney disease (ADPKD; OMIM number 173900), the most common inherited kidney disorder, affecting 1 in 400 to 1,000 people. 85% of ADPKD cases are caused by mutations in PKD1, and the majority of these mutations are loss-of-function. PKD1 is 13 kb long and therefore cannot be packaged into standard gene therapy vectors. Using the CRISPRa technology disclosed herein, we demonstrated that PKD1 can be upregulated by targeting the PKD1 promoter. Therefore, upregulation of PKD1 could be used as a treatment for autosomal dominant polycystic kidney disease.

[0139] We designed sgRNAs for the human PKD1 promoter (see SEQ ID NOs: 55-64). Using these guides, we tested whether dCas9 fused to VP64 (dCas9-VP64) could overexpress PKD1 in human HEK293T cells. Cells were transfected with dCas9-VP64 and various guides, and PKD1 mRNA levels were measured 48 hours later using quantitative PCR (qPCR). We identified at least three sgRNAs for the PKD1 promoter that could overexpress endogenous PKD1 by more than twofold (Figure 10).

[0140] L. Upregulation of PAX6 in vitro Loss-of-function mutations in PAX6 lead to aniridia 1 (OMIM number 106210) due to haploinsufficiency. Here, we show that PAX6 can be upregulated by targeting the PAX6 promoter. Therefore, upregulation of PAX6 could be used as a treatment for aniridia 1.

[0141] We designed an sgRNA for the human PAX6 promoter (SEQ ID NO:95). Using this guide, we tested whether dCas9 (Streptococcus pyogenes) fused to VP64 (dCas9-VP64) could overexpress PAX6 in human H1-ESC cells differentiated into neurons. Lentivirus carrying the guide was transfected into cells, and PAX6 mRNA levels were measured 48 hours later using quantitative PCR (qPCR). Our exemplary sgRNA for the PAX6 promoter was able to overexpress endogenous PAX6 by more than sixfold (Figure 13). Figure 13 also demonstrates that other neuronal markers (e.g., NES) also possess the neural induction potential of H1-ESCs.

[0142] III. Discussion CRISPR-based gene editing is a promising therapeutic technique for correcting genetic mutations. However, it is currently not a viable technique for haploinsufficiency due to limitations imposed by low non-homologous end joining (NHEJ) efficiency (i.e., only a small fraction of cells are edited) and the need to customize specific guide and donor sequences for each mutation. Furthermore, although over 200 microdeletions are known to cause human disease, primarily through haploinsufficiency (28), CRISPR-based gene editing is also not a viable therapeutic strategy for microdeletions. In this study, we address these challenges using a novel approach and demonstrate that haploinsufficiency diseases can be corrected by increasing transcriptional output from existing functional alleles with CRISPRa.

[0143] Using CRISPRa targeting either the promoter or enhancer of Sim1, we were able to rescue the obesity phenotype in mice haploinsufficient for Sim1 in a tissue-specific manner (Figure 6). Because this therapeutic approach utilizes existing functional alleles, it offers several advantages: (1) it eliminates the need to customize CRISPR gene editing approaches for different haploinsufficient mutations in the same gene; (2) this approach could potentially be used to target more than one gene. Therefore, it could be seen as a potential therapeutic strategy for microdeletion-associated diseases caused by heterozygous LoF of two or more genes; (3) CRISPRa-AAV could be used to rescue haploinsufficient diseases caused by genes longer than their optimal packaging capacity; and (4) CRISPR-based therapy can utilize cis-regulatory elements to guide tissue specificity. The availability of a large, tissue-specific map of gene regulatory elements will provide ample candidates for use in this therapeutic approach. In our studies, we observed clear differences in tissue-specific activation of Sim1, which can be attributed to the chromatin accessibility of the locus in various tissues. Previous large-scale Cas9 and dCas9 cell culture screens have shown a targeting preference for regions with low nucleosome occupancy. (29) Active promoters or enhancers have low nucleosome occupancy and are therefore likely to be amenable to dCas9 targeting.

[0144] Our dCas9-VP64 mouse and AAV vector could be a useful tool for in vivo targeted gene activation by delivering sgRNAs targeting specific genes in certain tissues / tissue types. This approach could be used to evaluate gene-gene interactions or to identify target genes of specific regulatory elements in vivo by measuring their expression levels after activation. Another potential area of research would be neural circuit manipulation. Discrepancies have been observed between acute and chronic neural circuit manipulation (30), which could be addressed by our AAV-CRISPRa and transgenic CRISPRa strategies, respectively.

[0145] Haploinsufficiency of Sim1 causes obesity in both mice (17) and humans (13). + / - Whether this is caused by the reduced PVN size observed in mice during development (17) or by disrupted energy homeostasis in adulthood has been a major area of investigation. The obese phenotype observed in postnatal conditional knockout of hypothalamic Sim1 (18) reinforced the hypothesis that Sim1 indeed plays a role in energy homeostasis later in adulthood. The rescue of the obese phenotype by injection of CRISPRa AAV into the hypothalamus of 4-week-old mice further corroborates this role. Abrogation of melanocortin 4 receptor (Mc4r) signaling is a hallmark of most polygenic and monogenic obesity phenotypes. Conditional postnatal deficiency of Sim1 leads to reduced levels of Mc4r signaling. Because Sim1 has been shown to be an essential downstream component of the leptin-Mc4r pathway ( 18 ), Sim1 CRISPRa targeting could be a potential treatment for conditions that disrupt the leptin signaling pathway.

[0146] Despite technological advances in CRISPR-based therapeutic interventions, our understanding of the long-term side effects of CRISPR expression in vivo and its off-target effects remains largely unknown, and this is also true for our current study. In the future, these concerns may be addressed by conditional activation or silencing of anti-CRISPR genes (31) or our CRISPRa system. Furthermore, CRISPRa / i tools must be developed to adjust gene dosage so that transcriptional output can be optimized for certain diseases, where higher or lower activation levels may be required. In this study, we used VP64 as our activator, as it is known to have weak activating activity (23), which is compatible with our need to achieve gene expression levels similar to those of two normal alleles. CRISPRa-based gene activation depends on the nature of the fused activator (23) and the sgRNA target (29), and may require optimization of the CRISPR system and delivery method.

[0147] As demonstrated in this study, CRISPRa can be used to activate genes not only by targeting gene promoters but also by targeting distal cis-regulatory elements, such as enhancers. Previous studies have shown that these elements are viable therapeutic targets. For example, activation of fetal hemoglobin was achieved in vitro by targeting a globin enhancer with a zinc-finger nuclease fused to the chromatin loop-forming factor LIM domain binding 1 (LDB1) gene, making it a potential treatment for sickle cell disease (37). In another study, reactivation of fetal hemoglobin was achieved by inactivating the enhancer of its repressor, B-cell CLL / lymphoma 11A (BCL11A), using CRISPR gene editing (38). Our study provides a novel approach to similarly exploiting cis-regulatory elements for therapeutic purposes. There are numerous diseases caused by reduced gene dosage that could be treated with CRISPRa therapy. In addition, some human diseases may be rescued by activating other genes with similar functions. These could include, for example, utrophin for Duchenne muscular dystrophy (39), survival of motor neuron 2 (SMA2) for spinal muscular atrophy (SMA) (40), or fetal globin for sickle cell disease. Further development of this technology may result in viable treatments for patients affected by these diseases.

[0148] III. Materials and Methods Plasmid The pMSCV-LTR-dCas9-VP64-BFP vector, encoding mammalian codon-optimized Streptococcus pyogenes dCas9 fused to two C-terminal SV40 NLSs and tagBFP with a VP64 domain, and the U6-sgRNA-CMV-mCherry-T2A-Puro plasmid were used for cell line transfection (both kindly provided by Dr. Stanley Qi). The sgRNA was cloned into the BstXI and XhoI sites using the In-Fusion HD-Cloning Kit (Clontech) according to the manufacturer's protocol. The mouse knock-in vector was generated by cloning the dCas9-VP64 expression cassette and the U6-sgRNA-CMV-mCherry expression cassette from the above vector into the TARGATT (CAG + polyA) plasmid (Applied StemCell). pcDNA-dCas9-VP64 (Addgene 47107) and U6-sgRNA-CMV-mCherry-WPREpA were cloned into the backbone of pAAV-Ef1a-FAS-hChR2(H134R)-mCherry-WPRE-pA (Addgene 37090), replacing Ef1a-FAS-hChR2(H134R)-mCherry-WPRE-pA with that of our U6-sgRNA-CMV-mCherry-WPREpA.

[0149] AAV production AAV DJ serotype particles were produced using the Stanford Neuroscience Viral Vector Core. Packaging loads were 5.4 kb for pCMV-dCas9-VP64 and 2.5 kb for pU6-Sim1Pr-CMV-mCherry and pU6-SCE2-CMV-mCherry. Genomic titers were 1.40 E for pCMV-dCas9-VP64 by WPRE and ITR probes. 10 viral genomes (vg) / ml, 3.30E for pU6-Sim1Pr-CMV-mCherry 13vg / ml, and 2.20E for pU6-SCE2-CMV-mCherry. 13 It was confirmed that the concentration was 0.05 vg / ml.

[0150] cell culture Neuroblastoma 2a cells (Neuro-2a; ATCC® CCL-131) were grown according to ATCC guidelines. Plasmids were transfected into Neuro-2a cells using X-tremeGENE HP DNA Transfection Reagent (Roche) according to the manufacturer's protocol. Neuro2a cells were infected with AAV particles at different MOIs. Neuro2a cells were harvested 48 hours post-transfection and 5 days post-infection, and RNA for qRT-PCR was isolated.

[0151] Human HEK293T cells were grown according to ATCC guidelines. Plasmids were transfected into these cells using X-tremeGENE HP DNA transfection reagent (Roche) according to the manufacturer's protocol.

[0152] Quantitative reverse transcription PCR RNA was isolated from cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. For mice, animals were euthanized and tissues were harvested directly into the RNA lysis buffer in the RNeasy Mini Kit. Hypothalamus was dissected using a Zivic Instruments Mouse Brain Matrix and slicer. cDNA was prepared using the SuperScript III First-Strand Synthesis System (Invitrogen) with DNase I digestion according to the manufacturer's protocol. qPCR was performed using SsoFast EvaGreen Supermix (Biorad). Results were expressed as fold-increase mRNA expression of the gene of interest normalized to either beta-actin, Rpl38, or Elf3 expression using the ΔΔCT method, followed by ANOVA and Tukey's test for statistical analysis. Values reported are the mean and standard error of the mean of three independent experiments (N = 3) performed on different days, with technical replicates averaged for each experiment.

[0153] mouse Sim1 with a mixed genetic background + / -Mice (17) were kindly provided by the laboratory of Dr. Jacques Michaud. In these mice, a 1-kb fragment containing the 750-bp 5' region, the initiation codon, and the sequence encoding the basic domain (first 17 amino acids) was replaced with a Pgk-neo cassette, which was then used for genotyping using the KAPA Mouse Genotyping Kit (KAPA Biosystems). To generate dCas9-VP64 and sgRNA mice, we used the TARGATT technique (24). DNA for injection was prepared and purified as minicircles using the TARGATT Transgenic Kit V6 (Applied StemCell). The injection mixture contained 3 ng / μL DNA and 48 ng / μL in vitro-transcribed φC31o mRNA in microinjection TE buffer (0.1 mM EDTA, 10 mM Tris, pH 7.5), and injections were performed using standard mouse transgenic protocols (41). dCas9-VP64 was inserted into the mouse Hipp11 locus, and the sgRNA was inserted into the Rosa26 locus. Mice were genotyped using the KAPA Mouse Genotyping Kit. F0 TARGATT knock-in was evaluated using PCR7+8 and PCR1 described in (PMID: 21464299) with vector insert-specific dCas9-VP64 primers and mCherry-specific primers. Throughout the study, all mice were fed ad libitum with Picolab Mouse Diet 20,5058, containing 20% protein, 9% lipid, and 4% fiber. The calorie breakdown from each component was 23.210% protein, 21.559% lipid (ether extract), and 55.231% carbohydrate. All animal experiments were approved by the UCSF Institutional Animal Care and Use Committee.

[0154] Mouse weight measurement To assess germline transmission, H11P CAG-dCas9-VP64 , ROSA26 Sim1Pr-sgRNA and ROSA26 SCE2En-sgRNA Mice were crossed with FVB mice for 3-5 generations. Three independent integrants were used for each line to set up the crosses.CAG-dCas9-VP64 Sim1 + / - and then Sim1 + / - ×H11P CAG-dCas9-VP64 Mouse ROSA26 Sim1Pr-sgRNA or ROSA26 SCE2En-sgRNA Mice were reared on Picodiet 5058 throughout the study, and all genotypes (wild-type littermates, Sim1 + / - , Sim1 + / - ×H11P CAG-dCas9-VP64 , Sim1 + / - ×H11P CAG-dCas9-VP64 ×ROSA26 Sim1Pr-sgRNA , Sim1 + / - ×H11P CAG-dCas9-VP64 ×ROSA26 SCE2En-sgRNA At least six females and six males from each group were weighed weekly between the ages of 4 and 16 weeks.

[0155] Mouse metabolic profiling Metabolic rates of individual mice were measured using the Columbus Instruments Comprehensive Laboratory Animal Monitoring System (CLAMS; Columbus Instruments). Mice were individually housed and acclimated with powdered picodiet 5058 for 3-4 days before metabolic monitoring. We housed mice individually in CLAMS units and performed measurements over 4-5 days. Temperature was maintained at 22°C, and oxygen and carbon dioxide were calibrated with an "air reference" set at 20.901 and 0.0049. For each genotype, wild-type littermates, Sim1 + / - , Sim1 + / - ×H11P CAG-dCas9-VP64 ×ROSA26 Sim1Pr-sgRNA , Sim1 + / - ×H11P CAG-dCas9-VP64 ×ROSA26 SCE2En-sgRNAThree males and three females from each study were measured for metabolic parameters (VCO2, VO2, RER, food intake, and activity monitoring). Metabolic data were analyzed using CLAX support software (Columbus Instruments).

[0156] body composition analysis Body composition was measured using either dual-energy X-ray absorptiometry (DEXA) or Echo Magnetic Resonance Imaging (EchoMRI; Echo Medical System). For DEXA, mice anesthetized with isoflurane were measured for bone mineral density and tissue composition (fat mass and lean mass) using a Lunar PIXImus. EchoMRI (Echo Medical System) was used to measure whole-body composition parameters, including total body fat, lean mass, body fluids, and total body water, in live mice without the need for anesthesia or sedation.

[0157] Stereotactic injection Sim1, 4 weeks old, weighing 22-26g + / - Males or females were housed individually in cages at least 2 days before surgical intervention. Mice were anesthetized with an intraperitoneal injection of 100 mg / kg avertin. The skull was fixed in a stereotaxic apparatus (David Kopf Instruments). The stereotaxic coordinates for injection into the PVN were 0.80 mm caudal to bregma, 0 mm above the midline, and 5.2 mm below the skull surface. A 1.5 mm hole was drilled in the skull using a circular motion with hand-held Dumont 5-45 tweezers (Fine Science Tools). A dose of 0.5 × 10 was administered using a 31-gauge, 1 μl Hamilton microsyringe. 7 2.5 x 10 vg / ml sgRNA-AAV 6The PVN was unilaterally injected with dCas-VP64-AAV at 100 mg / kg with a total injection volume of 1 μl per mouse over 10 minutes. The needle was left in place for 20 minutes after AAV delivery to prevent reflux and then slowly withdrawn in several steps over 10 minutes. Mice received two doses of buprenorphine (100 mg / kg) before and 24 hours after surgery. PVN injection coordinates were verified in some mice 2–12 weeks after injection using mCherry immunostaining, as described below. Mice were maintained on picodiet 5058 and weighed weekly.

[0158] immunostaining For immunostaining, mice were anesthetized with pentobarbital (7.5 mg / 0.15 ml, i.p.) and transcardially perfused with 10 ml of heparinized saline (10 U / ml, 2 ml / min) followed by 10 ml of phosphate-buffered 4% paraformaldehyde (PFA). Brains were removed and postfixed in 4% PFA for 24 h before equilibrating in 30% sucrose in PBS for 72 h. Brains were sectioned coronally (35 μm for immunostaining and 50 μm for stereology) using a sliding microtome (Leica SM 2000R). Immunohistochemistry was performed as previously described (19, 42, 43). Coronal brain sections stored in PBS at 4°C were permeabilized, blocked in 3% normal goat serum / 0.3% Triton X-100 for 1 hour, and incubated overnight at 4°C with a 1:500 dilution of mCherry antibody (Abcam ab167453). Sections were placed in 4,6-diamidino-2-phenylindole (DAPI) (0.2 g / ml; 236276; Roche) for 10 minutes, then mounted on plus-coated slides and coverslipped with Vectashield (H-1000; Vector Laboratories). Images of sections containing the PVN were captured on a Zeiss Apotome.

[0159] References TIFF2025118716000088.tif213161TIFF2025118716000089.tif220161TIFF2025118716000090.tif155160

[0160] Although the foregoing invention has been described in some detail, by way of illustration and examples for purposes of clarity and understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims. All patents, patent applications, and other publications referred to in this application, including publications referenced by GenBank accession number, Entrez gene ID, and pubmed ID (PMID), are incorporated herein by reference in their entirety for all purposes.

[0161] Sequence information SEQUENCE LISTING <110> The Regents of the University of California <120> GENE THERAPY FOR HAPLOINSUFFICIENCY <150> US 62 / 455,988 <151> 2017-02-07 <160> 95 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 1 gacacggaat tcattgccag 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 2 ctgcgggtta ggtctaccgg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 3 gttgagcgct cagtccagcg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 4 tcccgacgtc gtgcgcgacc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 5 gctctgaatc ttactacccg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 6 gctgttaact aaagacaggg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 7 gtggtctggg tgatctcatg 20 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 8 gacaaaggaa catctgagag g 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 9 gtgatctcat ggggaagagg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 10 ggctttgatc gtggtctggg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 11 gcgagcccag tcgcgtgggg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 12 gccaagaatt ggccaaaggg 20 <210> 13 <211> 159 <212> PRT <213> Homo sapiens <400> 13 Glu Lys Cys Leu Ser Val Ala Cys Leu Asp Lys Asn Glu Leu Ser Asp 1 5 10 15 His Leu Asp Ala Met Asp Ser Asn Leu Asp Asn Leu Gln Thr Met Leu 20 25 30 Ser Ser His Gly Phe Ser Val Asp Thr Ser Ala Leu Leu Asp Leu Phe 35 40 45 Ser Pro Ser Val Thr Val Pro Asp Met Ser Leu Pro Asp Leu Asp Ser 50 55 60 Ser Leu Ala Ser Ile Gln Glu Leu Leu Ser Pro Gln Glu Pro Pro Arg 65 70 75 80 Pro Pro Glu Ala Glu Asn Ser Ser Pro Asp Ser Gly Lys Gln Leu Val 85 90 95 His Tyr Thr Ala Gln Pro Leu Phe Leu Leu Asp Pro Gly Ser Val Asp 100 105 110 Thr Gly Ser Asn Asp Leu Pro Val Leu Phe Glu Leu Gly Glu Gly Ser 115 120 125 Tyr Phe Ser Glu Gly Asp Gly Phe Ala Glu Asp Pro Thr Ile Ser Leu 130 135 140 Leu Thr Gly Ser Glu Pro Pro Lys Ala Lys Asp Pro Thr Val Ser 145 150 155 <210> 14 <211> 11 <212> PRT <213> Unknown <220> <223> Herpes simplex virus <400> 14 Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu 1 5 10 <210> 15 <211> 50 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 15 Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu 1 5 10 15 Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe 20 25 30 Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp 35 40 45 Met Leu 50 <210> 16 <211> 261 <212> PRT <213> Homo sapiens <400> 16 Ser Gln Tyr Leu Pro Asp Thr Asp Asp Arg His Arg Ile Glu Glu Lys 1 5 10 15 Arg Lys Arg Thr Tyr Glu Thr Phe Lys Ser Ile Met Lys Lys Ser Pro 20 25 30 Phe Ser Gly Pro Thr Asp Pro Arg Pro Pro Pro Arg Arg Ile Ala Val 35 40 45 Pro Ser Arg Ser Ser Ala Ser Val Pro Lys Pro Ala Pro Gln Pro Tyr 50 55 60 Pro Phe Thr Ser Ser Leu Ser Thr Ile Asn Tyr Asp Glu Phe Pro Thr 65 70 75 80 Met Val Phe Pro Ser Gly Gln Ile Ser Gln Ala Ser Ala Leu Ala Pro 85 90 95 Ala Pro Pro Gln Val Leu Pro Gln Ala Pro Ala Pro Ala Pro Ala Pro 100 105 110 Ala Met Val Ser Ala Leu Ala Gln Ala Pro Ala Pro Val Pro Val Leu 115 120 125 Ala Pro Gly Pro Pro Gln Ala Val Ala Pro Pro Ala Pro Lys Pro Thr 130 135 140 Gln Ala Gly Glu Gly Thr Leu Ser Glu Ala Leu Leu Gln Leu Gln Phe 145 150 155 160 Asp Asp Glu Asp Leu Gly Ala Leu Leu Gly Asn Ser Thr Asp Pro Ala 165 170 175 Val Phe Thr Asp Leu Ala Ser Val Asp Asn Ser Glu Phe Gln Gln Leu 180 185 190 Leu Asn Gln Gly Ile Pro Val Ala Pro His Thr Thr Glu Pro Met Leu 195 200 205 Met Glu Tyr Pro Glu Ala Ile Thr Arg Leu Val Thr Gly Ala Gln Arg 210 215 220 Pro Pro Asp Pro Ala Pro Ala Pro Leu Gly Ala Pro Gly Leu Pro Asn 225 230 235 240 Gly Leu Leu Ser Gly Asp Glu Asp Phe Ser Ser Ile Ala Asp Met Asp 245 250 255 Phe Ser Ala Leu Leu 260 <210> 17 <211> 318 <212> PRT <213> Mus musculus <400> 17 Met Glu Leu Leu Ser Pro Pro Leu Arg Asp Ile Asp Leu Thr Gly Pro 1 5 10 15 Asp Gly Ser Leu Cys Ser Phe Glu Thr Ala Asp Asp Phe Tyr Asp Asp 20 25 30 Pro Cys Phe Asp Ser Pro Asp Leu Arg Phe Phe Glu Asp Leu Asp Pro 35 40 45 Arg Leu Val His Met Gly Ala Leu Leu Lys Pro Glu Glu His Ala His 50 55 60 Phe Pro Thr Ala Val His Pro Gly Pro Gly Ala Arg Glu Asp Glu His 65 70 75 80 Val Arg Ala Pro Ser Gly His His Gln Ala Gly Arg Cys Leu Leu Trp 85 90 95 Ala Cys Lys Ala Cys Lys Arg Lys Thr Thr Asn Ala Asp Arg Arg Lys 100 105 110 Ala Ala Thr Met Arg Glu Arg Arg Arg Leu Ser Lys Val Asn Glu Ala 115 120 125 Phe Glu Thr Leu Lys Arg Cys Thr Ser Ser Asn Pro Asn Gln Arg Leu 130 135 140 Pro Lys Val Glu Ile Leu Arg Asn Ala Ile Arg Tyr Ile Glu Gly Leu 145 150 155 160 Gln Ala Leu Leu Arg Asp Gln Asp Ala Ala Pro Pro Gly Ala Ala Ala 165 170 175 Phe Tyr Ala Pro Gly Pro Leu Pro Pro Gly Arg Gly Ser Glu His Tyr 180 185 190 Ser Gly Asp Ser Asp Ala Ser Ser Pro Arg Ser Asn Cys Ser Asp Gly 195 200 205 Met Met Asp Tyr Ser Gly Pro Pro Ser Gly Pro Arg Arg Gln Asn Gly 210 215 220 Tyr Asp Thr Ala Tyr Tyr Ser Glu Ala Ala Arg Glu Ser Arg Pro Gly 225 230 235 240 Lys Ser Ala Ala Val Ser Ser Leu Asp Cys Leu Ser Ser Ile Val Glu 245 250 255 Arg Ile Ser Thr Asp Ser Pro Ala Ala Pro Ala Leu Leu Leu Ala Asp 260 265 270 Ala Pro Pro Glu Ser Pro Pro Gly Pro Pro Glu Gly Ala Ser Leu Ser 275 280 285 Asp Thr Glu Gln Gly Thr Gln Thr Pro Ser Pro Asp Ala Ala Pro Gln 290 295 300 Cys Pro Ala Gly Ser Asn Pro Asn Ala Ile Tyr Gln Val Leu 305 310 315 <210> 18 <211> 190 <212> PRT <213> Unknown <220> <223> Epstein-Barr virus <400> 18 Arg Asp Ser Arg Glu Gly Met Phe Leu Pro Lys Pro Glu Ala Gly Ser 1 5 10 15 Ala Ile Ser Asp Val Phe Glu Gly Arg Glu Val Cys Gln Pro Lys Arg 20 25 30 Ile Arg Pro Phe His Pro Pro Gly Ser Pro Trp Ala Asn Arg Pro Leu 35 40 45 Pro Ala Ser Leu Ala Pro Thr Pro Thr Gly Pro Val His Glu Pro Val 50 55 60 Gly Ser Leu Thr Pro Ala Pro Val Pro Gln Pro Leu Asp Pro Ala Pro 65 70 75 80 Ala Val Thr Pro Glu Ala Ser His Leu Leu Glu Asp Pro Asp Glu Glu 85 90 95 Thr Ser Gln Ala Val Lys Ala Leu Arg Glu Met Ala Asp Thr Val Ile 100 105 110 Pro Gln Lys Glu Glu Ala Ala Ile Cys Gly Gln Met Asp Leu Ser His 115 120 125 Pro Pro Pro Arg Gly His Leu Asp Glu Leu Thr Thr Thr Leu Glu Ser 130 135 140 Met Thr Glu Asp Leu Asn Leu Asp Ser Pro Leu Thr Pro Glu Leu Asn 145 150 155 160 Glu Ile Leu Asp Thr Phe Leu Asn Asp Glu Cys Leu Leu His Ala Met 165 170 175 His Ile Ser Thr Gly Leu Ser Ile Phe Asp Thr Ser Leu Phe 180 185 190 <210> 19 <211> 366 <212> PRT <213> Homo sapiens <400> 19 Met Asp Ser Asp Asp Glu Met Val Glu Glu Ala Val Glu Gly His Leu 1 5 10 15 Asp Asp Asp Gly Leu Pro His Gly Phe Cys Thr Val Thr Tyr Ser Ser 20 25 30 Thr Asp Arg Phe Glu Gly Asn Phe Val His Gly Glu Lys Asn Gly Arg 35 40 45 Gly Lys Phe Phe Phe Phe Asp Gly Ser Thr Leu Glu Gly Tyr Tyr Val 50 55 60 Asp Asp Ala Leu Gln Gly Gln Gly Val Tyr Thr Tyr Glu Asp Gly Gly 65 70 75 80 Val Leu Gln Gly Thr Tyr Val Asp Gly Glu Leu Asn Gly Pro Ala Gln 85 90 95 Glu Tyr Asp Thr Asp Gly Arg Leu Ile Phe Lys Gly Gln Tyr Lys Asp 100 105 110 Asn Ile Arg His Gly Val Cys Trp Ile Tyr Tyr Pro Asp Gly Gly Ser 115 120 125 Leu Val Gly Glu Val Asn Glu Asp Gly Glu Met Thr Gly Glu Lys Ile 130 135 140 Ala Tyr Val Tyr Pro Asp Glu Arg Thr Ala Leu Tyr Gly Lys Phe Ile 145 150 155 160 Asp Gly Glu Met Ile Glu Gly Lys Leu Ala Thr Leu Met Ser Thr Glu 165 170 175 Glu Gly Arg Pro His Phe Glu Leu Met Pro Gly Asn Ser Val Tyr His 180 185 190 Phe Asp Lys Ser Thr Ser Ser Cys Ile Ser Thr Asn Ala Leu Leu Pro 195 200 205 Asp Pro Tyr Glu Ser Glu Arg Val Tyr Val Ala Glu Ser Leu Ile Ser 210 215 220 Ser Ala Gly Glu Gly Leu Phe Ser Lys Val Ala Val Gly Pro Asn Thr 225 230 235 240 Val Met Ser Phe Tyr Asn Gly Val Arg Ile Thr His Gln Glu Val Asp 245 250 255 Ser Arg Asp Trp Ala Leu Asn Gly Asn Thr Leu Ser Leu Asp Glu Glu 260 265 270 Thr Val Ile Asp Val Pro Glu Pro Tyr Asn His Val Ser Lys Tyr Cys 275 280 285 Ala Ser Leu Gly His Lys Ala Asn His Ser Phe Thr Pro Asn Cys Ile 290 295 300 Tyr Asp Met Phe Val His Pro Arg Phe Gly Pro Ile Lys Cys Ile Arg 305 310 315 320 Thr Leu Arg Ala Val Glu Ala Asp Glu Glu Leu Thr Val Ala Tyr Gly 325 330 335 Tyr Asp His Ser Pro Pro Gly Lys Ser Gly Pro Glu Ala Pro Glu Trp 340 345 350 Tyr Gln Val Glu Leu Lys Ala Phe Gln Ala Thr Gln Gln Lys 355 360 365 <210> 20 <211> 531 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 20 Glu Ala Ser Gly Ser Gly Arg Ala Asp Ala Leu Asp Asp Phe Asp Leu 1 5 10 15 Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu 20 25 30 Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp 35 40 45 Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Ile Asn Ser Arg Ser Ser 50 55 60 Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Ser Gln Tyr Leu Pro Asp 65 70 75 80 Thr Asp Asp Arg His Arg Ile Glu Glu Lys Arg Lys Arg Thr Tyr Glu 85 90 95 Thr Phe Lys Ser Ile Met Lys Lys Ser Pro Phe Ser Gly Pro Thr Asp 100 105 110 Pro Arg Pro Pro Pro Pro Arg Arg Ile Ala Val Pro Ser Arg Ser Ser Ala 115 120 125 Ser Val Pro Lys Pro Ala Pro Gln Pro Tyr Pro Phe Thr Ser Ser Leu 130 135 140 Ser Thr Ile Asn Tyr Asp Glu Phe Pro Thr Met Val Phe Pro Ser Gly 145 150 155 160 Gln Ile Ser Gln Ala Ser Ala Leu Ala Pro Ala Pro Pro Gln Val Leu 165 170 175 Pro Gln Ala Pro Ala Pro Ala Pro Ala Pro Ala Met Val Ser Ala Leu 180 185 190 Ala Gln Ala Pro Ala Pro Val Pro Val Leu Ala Pro Gly Pro Pro Gln 195 200 205 Ala Val Ala Pro Pro Ala Pro Lys Pro Thr Gln Ala Gly Glu Gly Thr 210 215 220 Leu Ser Glu Ala Leu Leu Gln Leu Gln Phe Asp Asp Glu Asp Leu Gly 225 230 235 240 Ala Leu Leu Gly Asn Ser Thr Asp Pro Ala Val Phe Thr Asp Leu Ala 245 250 255 Ser Val Asp Asn Ser Glu Phe Gln Gln Leu Leu Asn Gln Gly Ile Pro 260 265 270 Val Ala Pro His Thr Thr Glu Pro Met Leu Met Glu Tyr Pro Glu Ala 275 280 285 Ile Thr Arg Leu Val Thr Gly Ala Gln Arg Pro Pro Asp Pro Ala Pro 290 295 300 Ala Pro Leu Gly Ala Pro Gly Leu Pro Asn Gly Leu Leu Ser Gly Asp 305 310 315 320 Glu Asp Phe Ser Ser Ile Ala Asp Met Asp Phe Ser Ala Leu Leu Gly 325 330 335 Ser Gly Ser Gly Ser Arg Asp Ser Arg Glu Gly Met Phe Leu Pro Lys 340 345 350 Pro Glu Ala Gly Ser Ala Ile Ser Asp Val Phe Glu Gly Arg Glu Val 355 360 365 Cys Gln Pro Lys Arg Ile Arg Pro Phe His Pro Pro Gly Ser Pro Trp 370 375 380 Ala Asn Arg Pro Leu Pro Ala Ser Leu Ala Pro Thr Pro Thr Gly Pro 385 390 395 400 Val His Glu Pro Val Gly Ser Leu Thr Pro Ala Pro Val Pro Gln Pro 405 410 415 Leu Asp Pro Ala Pro Ala Val Thr Pro Glu Ala Ser His Leu Leu Glu 420 425 430 Asp Pro Asp Glu Glu Thr Ser Gln Ala Val Lys Ala Leu Arg Glu Met 435 440 445 Ala Asp Thr Val Ile Pro Gln Lys Glu Glu Ala Ala Ile Cys Gly Gln 450 455 460 Met Asp Leu Ser His Pro Pro Pro Arg Gly His Leu Asp Glu Leu Thr 465 470 475 480 Thr Thr Leu Glu Ser Met Thr Glu Asp Leu Asn Leu Asp Ser Pro Leu 485 490 495 Thr Pro Glu Leu Asn Glu Ile Leu Asp Thr Phe Leu Asn Asp Glu Cys 500 505 510 Leu Leu His Ala Met His Ile Ser Thr Gly Leu Ser Ile Phe Asp Thr 515 520 525 Serum Leu Phe 530 <210> 21 <211> 377 <212> PRT <213> Homo sapiens <400> 21 Lys Phe Ser Ala Lys Arg Leu Pro Ser Thr Arg Leu Gly Thr Phe Leu 1 5 10 15 Glu Asn Arg Val Asn Asp Phe Leu Arg Arg Gln Asn His Pro Glu Ser 20 25 30 Gly Glu Val Thr Val Arg Val Val His Ala Ser Asp Lys Thr Val Glu 35 40 45 Val Lys Pro Gly Met Lys Ala Arg Phe Val Asp Ser Gly Glu Met Ala 50 55 60 Glu Ser Phe Pro Tyr Arg Thr Lys Ala Leu Phe Ala Phe Glu Glu Ile 65 70 75 80 Asp Gly Val Asp Leu Cys Phe Phe Gly Met His Val Gln Glu Tyr Gly 85 90 95 Ser Asp Cys Pro Pro Pro Asn Gln Arg Arg Val Tyr Ile Ser Tyr Leu 100 105 110 Asp Ser Val His Phe Phe Arg Pro Lys Cys Leu Arg Thr Ala Val Tyr 115 120 125 His Glu Ile Leu Ile Gly Tyr Leu Glu Tyr Val Lys Lys Leu Gly Tyr 130 135 140 Thr Thr Gly His Ile Trp Ala Cys Pro Pro Ser Glu Gly Asp Asp Tyr 145 150 155 160 Ile Phe His Cys His Pro Pro Asp Gln Lys Ile Pro Lys Pro Lys Arg 165 170 175 Leu Gln Glu Trp Tyr Lys Lys Met Leu Asp Lys Ala Val Ser Glu Arg 180 185 190 Ile Val His Asp Tyr Lys Asp Ile Phe Lys Gln Ala Thr Glu Asp Arg 195 200 205 Leu Thr Ser Ala Lys Glu Leu Pro Tyr Phe Glu Gly Asp Phe Trp Pro 210 215 220 Asn Val Leu Glu Glu Ser Ile Lys Glu Leu Glu Gln Glu Glu Glu Glu 225 230 235 240 Arg Lys Arg Glu Glu Asn Thr Ser Asn Glu Ser Thr Asp Val Thr Lys 245 250 255 Gly Asp Ser Lys Asn Ala Lys Lys Lys Asn Asn Lys Lys Thr Ser Lys 260 265 270 Asn Lys Ser Ser Leu Ser Arg Gly Asn Lys Lys Lys Pro Gly Met Pro 275 280 285 Asn Val Ser Asn Asp Leu Ser Gln Lys Leu Tyr Ala Thr Met Glu Lys 290 295 300 His Lys Glu Val Phe Phe Val Ile Arg Leu Ile Ala Gly Pro Ala Ala 305 310 315 320 Asn Ser Leu Pro Pro Ile Val Asp Pro Asp Pro Leu Ile Pro Cys Asp 325 330 335 Leu Met Asp Gly Arg Asp Ala Phe Leu Thr Leu Ala Arg Asp Lys His 340 345 350 Leu Glu Phe Ser Ser Leu Arg Arg Ala Gln Trp Ser Thr Met Cys Met 355 360 365 Leu Val Glu Leu His Thr Gln Ser Gln 370 375 <210> 22 <211> 2136 <212> PRT <213> Homo sapiens <400> 22 Met Ser Arg Ser Arg His Ala Arg Pro Ser Arg Leu Val Arg Lys Glu 1 5 10 15 Asp Val Asn Lys Lys Lys Asn Ser Gln Leu Arg Lys Thr Thr Lys 20 25 30 Gly Ala Asn Lys Asn Val Ala Ser Val Lys Thr Leu Ser Pro Gly Lys 35 40 45 Leu Lys Gln Leu Ile Gln Glu Arg Asp Val Lys Lys Lys Thr Glu Pro 50 55 60 Lys Pro Pro Val Pro Val Arg Ser Leu Leu Thr Arg Ala Gly Ala Ala 65 70 75 80 Arg Met Asn Leu Asp Arg Thr Glu Val Leu Phe Gln Asn Pro Glu Ser 85 90 95 Leu Thr Cys Asn Gly Phe Thr Met Ala Leu Arg Ser Thr Ser Leu Ser 100 105 110 Arg Arg Leu Ser Gln Pro Pro Leu Val Val Ala Lys Ser Lys Lys Val 115 120 125 Pro Leu Ser Lys Gly Leu Glu Lys Gln His Asp Cys Asp Tyr Lys Ile 130 135 140 Leu Pro Ala Leu Gly Val Lys His Ser Glu Asn Asp Ser Val Pro Met 145 150 155 160 Gln Asp Thr Gln Val Leu Pro Asp Ile Glu Thr Leu Ile Gly Val Gln 165 170 175 Asn Pro Ser Leu Leu Lys Gly Lys Ser Gln Glu Thr Thr Gln Phe Trp 180 185 190 Ser Gln Arg Val Glu Asp Ser Lys Ile Asn Ile Pro Thr His Ser Gly 195 200 205 Pro Ala Ala Glu Ile Leu Pro Gly Pro Leu Glu Gly Thr Arg Cys Gly 210 215 220 Glu Gly Leu Phe Ser Glu Glu Thr Leu Asn Asp Thr Ser Gly Ser Pro 225 230 235 240 Lys Met Phe Ala Gln Asp Thr Val Cys Ala Pro Phe Pro Gln Arg Ala 245 250 255 Thr Pro Lys Val Thr Ser Gln Gly Asn Pro Ser Ile Gln Leu Glu Glu 260 265 270 Leu Gly Ser Arg Val Glu Ser Leu Lys Leu Ser Asp Ser Tyr Leu Asp 275 280 285 Pro Ile Lys Ser Glu His Asp Cys Tyr Pro Thr Ser Ser Leu Asn Lys 290 295 300 Val Ile Pro Asp Leu Asn Leu Arg Asn Cys Leu Ala Leu Gly Gly Ser 305 310 315 320 Thr Ser Pro Thr Ser Val Ile Lys Phe Leu Leu Ala Gly Ser Lys Gln 325 330 335 Ala Thr Leu Gly Ala Lys Pro Asp His Gln Glu Ala Phe Glu Ala Thr 340 345 350 Ala Asn Gln Gln Glu Val Ser Asp Thr Thr Ser Phe Leu Gly Gln Ala 355 360 365 Phe Gly Ala Ile Pro His Gln Trp Glu Leu Pro Gly Ala Asp Pro Val 370 375 380 His Gly Glu Ala Leu Gly Glu Thr Pro Asp Leu Pro Glu Ile Pro Gly 385 390 395 400 Ala Ile Pro Val Gln Gly Glu Val Phe Gly Thr Ile Leu Asp Gln Gln 405 410 415 Glu Thr Leu Gly Met Ser Gly Ser Val Val Pro Asp Leu Pro Val Phe 420 425 430 Leu Pro Val Pro Pro Asn Pro Ile Ala Thr Phe Asn Ala Pro Ser Lys 435 440 445 Trp Pro Glu Pro Gln Ser Thr Val Ser Tyr Gly Leu Ala Val Gln Gly 450 455 460 Ala Ile Gln Ile Leu Pro Leu Gly Ser Gly His Thr Pro Gln Ser Ser 465 470 475 480 Ser Asn Ser Glu Lys Asn Ser Leu Pro Pro Val Met Ala Ile Ser Asn 485 490 495 Val Glu Asn Glu Lys Gln Val His Ile Ser Phe Leu Pro Ala Asn Thr 500 505 510 Gln Gly Phe Pro Leu Ala Pro Glu Arg Gly Leu Phe His Ala Ser Leu 515 520 525 Gly Ile Ala Gln Leu Ser Gln Ala Gly Pro Ser Lys Ser Asp Arg Gly 530 535 540 Ser Ser Gln Val Ser Val Thr Ser Thr Val His Val Val Asn Thr Thr 545 550 555 560 Val Val Thr Met Pro Val Pro Met Val Ser Thr Ser Ser Ser Ser Tyr 565 570 575 Thr Thr Leu Leu Pro Thr Leu Glu Lys Lys Lys Arg Lys Arg Cys Gly 580 585 590 Val Cys Glu Pro Cys Gln Gln Lys Thr Asn Cys Gly Glu Cys Thr Tyr 595 600 605 Cys Lys Asn Arg Lys Asn Ser His Gln Ile Cys Lys Lys Arg Lys Cys 610 615 620 Glu Glu Leu Lys Lys Lys Pro Ser Val Val Val Pro Leu Glu Val Ile 625 630 635 640 Lys Glu Asn Lys Arg Pro Gln Arg Glu Lys Lys Pro Lys Val Leu Lys 645 650 655 Ala Asp Phe Asp Asn Lys Pro Val Asn Gly Pro Lys Ser Glu Ser Met 660 665 670 Asp Tyr Ser Arg Cys Gly His Gly Glu Glu Gln Lys Leu Glu Leu Asn 675 680 685 Pro His Thr Val Glu Asn Val Thr Lys Asn Glu Asp Ser Met Thr Gly 690 695 700 Ile Glu Val Glu Lys Trp Thr Gln Asn Lys Lys Ser Gln Leu Thr Asp 705 710 715 720 His Val Lys Gly Asp Phe Ser Ala Asn Val Pro Glu Ala Glu Lys Ser 725 730 735 Lys Asn Ser Glu Val Asp Lys Lys Arg Thr Lys Ser Pro Lys Leu Phe 740 745 750 Val Gln Thr Val Arg Asn Gly Ile Lys His Val His Cys Leu Pro Ala 755 760 765 Glu Thr Asn Val Ser Phe Lys Lys Phe Asn Ile Glu Glu Phe Gly Lys 770 775 780 Thr Leu Glu Asn Asn Ser Tyr Lys Phe Leu Lys Asp Thr Ala Asn His 785 790 795 800 Lys Asn Ala Met Ser Ser Val Ala Thr Asp Met Ser Cys Asp His Leu 805 810 815 Lys Gly Arg Ser Asn Val Leu Val Phe Gln Gln Pro Gly Phe Asn Cys 820 825 830 Ser Ser Ile Pro His Ser Ser His Ser Ile Ile Asn His His Ala Ser 835 840 845 Ile His Asn Glu Gly Asp Gln Pro Lys Thr Pro Glu Asn Ile Pro Ser 850 855 860 Lys Glu Pro Lys Asp Gly Ser Pro Val Gln Pro Ser Leu Leu Ser Leu 865 870 875 880 Met Lys Asp Arg Arg Leu Thr Leu Glu Gln Val Val Ala Ile Glu Ala 885 890 895 Leu Thr Gln Leu Ser Glu Ala Pro Ser Glu Asn Ser Ser Pro Ser Lys 900 905 910 Ser Glu Lys Asp Glu Glu Ser Glu Gln Arg Thr Ala Ser Leu Leu Asn 915 920 925 Ser Cys Lys Ala Ile Leu Tyr Thr Val Arg Lys Asp Leu Gln Asp Pro 930 935 940 Asn Leu Gln Gly Glu Pro Pro Lys Leu Asn His Cys Pro Ser Leu Glu 945 950 955 960 Lys Gln Ser Ser Cys Asn Thr Val Val Phe Asn Gly Gln Thr Thr Thr 965 970 975 Leu Ser Asn Ser His Ile Asn Ser Ala Thr Asn Gln Ala Ser Thr Lys 980 985 990 Ser His Glu Tyr Ser Lys Val Thr Asn Ser Leu Ser Leu Phe Ile Pro 995 1000 1005 Lys Ser Asn Ser Ser Lys Ile Asp Thr Asn Lys Ser Ile Ala Gln 1010 1015 1020 Gly Ile Ile Thr Leu Asp Asn Cys Ser Asn Asp Leu His Gln Leu 1025 1030 1035 Pro Pro Arg Asn Asn Glu Val Glu Tyr Cys Asn Gln Leu Leu Asp 1040 1045 1050 Ser Ser Lys Lys Leu Asp Ser Asp Asp Leu Ser Cys Gln Asp Ala 1055 1060 1065 Thr His Thr Gln Ile Glu Glu Asp Val Ala Thr Gln Leu Thr Gln 1070 1075 1080 Leu Ala Ser Ile Ile Lys Ile Asn Tyr Ile Lys Pro Glu Asp Lys 1085 1090 1095 Lys Val Glu Ser Thr Pro Thr Ser Leu Val Thr Cys Asn Val Gln 1100 1105 1110 Gln Lys Tyr Asn Gln Glu Lys Gly Thr Ile Gln Gln Lys Pro Pro 1115 1120 1125 Ser Ser Val His Asn Asn His Gly Ser Ser Leu Thr Lys Gln Lys 1130 1135 1140 Asn Pro Thr Gln Lys Lys Thr Lys Ser Thr Pro Ser Arg Asp Arg 1145 1150 1155 Arg Lys Lys Lys Pro Thr Val Val Ser Tyr Gln Glu Asn Asp Arg 1160 1165 1170 Gln Lys Trp Glu Lys Leu Ser Tyr Met Tyr Gly Thr Ile Cys Asp 1175 1180 1185 Ile Trp Ile Ala Ser Lys Phe Gln Asn Phe Gly Gln Phe Cys Pro 1190 1195 1200 His Asp Phe Pro Thr Val Phe Gly Lys Ile Ser Ser Ser Thr Lys 1205 1210 1215 Ile Trp Lys Pro Leu Ala Gln Thr Arg Ser Ile Met Gln Pro Lys 1220 1225 1230 Thr Val Phe Pro Pro Leu Thr Gln Ile Lys Leu Gln Arg Tyr Pro 1235 1240 1245 Glu Ser Ala Glu Glu Lys Val Lys Val Glu Pro Leu Asp Ser Leu 1250 1255 1260 Ser Leu Phe His Leu Lys Thr Glu Ser Asn Gly Lys Ala Phe Thr 1265 1270 1275 Asp Lys Ala Tyr Asn Ser Gln Val Gln Leu Thr Val Asn Ala Asn 1280 1285 1290 Gln Lys Ala His Pro Leu Thr Gln Pro Ser Ser Pro Pro Asn Gln 1295 1300 1305 Cys Ala Asn Val Met Ala Gly Asp Asp Gln Ile Arg Phe Gln Gln 1310 1315 1320 Val Val Lys Glu Gln Leu Met His Gln Arg Leu Pro Thr Leu Pro 1325 1330 1335 Gly Ile Ser His Glu Thr Pro Leu Pro Glu Ser Ala Leu Thr Leu 1340 1345 1350 Arg Asn Val Asn Val Val Cys Ser Gly Gly Ile Thr Val Val Ser 1355 1360 1365 Thr Lys Ser Glu Glu Glu Val Cys Ser Ser Ser Phe Gly Thr Ser 1370 1375 1380 Glu Phe Ser Thr Val Asp Ser Ala Gln Lys Asn Phe Asn Asp Tyr 1385 1390 1395 Ala Met Asn Phe Phe Thr Asn Pro Thr Lys Asn Leu Val Ser Ile 1400 1405 1410 Thr Lys Asp Ser Glu Leu Pro Thr Cys Ser Cys Leu Asp Arg Val 1415 1420 1425 Ile Gln Lys Asp Lys Gly Pro Tyr Tyr Thr His Leu Gly Ala Gly 1430 1435 1440 Pro Ser Val Ala Ala Val Arg Glu Ile Met Glu Asn Arg Tyr Gly 1445 1450 1455 Gln Lys Gly Asn Ala Ile Arg Ile Glu Ile Val Val Tyr Thr Gly 1460 1465 1470 Lys Glu Gly Lys Ser Ser His Gly Cys Pro Ile Ala Lys Trp Val 1475 1480 1485 Leu Arg Arg Ser Ser Asp Glu Glu Lys Val Leu Cys Leu Val Arg 1490 1495 1500 Gln Arg Thr Gly His His Cys Pro Thr Ala Val Met Val Val Leu 1505 1510 1515 Ile Met Val Trp Asp Gly Ile Pro Leu Pro Met Ala Asp Arg Leu 1520 1525 1530 Tyr Thr Glu Leu Thr Glu Asn Leu Lys Ser Tyr Asn Gly His Pro 1535 1540 1545 Thr Asp Arg Arg Cys Thr Leu Asn Glu Asn Arg Thr Cys Thr Cys 1550 1555 1560 Gln Gly Ile Asp Pro Glu Thr Cys Gly Ala Ser Phe Ser Phe Gly 1565 1570 1575 Cys Ser Trp Ser Met Tyr Phe Asn Gly Cys Lys Phe Gly Arg Ser 1580 1585 1590 Pro Ser Pro Arg Arg Phe Arg Ile Asp Pro Ser Ser Pro Leu His 1595 1600 1605 Glu Lys Asn Leu Glu Asp Asn Leu Gln Ser Leu Ala Thr Arg Leu 1610 1615 1620 Ala Pro Ile Tyr Lys Gln Tyr Ala Pro Val Ala Tyr Gln Asn Gln 1625 1630 1635 Val Glu Tyr Glu Asn Val Ala Arg Glu Cys Arg Leu Gly Ser Lys 1640 1645 1650 Glu Gly Arg Pro Phe Ser Gly Val Thr Ala Cys Leu Asp Phe Cys 1655 1660 1665 Ala His Pro His Arg Asp Ile His Asn Met Asn Asn Gly Ser Thr 1670 1675 1680 Val Val Cys Thr Leu Thr Arg Glu Asp Asn Arg Ser Leu Gly Val 1685 1690 1695 Ile Pro Gln Asp Glu Gln Leu His Val Leu Pro Leu Tyr Lys Leu 1700 1705 1710 Ser Asp Thr Asp Glu Phe Gly Ser Lys Glu Gly Met Glu Ala Lys 1715 1720 1725 Ile Lys Ser Gly Ala Ile Glu Val Leu Ala Pro Arg Arg Lys Lys 1730 1735 1740 Arg Thr Cys Phe Thr Gln Pro Val Pro Arg Ser Gly Lys Lys Arg 1745 1750 1755 Ala Ala Met Met Thr Glu Val Leu Ala His Lys Ile Arg Ala Val 1760 1765 1770 Glu Lys Lys Pro Ile Pro Arg Ile Lys Arg Lys Asn Asn Ser Thr 1775 1780 1785 Thr Thr Asn Asn Ser Lys Pro Ser Ser Leu Pro Thr Leu Gly Ser 1790 1795 1800 Asn Thr Glu Thr Val Gln Pro Glu Val Lys Ser Glu Thr Glu Pro 1805 1810 1815 His Phe Ile Leu Lys Ser Ser Asp Asn Thr Lys Thr Tyr Ser Leu 1820 1825 1830 Met Pro Ser Ala Pro His Pro Val Lys Glu Ala Ser Pro Gly Phe 1835 1840 1845 Ser Trp Ser Pro Lys Thr Ala Ser Ala Thr Pro Ala Pro Leu Lys 1850 1855 1860 Asn Asp Ala Thr Ala Ser Cys Gly Phe Ser Glu Arg Ser Ser Thr 1865 1870 1875 Pro His Cys Thr Met Pro Ser Gly Arg Leu Ser Gly Ala Asn Ala 1880 1885 1890 Ala Ala Ala Asp Gly Pro Gly Ile Ser Gln Leu Gly Glu Val Ala 1895 1900 1905 Pro Leu Pro Thr Leu Ser Ala Pro Val Met Glu Pro Leu Ile Asn 1910 1915 1920 Ser Glu Pro Ser Thr Gly Val Thr Glu Pro Leu Thr Pro His Gln 1925 1930 1935 Pro Asn His Gln Pro Ser Phe Leu Thr Ser Pro Gln Asp Leu Ala 1940 1945 1950 Ser Ser Pro Met Glu Glu Asp Glu Gln His Ser Glu Ala Asp Glu 1955 1960 1965 Pro Pro Ser Asp Glu Pro Leu Ser Asp Asp Pro Leu Ser Pro Ala 1970 1975 1980 Glu Glu Lys Leu Pro His Ile Asp Glu Tyr Trp Ser Asp Ser Glu 1985 1990 1995 His Ile Phe Leu Asp Ala Asn Ile Gly Gly Val Ala Ile Ala Pro 2000 2005 2010 Ala His Gly Ser Val Leu Ile Glu Cys Ala Arg Arg Glu Leu His 2015 2020 2025 Ala Thr Thr Pro Val Glu His Pro Asn Arg Asn His Pro Thr Arg 2030 2035 2040 Leu Ser Leu Val Phe Tyr Gln His Lys Asn Leu Asn Lys Pro Gln 2045 2050 2055 His Gly Phe Glu Leu Asn Lys Ile Lys Phe Glu Ala Lys Glu Ala 2060 2065 2070 Lys Asn Lys Lys Met Lys Ala Ser Glu Gln Lys Asp Gln Ala Ala 2075 2080 2085 Asn Glu Gly Pro Glu Gln Ser Ser Glu Val Asn Glu Leu Asn Gln 2090 2095 2100 Ile Pro Ser His Lys Ala Leu Thr Leu Thr His Asp Asn Val Val 2105 2110 2115 Thr Val Ser Pro Tyr Ala Leu Thr His Val Ala Gly Pro Tyr Asn 2120 2125 2130 His Trp Val 2135 <210> 23 <211> 552 <212> PRT <213> Mus musculus <400> 23 Gly Met Asp Val Thr Leu Leu Glu Ala Arg Asp Arg Val Gly Gly Arg 1 5 10 15 Val Ala Thr Phe Arg Lys Gly Asn Tyr Val Ala Asp Leu Gly Ala Met 20 25 30 Val Val Thr Gly Leu Gly Gly Asn Pro Met Ala Val Val Ser Lys Gln 35 40 45 Val Asn Met Glu Leu Ala Lys Ile Lys Gln Lys Cys Pro Leu Tyr Glu 50 55 60 Ala Asn Gly Gln Ala Val Pro Lys Glu Lys Asp Glu Met Val Glu Gln 65 70 75 80 Glu Phe Asn Arg Leu Leu Glu Ala Thr Ser Tyr Leu Ser His Gln Leu 85 90 95 Asp Phe Asn Val Leu Asn Asn Lys Pro Val Ser Leu Gly Gln Ala Leu 100 105 110 Glu Val Val Ile Gln Leu Gln Glu Lys His Val Lys Asp Glu Gln Ile 115 120 125 Glu His Trp Lys Lys Ile Val Lys Thr Gln Glu Glu Leu Lys Glu Leu 130 135 140 Leu Asn Lys Met Val Asn Leu Lys Glu Lys Ile Lys Glu Leu His Gln 145 150 155 160 Gln Tyr Lys Glu Ala Ser Glu Val Lys Pro Pro Arg Asp Ile Thr Ala 165 170 175 Glu Phe Leu Val Lys Ser Lys His Arg Asp Leu Thr Ala Leu Cys Lys 180 185 190 Glu Tyr Asp Glu Leu Ala Glu Thr Gln Gly Lys Leu Glu Glu Lys Leu 195 200 205 Gln Glu Leu Glu Ala Asn Pro Pro Ser Asp Val Tyr Leu Ser Ser Arg 210 215 220 Asp Arg Gln Ile Leu Asp Trp His Phe Ala Asn Leu Glu Phe Ala Asn 225 230 235 240 Ala Thr Pro Leu Ser Thr Leu Ser Leu Lys His Trp Asp Gln Asp Asp 245 250 255 Asp Phe Glu Phe Thr Gly Ser His Leu Thr Val Arg Asn Gly Tyr Ser 260 265 270 Cys Val Pro Val Ala Leu Ala Glu Gly Leu Asp Ile Lys Leu Asn Thr 275 280 285 Ala Val Arg Gln Val Arg Tyr Thr Ala Ser Gly Cys Glu Val Ile Ala 290 295 300 Val Asn Thr Arg Ser Thr Ser Gln Thr Phe Ile Tyr Lys Cys Asp Ala 305 310 315 320 Val Leu Cys Thr Leu Pro Leu Gly Val Leu Lys Gln Gln Pro Pro Ala 325 330 335 Val Gln Phe Val Pro Pro Leu Pro Glu Trp Lys Thr Ser Ala Val Gln 340 345 350 Arg Met Gly Phe Gly Asn Leu Asn Lys Val Val Leu Cys Phe Asp Arg 355 360 365 Val Phe Trp Asp Pro Ser Val Asn Leu Phe Gly His Val Gly Ser Thr 370 375 380 Thr Ala Ser Arg Gly Glu Leu Phe Leu Phe Trp Asn Leu Tyr Lys Ala 385 390 395 400 Pro Ile Leu Leu Ala Leu Val Ala Gly Glu Ala Ala Gly Ile Met Glu 405 410 415 Asn Ile Ser Asp Asp Val Ile Val Gly Arg Cys Leu Ala Ile Leu Lys 420 425 430 Gly Ile Phe Gly Ser Ser Ala Val Pro Gln Pro Lys Glu Thr Val Val 435 440 445 Ser Arg Trp Arg Ala Asp Pro Trp Ala Arg Gly Ser Tyr Ser Tyr Val 450 455 460 Ala Ala Gly Ser Ser Gly Asn Asp Tyr Asp Leu Met Ala Gln Pro Ile 465 470 475 480 Thr Pro Gly Pro Ser Ile Pro Gly Ala Pro Gln Pro Ile Pro Arg Leu 485 490 495 Phe Phe Ala Gly Glu His Thr Ile Arg Asn Tyr Pro Ala Thr Val His 500 505 510 Gly Ala Leu Leu Ser Gly Leu Arg Glu Ala Gly Arg Ile Ala Asp Gln 515 520 525 Phe Leu Gly Ala Met Tyr Thr Leu Pro Arg Gln Ala Thr Pro Gly Val 530 535 540 Pro Ala Gln Gln Ser Pro Ser Met 545 550 <210> 24 <211> 191 <212> PRT <213> Homo sapiens <400> 24 Met Gly Gly Ser Gly Ser Arg Leu Ser Lys Glu Leu Leu Ala Glu Tyr 1 5 10 15 Gln Asp Leu Thr Phe Leu Thr Lys Gln Glu Ile Leu Leu Ala His Arg 20 25 30 Arg Phe Cys Glu Leu Leu Pro Gln Glu Gln Arg Ser Val Glu Ser Ser 35 40 45 Leu Arg Ala Gln Val Pro Phe Glu Gln Ile Leu Ser Leu Pro Glu Leu 50 55 60 Lys Ala Asn Pro Phe Lys Glu Arg Ile Cys Arg Val Phe Ser Thr Ser 65 70 75 80 Pro Ala Lys Asp Ser Leu Ser Phe Glu Asp Phe Leu Asp Leu Leu Ser 85 90 95 Val Phe Ser Asp Thr Ala Thr Pro Asp Ile Lys Ser His Tyr Ala Phe 100 105 110 Arg Ile Phe Asp Phe Asp Asp Asp Gly Thr Leu Asn Arg Glu Asp Leu 115 120 125 Ser Arg Leu Val Asn Cys Leu Thr Gly Glu Gly Glu Asp Thr Arg Leu 130 135 140 Ser Ala Ser Glu Met Lys Gln Leu Ile Asp Asn Ile Leu Glu Glu Ser 145 150 155 160 Asp Ile Asp Arg Asp Gly Thr Ile Asn Leu Ser Glu Phe Gln His Val 165 170 175 Ile Ser Arg Ser Pro Asp Phe Ala Ser Ser Phe Lys Ile Val Leu 180 185 190 <210> 25 <211> 654 <212> PRT <213> Homo sapiens <400> 25 Met Asn Gln Pro Gln Arg Met Ala Pro Val Gly Thr Asp Lys Glu Leu 1 5 10 15 Ser Asp Leu Leu Asp Phe Ser Met Met Phe Pro Leu Pro Val Thr Asn 20 25 30 Gly Lys Gly Arg Pro Ala Ser Leu Ala Gly Ala Gln Phe Gly Gly Ser 35 40 45 Gly Leu Glu Asp Arg Pro Ser Ser Gly Ser Trp Gly Ser Gly Asp Gln 50 55 60 Ser Ser Ser Ser Phe Asp Pro Ser Arg Thr Phe Ser Glu Gly Thr His 65 70 75 80 Phe Thr Glu Ser His Ser Ser Leu Ser Ser Ser Thr Phe Leu Gly Pro 85 90 95 Gly Leu Gly Gly Lys Ser Gly Glu Arg Gly Ala Tyr Ala Ser Phe Gly 100 105 110 Arg Asp Ala Gly Val Gly Gly Leu Thr Gln Ala Gly Phe Leu Ser Gly 115 120 125 Glu Leu Ala Leu Asn Ser Pro Gly Pro Leu Ser Pro Ser Gly Met Lys 130 135 140 Gly Thr Ser Gln Tyr Tyr Pro Ser Tyr Ser Gly Ser Ser Arg Arg Arg 145 150 155 160 Ala Ala Asp Gly Ser Leu Asp Thr Gln Pro Lys Lys Val Arg Lys Val 165 170 175 Pro Pro Gly Leu Pro Ser Ser Val Tyr Pro Pro Ser Ser Gly Glu Asp 180 185 190 Tyr Gly Arg Asp Ala Thr Ala Tyr Pro Ser Ala Lys Thr Pro Ser Ser 195 200 205 Thr Tyr Pro Ala Pro Phe Tyr Val Ala Asp Gly Ser Leu His Pro Ser 210 215 220 Ala Glu Leu Trp Ser Pro Pro Gly Gln Ala Gly Phe Gly Pro Met Leu 225 230 235 240 Gly Gly Gly Ser Ser Pro Leu Pro Leu Pro Pro Gly Ser Gly Pro Val 245 250 255 Gly Ser Ser Gly Ser Ser Ser Thr Phe Gly Gly Leu His Gln His Glu 260 265 270 Arg Met Gly Tyr Gln Leu His Gly Ala Glu Val Asn Gly Gly Leu Pro 275 280 285 Ser Ala Ser Ser Phe Ser Ser Ala Pro Gly Ala Thr Tyr Gly Gly Val 290 295 300 Ser Ser His Thr Pro Pro Val Ser Gly Ala Asp Ser Leu Leu Gly Ser 305 310 315 320 Arg Gly Thr Thr Ala Gly Ser Ser Gly Asp Ala Leu Gly Lys Ala Leu 325 330 335 Ala Ser Ile Tyr Ser Pro Asp His Ser Ser Asn Asn Phe Ser Ser Ser 340 345 350 Pro Ser Thr Pro Val Gly Ser Pro Gln Gly Leu Ala Gly Thr Ser Gln 355 360 365 Trp Pro Arg Ala Gly Ala Pro Gly Ala Leu Ser Pro Ser Tyr Asp Gly 370 375 380 Gly Leu His Gly Leu Gln Ser Lys Ile Glu Asp His Leu Asp Glu Ala 385 390 395 400 Ile His Val Leu Arg Ser His Ala Val Gly Thr Ala Gly Asp Met His 405 410 415 Thr Leu Leu Pro Gly His Gly Ala Leu Ala Ser Gly Phe Thr Gly Pro 420 425 430 Met Ser Leu Gly Gly Arg His Ala Gly Leu Val Gly Gly Ser His Pro 435 440 445 Glu Asp Gly Leu Ala Gly Ser Thr Ser Leu Met His Asn His Ala Ala 450 455 460 Leu Pro Ser Gln Pro Gly Thr Leu Pro Asp Leu Ser Arg Pro Pro Asp 465 470 475 480 Ser Tyr Ser Gly Leu Gly Arg Ala Gly Ala Thr Ala Ala Ala Ser Glu 485 490 495 Ile Lys Arg Glu Glu Lys Glu Asp Glu Glu Asn Thr Ser Ala Ala Asp 500 505 510 His Ser Glu Glu Glu Lys Lys Glu Leu Lys Ala Pro Arg Ala Arg Thr 515 520 525 Ser Pro Asp Glu Asp Glu Asp Asp Leu Leu Pro Pro Glu Gln Lys Ala 530 535 540 Glu Arg Glu Lys Glu Arg Arg Val Ala Asn Asn Ala Arg Glu Arg Leu 545 550 555 560 Arg Val Arg Asp Ile Asn Glu Ala Phe Lys Glu Leu Gly Arg Met Cys 565 570 575 Gln Leu His Leu Asn Ser Glu Lys Pro Gln Thr Lys Leu Leu Ile Leu 580 585 590 His Gln Ala Val Ser Val Ile Leu Asn Leu Glu Gln Gln Val Arg Glu 595 600 605 Arg Asn Leu Asn Pro Lys Ala Ala Cys Leu Lys Arg Arg Glu Glu Glu 610 615 620 Lys Val Ser Gly Val Val Gly Asp Pro Gln Met Val Leu Ser Ala Pro 625 630 635 640 His Pro Gly Leu Ser Glu Ala His Asn Pro Ala Gly His Met 645 650 <210> 26 <211> 1663 <212> PRT <213> Homo sapiens <400> 26 Met Gly Pro Thr Ser Gly Pro Ser Leu Leu Leu Leu Leu Thr His 1 5 10 15 Leu Pro Leu Ala Leu Gly Ser Pro Met Tyr Ser Ile Ile Thr Pro Asn 20 25 30 Ile Leu Arg Leu Glu Ser Glu Glu Thr Met Val Leu Glu Ala His Asp 35 40 45 Ala Gln Gly Asp Val Pro Val Thr Val Thr Val His Asp Phe Pro Gly 50 55 60 Lys Lys Leu Val Leu Ser Ser Glu Lys Thr Val Leu Thr Pro Ala Thr 65 70 75 80 Asn His Met Gly Asn Val Thr Phe Thr Ile Pro Ala Asn Arg Glu Phe 85 90 95 Lys Ser Glu Lys Gly Arg Asn Lys Phe Val Thr Val Gln Ala Thr Phe 100 105 110 Gly Thr Gln Val Val Glu Lys Val Val Leu Val Ser Leu Gln Ser Gly 115 120 125 Tyr Leu Phe Ile Gln Thr Asp Lys Thr Ile Tyr Thr Pro Gly Ser Thr 130 135 140 Val Leu Tyr Arg Ile Phe Thr Val Asn His Lys Leu Leu Pro Val Gly 145 150 155 160 Arg Thr Val Met Val Asn Ile Glu Asn Pro Glu Gly Ile Pro Val Lys 165 170 175 Gln Asp Ser Leu Ser Ser Gln Asn Gln Leu Gly Val Leu Pro Leu Ser 180 185 190 Trp Asp Ile Pro Glu Leu Val Asn Met Gly Gln Trp Lys Ile Arg Ala 195 200 205 Tyr Tyr Glu Asn Ser Pro Gln Gln Val Phe Ser Thr Glu Phe Glu Val 210 215 220 Lys Glu Tyr Val Leu Pro Ser Phe Glu Val Ile Val Glu Pro Thr Glu 225 230 235 240 Lys Phe Tyr Tyr Ile Tyr Asn Glu Lys Gly Leu Glu Val Thr Ile Thr 245 250 255 Ala Arg Phe Leu Tyr Gly Lys Lys Val Glu Gly Thr Ala Phe Val Ile 260 265 270 Phe Gly Ile Gln Asp Gly Glu Gln Arg Ile Ser Leu Pro Glu Ser Leu 275 280 285 Lys Arg Ile Pro Ile Glu Asp Gly Ser Gly Glu Val Val Leu Ser Arg 290 295 300 Lys Val Leu Leu Asp Gly Val Gln Asn Pro Arg Ala Glu Asp Leu Val 305 310 315 320 Gly Lys Ser Leu Tyr Val Ser Ala Thr Val Ile Leu His Ser Gly Ser 325 330 335 Asp Met Val Gln Ala Glu Arg Ser Gly Ile Pro Ile Val Thr Ser Pro 340 345 350 Tyr Gln Ile His Phe Thr Lys Thr Pro Lys Tyr Phe Lys Pro Gly Met 355 360 365 Pro Phe Asp Leu Met Val Phe Val Thr Asn Pro Asp Gly Ser Pro Ala 370 375 380 Tyr Arg Val Pro Val Ala Val Gln Gly Glu Asp Thr Val Gln Ser Leu 385 390 395 400 Thr Gln Gly Asp Gly Val Ala Lys Leu Ser Ile Asn Thr His Pro Ser 405 410 415 Gln Lys Pro Leu Ser Ile Thr Val Arg Thr Lys Lys Gln Glu Leu Ser 420 425 430 Glu Ala Glu Gln Ala Thr Arg Thr Met Gln Ala Leu Pro Tyr Ser Thr 435 440 445 Val Gly Asn Ser Asn Asn Tyr Leu His Leu Ser Val Leu Arg Thr Glu 450 455 460 Leu Arg Pro Gly Glu Thr Leu Asn Val Asn Phe Leu Leu Arg Met Asp 465 470 475 480 Arg Ala His Glu Ala Lys Ile Arg Tyr Tyr Thr Tyr Leu Ile Met Asn 485 490 495 Lys Gly Arg Leu Leu Lys Ala Gly Arg Gln Val Arg Glu Pro Gly Gln 500 505 510 Asp Leu Val Val Leu Pro Leu Ser Ile Thr Thr Asp Phe Ile Pro Ser 515 520 525 Phe Arg Leu Val Ala Tyr Tyr Thr Leu Ile Gly Ala Ser Gly Gln Arg 530 535 540 Glu Val Val Ala Asp Ser Val Trp Val Asp Val Lys Asp Ser Cys Val 545 550 555 560 Gly Ser Leu Val Val Lys Ser Gly Gln Ser Glu Asp Arg Gln Pro Val 565 570 575 Pro Gly Gln Gln Met Thr Leu Lys Ile Glu Gly Asp His Gly Ala Arg 580 585 590 Val Val Leu Val Ala Val Asp Lys Gly Val Phe Val Leu Asn Lys Lys 595 600 605 Asn Lys Leu Thr Gln Ser Lys Ile Trp Asp Val Val Glu Lys Ala Asp 610 615 620 Ile Gly Cys Thr Pro Gly Ser Gly Lys Asp Tyr Ala Gly Val Phe Ser 625 630 635 640 Asp Ala Gly Leu Thr Phe Thr Ser Ser Ser Gly Gln Gln Thr Ala Gln 645 650 655 Arg Ala Glu Leu Gln Cys Pro Gln Pro Ala Ala Arg Arg Arg Arg Ser 660 665 670 Val Gln Leu Thr Glu Lys Arg Met Asp Lys Val Gly Lys Tyr Pro Lys 675 680 685 Glu Leu Arg Lys Cys Cys Glu Asp Gly Met Arg Glu Asn Pro Met Arg 690 695 700 Phe Ser Cys Gln Arg Arg Thr Arg Phe Ile Ser Leu Gly Glu Ala Cys 705 710 715 720 Lys Lys Val Phe Leu Asp Cys Cys Asn Tyr Ile Thr Glu Leu Arg Arg 725 730 735 Gln His Ala Arg Ala Ser His Leu Gly Leu Ala Arg Ser Asn Leu Asp 740 745 750 Glu Asp Ile Ile Ala Glu Glu Asn Ile Val Ser Arg Ser Glu Phe Pro 755 760 765 Glu Ser Trp Leu Trp Asn Val Glu Asp Leu Lys Glu Pro Pro Lys Asn 770 775 780 Gly Ile Ser Thr Lys Leu Met Asn Ile Phe Leu Lys Asp Ser Ile Thr 785 790 795 800 Thr Trp Glu Ile Leu Ala Val Ser Met Ser Asp Lys Lys Gly Ile Cys 805 810 815 Val Ala Asp Pro Phe Glu Val Thr Val Met Gln Asp Phe Phe Ile Asp 820 825 830 Leu Arg Leu Pro Tyr Ser Val Val Arg Asn Glu Gln Val Glu Ile Arg 835 840 845 Ala Val Leu Tyr Asn Tyr Arg Gln Asn Gln Glu Leu Lys Val Arg Val 850 855 860 Glu Leu Leu His Asn Pro Ala Phe Cys Ser Leu Ala Thr Thr Lys Arg 865 870 875 880 Arg His Gln Gln Thr Val Thr Ile Pro Pro Lys Ser Ser Leu Ser Val 885 890 895 Pro Tyr Val Ile Val Pro Leu Lys Thr Gly Leu Gln Glu Val Glu Val 900 905 910 Lys Ala Ala Val Tyr His His Phe Ile Ser Asp Gly Val Arg Lys Ser 915 920 925 Leu Lys Val Val Pro Glu Gly Ile Arg Met Asn Lys Thr Val Ala Val 930 935 940 Arg Thr Leu Asp Pro Glu Arg Leu Gly Arg Glu Gly Val Gln Lys Glu 945 950 955 960 Asp Ile Pro Pro Ala Asp Leu Ser Asp Gln Val Pro Asp Thr Glu Ser 965 970 975 Glu Thr Arg Ile Leu Leu Gln Gly Thr Pro Val Ala Gln Met Thr Glu 980 985 990 Asp Ala Val Asp Ala Glu Arg Leu Lys His Leu Ile Val Thr Pro Ser 995 1000 1005 Gly Cys Gly Glu Gln Asn Met Ile Gly Met Thr Pro Thr Val Ile 1010 1015 1020 Ala Val His Tyr Leu Asp Glu Thr Glu Gln Trp Glu Lys Phe Gly 1025 1030 1035 Leu Glu Lys Arg Gln Gly Ala Leu Glu Leu Ile Lys Lys Gly Tyr 1040 1045 1050 Thr Gln Gln Leu Ala Phe Arg Gln Pro Ser Ser Ala Phe Ala Ala 1055 1060 1065 Phe Val Lys Arg Ala Pro Ser Thr Trp Leu Thr Ala Tyr Val Val 1070 1075 1080 Lys Val Phe Ser Leu Ala Val Asn Leu Ile Ala Ile Asp Ser Gln 1085 1090 1095 Val Leu Cys Gly Ala Val Lys Trp Leu Ile Leu Glu Lys Gln Lys 1100 1105 1110 Pro Asp Gly Val Phe Gln Glu Asp Ala Pro Val Ile His Gln Glu 1115 1120 1125 Met Ile Gly Gly Leu Arg Asn Asn Asn Glu Lys Asp Met Ala Leu 1130 1135 1140 Thr Ala Phe Val Leu Ile Ser Leu Gln Glu Ala Lys Asp Ile Cys 1145 1150 1155 Glu Glu Gln Val Asn Ser Leu Pro Gly Ser Ile Thr Lys Ala Gly 1160 1165 1170 Asp Phe Leu Glu Ala Asn Tyr Met Asn Leu Gln Arg Ser Tyr Thr 1175 1180 1185 Val Ala Ile Ala Gly Tyr Ala Leu Ala Gln Met Gly Arg Leu Lys 1190 1195 1200 Gly Pro Leu Leu Asn Lys Phe Leu Thr Thr Ala Lys Asp Lys Asn 1205 1210 1215 Arg Trp Glu Asp Pro Gly Lys Gln Leu Tyr Asn Val Glu Ala Thr 1220 1225 1230 Ser Tyr Ala Leu Leu Ala Leu Leu Gln Leu Lys Asp Phe Asp Phe 1235 1240 1245 Val Pro Pro Val Val Arg Trp Leu Asn Glu Gln Arg Tyr Tyr Gly 1250 1255 1260 Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe Met Val Phe Gln Ala 1265 1270 1275 Leu Ala Gln Tyr Gln Lys Asp Ala Pro Asp His Gln Glu Leu Asn 1280 1285 1290 Leu Asp Val Ser Leu Gln Leu Pro Ser Arg Ser Ser Lys Ile Thr 1295 1300 1305 His Arg Ile His Trp Glu Ser Ala Ser Leu Leu Arg Ser Glu Glu 1310 1315 1320 Thr Lys Glu Asn Glu Gly Phe Thr Val Thr Ala Glu Gly Lys Gly 1325 1330 1335 Gln Gly Thr Leu Ser Val Val Thr Met Tyr His Ala Lys Ala Lys 1340 1345 1350 Asp Gln Leu Thr Cys Asn Lys Phe Asp Leu Lys Val Thr Ile Lys 1355 1360 1365 Pro Ala Pro Glu Thr Glu Lys Arg Pro Gln Asp Ala Lys Asn Thr 1370 1375 1380 Met Ile Leu Glu Ile Cys Thr Arg Tyr Arg Gly Asp Gln Asp Ala 1385 1390 1395 Thr Met Ser Ile Leu Asp Ile Ser Met Met Thr Gly Phe Ala Pro 1400 1405 1410 Asp Thr Asp Asp Leu Lys Gln Leu Ala Asn Gly Val Asp Arg Tyr 1415 1420 1425 Ile Ser Lys Tyr Glu Leu Asp Lys Ala Phe Ser Asp Arg Asn Thr 1430 1435 1440 Leu Ile Ile Tyr Leu Asp Lys Val Ser His Ser Glu Asp Asp Cys 1445 1450 1455 Leu Ala Phe Lys Val His Gln Tyr Phe Asn Val Glu Leu Ile Gln 1460 1465 1470 Pro Gly Ala Val Lys Val Tyr Ala Tyr Tyr Asn Leu Glu Glu Ser 1475 1480 1485 Cys Thr Arg Phe Tyr His Pro Glu Lys Glu Asp Gly Lys Leu Asn 1490 1495 1500 Lys Leu Cys Arg Asp Glu Leu Cys Arg Cys Ala Glu Glu Asn Cys 1505 1510 1515 Phe Ile Gln Lys Ser Asp Asp Lys Val Thr Leu Glu Glu Arg Leu 1520 1525 1530 Asp Lys Ala Cys Glu Pro Gly Val Asp Tyr Val Tyr Lys Thr Arg 1535 1540 1545 Leu Val Lys Val Gln Leu Ser Asn Asp Phe Asp Glu Tyr Ile Met 1550 1555 1560 Ala Ile Glu Gln Thr Ile Lys Ser Gly Ser Asp Glu Val Gln Val 1565 1570 1575 Gly Gln Gln Arg Thr Phe Ile Ser Pro Ile Lys Cys Arg Glu Ala 1580 1585 1590 Leu Lys Leu Glu Glu Lys Lys His Tyr Leu Met Trp Gly Leu Ser 1595 1600 1605 Ser Asp Phe Trp Gly Glu Lys Pro Asn Leu Ser Tyr Ile Ile Gly 1610 1615 1620 Lys Asp Thr Trp Val Glu His Trp Pro Glu Glu Asp Glu Cys Gln 1625 1630 1635 Asp Glu Glu Asn Gln Lys Gln Cys Gln Asp Leu Gly Ala Phe Thr 1640 1645 1650 Glu Ser Met Val Val Phe Gly Cys Pro Asn 1655 1660 <210> 27 <211> 442 <212> PRT <213> Homo sapiens <400> 27 Met Tyr Gln Ser Leu Ala Met Ala Ala Asn His Gly Pro Pro Pro Gly 1 5 10 15 Ala Tyr Glu Ala Gly Gly Pro Gly Ala Phe Met His Gly Ala Gly Ala 20 25 30 Ala Ser Ser Pro Val Tyr Val Pro Thr Pro Arg Val Pro Ser Ser Val 35 40 45 Leu Gly Leu Ser Tyr Leu Gln Gly Gly Gly Ala Gly Ser Ala Ser Gly 50 55 60 Gly Ala Ser Gly Gly Ser Ser Gly Gly Ala Ala Ser Gly Ala Gly Pro 65 70 75 80 Gly Thr Gln Gln Gly Ser Pro Gly Trp Ser Gln Ala Gly Ala Asp Gly 85 90 95 Ala Ala Tyr Thr Pro Pro Pro Val Ser Pro Arg Phe Ser Phe Pro Gly 100 105 110 Thr Thr Gly Ser Leu Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg Glu 115 120 125 Ala Ala Ala Tyr Ser Ser Gly Gly Gly Ala Ala Gly Ala Gly Leu Ala 130 135 140 Gly Arg Glu Gln Tyr Gly Arg Ala Gly Phe Ala Gly Ser Tyr Ser Ser 145 150 155 160 Pro Tyr Pro Ala Tyr Met Ala Asp Val Gly Ala Ser Trp Ala Ala Ala 165 170 175 Ala Ala Ala Ser Ala Gly Pro Phe Asp Ser Pro Val Leu His Ser Leu 180 185 190 Pro Gly Arg Ala Asn Pro Ala Ala Arg His Pro Asn Leu Asp Met Phe 195 200 205 Asp Asp Phe Ser Glu Gly Arg Glu Cys Val Asn Cys Gly Ala Met Ser 210 215 220 Thr Pro Leu Trp Arg Arg Asp Gly Thr Gly His Tyr Leu Cys Asn Ala 225 230 235 240 Cys Gly Leu Tyr His Lys Met Asn Gly Ile Asn Arg Pro Leu Ile Lys 245 250 255 Pro Gln Arg Arg Leu Ser Ala Ser Arg Arg Val Gly Leu Ser Cys Ala 260 265 270 Asn Cys Gln Thr Thr Thr Thr Thr Leu Trp Arg Arg Asn Ala Glu Gly 275 280 285 Glu Pro Val Cys Asn Ala Cys Gly Leu Tyr Met Lys Leu His Gly Val 290 295 300 Pro Arg Pro Leu Ala Met Arg Lys Glu Gly Ile Gln Thr Arg Lys Arg 305 310 315 320 Lys Pro Lys Asn Leu Asn Lys Ser Lys Thr Pro Ala Ala Pro Ser Gly 325 330 335 Ser Glu Ser Leu Pro Pro Ala Ser Gly Ala Ser Ser Asn Ser Ser Asn 340 345 350 Ala Thr Thr Ser Ser Ser Glu Glu Met Arg Pro Ile Lys Thr Glu Pro 355 360 365 Gly Leu Ser Ser His Tyr Gly His Ser Ser Ser Val Ser Gln Thr Phe 370 375 380 Ser Val Ser Ala Met Ser Gly His Gly Pro Ser Ile His Pro Val Leu 385 390 395 400 Ser Ala Leu Lys Leu Ser Pro Gln Gly Tyr Ala Ser Pro Val Ser Gln 405 410 415 Ser Pro Gln Thr Ser Ser Lys Gln Asp Ser Trp Asn Ser Leu Val Leu 420 425 430 Ala Asp Ser His Gly Asp Ile Ile Thr Ala 435 440 <210> 28 <211> 393 <212> PRT <213> Homo sapiens <400> 28 Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln 1 5 10 15 Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu 20 25 30 Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp 35 40 45 Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro 50 55 60 Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala Pro Ala Ala Pro 65 70 75 80 Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser 85 90 95 Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly 100 105 110 Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro 115 120 125 Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln 130 135 140 Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met 145 150 155 160 Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys 165 170 175 Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln 180 185 190 His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp 195 200 205 Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu 210 215 220 Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser 225 230 235 240 Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr 245 250 255 Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val 260 265 270 Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn 275 280 285 Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr 290 295 300 Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys 305 310 315 320 Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu 325 330 335 Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp 340 345 350 Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His 355 360 365 Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met 370 375 380 Phe Lys Thr Glu Gly Pro Asp Ser Asp 385 390 <210> 29 <211> 785 <212> PRT <213> Homo sapiens <400> 29 Met Ser Asp Gln Asp His Ser Met Asp Glu Met Thr Ala Val Val Lys 1 5 10 15 Ile Glu Lys Gly Val Gly Gly Asn Asn Gly Gly Asn Gly Asn Gly Gly 20 25 30 Gly Ala Phe Ser Gln Ala Arg Ser Ser Ser Thr Gly Ser Ser Ser Ser 35 40 45 Thr Gly Gly Gly Gly Gln Glu Ser Gln Pro Ser Pro Leu Ala Leu Leu 50 55 60 Ala Ala Thr Cys Ser Arg Ile Glu Ser Pro Asn Glu Asn Ser Asn Asn 65 70 75 80 Ser Gln Gly Pro Ser Gln Ser Gly Gly Thr Gly Glu Leu Asp Leu Thr 85 90 95 Ala Thr Gln Leu Ser Gln Gly Ala Asn Gly Trp Gln Ile Ile Ser Ser 100 105 110 Ser Ser Gly Ala Thr Pro Thr Ser Lys Glu Gln Ser Gly Ser Ser Thr 115 120 125 Asn Gly Ser Asn Gly Ser Glu Ser Ser Lys Asn Arg Thr Val Ser Gly 130 135 140 Gly Gln Tyr Val Val Ala Ala Ala Pro Asn Leu Gln Asn Gln Gln Val 145 150 155 160 Leu Thr Gly Leu Pro Gly Val Met Pro Asn Ile Gln Tyr Gln Val Ile 165 170 175 Pro Gln Phe Gln Thr Val Asp Gly Gln Gln Leu Gln Phe Ala Ala Thr 180 185 190 Gly Ala Gln Val Gln Gln Asp Gly Ser Gly Gln Ile Gln Ile Ile Pro 195 200 205 Gly Ala Asn Gln Gln Ile Ile Thr Asn Arg Gly Ser Gly Gly Asn Ile 210 215 220 Ile Ala Ala Met Pro Asn Leu Leu Gln Gln Ala Val Pro Leu Gln Gly 225 230 235 240 Leu Ala Asn Asn Val Leu Ser Gly Gln Thr Gln Tyr Val Thr Asn Val 245 250 255 Pro Val Ala Leu Asn Gly Asn Ile Thr Leu Leu Pro Val Asn Ser Val 260 265 270 Ser Ala Ala Thr Leu Thr Pro Ser Ser Gln Ala Val Thr Ile Ser Ser 275 280 285 Ser Gly Ser Gln Glu Ser Gly Ser Gln Pro Val Thr Ser Gly Thr Thr 290 295 300 Ile Ser Ser Ala Ser Leu Val Ser Ser Gln Ala Ser Ser Ser Ser Phe 305 310 315 320 Phe Thr Asn Ala Asn Ser Tyr Ser Thr Thr Thr Thr Thr Ser Asn Met 325 330 335 Gly Ile Met Asn Phe Thr Thr Ser Gly Ser Ser Gly Thr Asn Ser Gln 340 345 350 Gly Gln Thr Pro Gln Arg Val Ser Gly Leu Gln Gly Ser Asp Ala Leu 355 360 365 Asn Ile Gln Gln Asn Gln Thr Ser Gly Gly Ser Leu Gln Ala Gly Gln 370 375 380 Gln Lys Glu Gly Glu Gln Asn Gln Gln Thr Gln Gln Gln Gln Ile Leu 385 390 395 400 Ile Gln Pro Gln Leu Val Gln Gly Gly Gln Ala Leu Gln Ala Leu Gln 405 410 415 Ala Ala Pro Leu Ser Gly Gln Thr Phe Thr Thr Gln Ala Ile Ser Gln 420 425 430 Glu Thr Leu Gln Asn Leu Gln Leu Gln Ala Val Pro Asn Ser Gly Pro 435 440 445 Ile Ile Ile Arg Thr Pro Thr Val Gly Pro Asn Gly Gln Val Ser Trp 450 455 460 Gln Thr Leu Gln Leu Gln Asn Leu Gln Val Gln Asn Pro Gln Ala Gln 465 470 475 480 Thr Ile Thr Leu Ala Pro Met Gln Gly Val Ser Leu Gly Gln Thr Ser 485 490 495 Ser Ser Asn Thr Thr Leu Thr Pro Ile Ala Ser Ala Ala Ser Ile Pro 500 505 510 Ala Gly Thr Val Thr Val Asn Ala Ala Gln Leu Ser Ser Met Pro Gly 515 520 525 Leu Gln Thr Ile Asn Leu Ser Ala Leu Gly Thr Ser Gly Ile Gln Val 530 535 540 His Pro Ile Gln Gly Leu Pro Leu Ala Ile Ala Asn Ala Pro Gly Asp 545 550 555 560 His Gly Ala Gln Leu Gly Leu His Gly Ala Gly Gly Asp Gly Ile His 565 570 575 Asp Asp Thr Ala Gly Gly Glu Glu Gly Glu Asn Ser Pro Asp Ala Gln 580 585 590 Pro Gln Ala Gly Arg Arg Thr Arg Arg Glu Ala Cys Thr Cys Pro Tyr 595 600 605 Cys Lys Asp Ser Glu Gly Arg Gly Ser Gly Asp Pro Gly Lys Lys Lys 610 615 620 Gln His Ile Cys His Ile Gln Gly Cys Gly Lys Val Tyr Gly Lys Thr 625 630 635 640 Ser His Leu Arg Ala His Leu Arg Trp His Thr Gly Glu Arg Pro Phe 645 650 655 Met Cys Thr Trp Ser Tyr Cys Gly Lys Arg Phe Thr Arg Ser Asp Glu 660 665 670 Leu Gln Arg His Lys Arg Thr His Thr Gly Glu Lys Lys Phe Ala Cys 675 680 685 Pro Glu Cys Pro Lys Arg Phe Met Arg Ser Asp His Leu Ser Lys His 690 695 700 Ile Lys Thr His Gln Asn Lys Lys Gly Gly Pro Gly Val Ala Leu Ser 705 710 715 720 Val Gly Thr Leu Pro Leu Asp Ser Gly Ala Gly Ser Glu Gly Ser Gly 725 730 735 Thr Ala Thr Pro Ser Ala Leu Ile Thr Thr Asn Met Val Ala Met Glu 740 745 750 Ala Ile Cys Pro Glu Gly Ile Ala Arg Leu Ala Asn Ser Gly Ile Asn 755 760 765 Val Met Gln Val Ala Asp Leu Gln Ser Ile Asn Ile Ser Gly Asn Gly 770 775 780 Phe 785 <210> 30 <211> 473 <212> PRT <213> Homo sapiens <400> 30 Met Gly Arg Lys Lys Ile Gln Ile Thr Arg Ile Met Asp Glu Arg Asn 1 5 10 15 Arg Gln Val Thr Phe Thr Lys Arg Lys Phe Gly Leu Met Lys Lys Ala 20 25 30 Tyr Glu Leu Ser Val Leu Cys Asp Cys Glu Ile Ala Leu Ile Ile Phe 35 40 45 Asn Ser Thr Asn Lys Leu Phe Gln Tyr Ala Ser Thr Asp Met Asp Lys 50 55 60 Val Leu Leu Lys Tyr Thr Glu Tyr Asn Glu Pro His Glu Ser Arg Thr 65 70 75 80 Asn Ser Asp Ile Val Glu Thr Leu Arg Lys Lys Gly Leu Asn Gly Cys 85 90 95 Asp Ser Pro Asp Pro Asp Ala Asp Asp Ser Val Gly His Ser Pro Glu 100 105 110 Ser Glu Asp Lys Tyr Arg Lys Ile Asn Glu Asp Ile Asp Leu Met Ile 115 120 125 Ser Arg Gln Arg Leu Cys Ala Val Pro Pro Pro Asn Phe Glu Met Pro 130 135 140 Val Ser Ile Pro Val Ser Ser His Asn Ser Leu Val Tyr Ser Asn Pro 145 150 155 160 Val Ser Ser Leu Gly Asn Pro Asn Leu Leu Pro Leu Ala His Pro Ser 165 170 175 Leu Gln Arg Asn Ser Met Ser Pro Gly Val Thr His Arg Pro Pro Ser 180 185 190 Ala Gly Asn Thr Gly Gly Leu Met Gly Gly Asp Leu Thr Ser Gly Ala 195 200 205 Gly Thr Ser Ala Gly Asn Gly Tyr Gly Asn Pro Arg Asn Ser Pro Gly 210 215 220 Leu Leu Val Ser Pro Gly Asn Leu Asn Lys Asn Met Gln Ala Lys Ser 225 230 235 240 Pro Pro Pro Met Asn Leu Gly Met Asn Asn Arg Lys Pro Asp Leu Arg 245 250 255 Val Leu Ile Pro Pro Gly Ser Lys Asn Thr Met Pro Ser Val Ser Glu 260 265 270 Asp Val Asp Leu Leu Leu Asn Gln Arg Ile Asn Asn Ser Gln Ser Ala 275 280 285 Gln Ser Leu Ala Thr Pro Val Val Ser Val Ala Thr Pro Thr Leu Pro 290 295 300 Gly Gln Gly Met Gly Gly Tyr Pro Ser Ala Ile Ser Thr Thr Tyr Gly 305 310 315 320 Thr Glu Tyr Ser Leu Ser Ser Ala Asp Leu Ser Ser Leu Ser Gly Phe 325 330 335 Asn Thr Ala Ser Ala Leu His Leu Gly Ser Val Thr Gly Trp Gln Gln 340 345 350 Gln His Leu His Asn Met Pro Pro Ser Ala Leu Ser Gln Leu Gly Ala 355 360 365 Cys Thr Ser Thr His Leu Ser Gln Ser Ser Asn Leu Ser Leu Pro Ser 370 375 380 Thr Gln Ser Leu Asn Ile Lys Ser Glu Pro Val Ser Pro Pro Arg Asp 385 390 395 400 Arg Thr Thr Thr Pro Ser Arg Tyr Pro Gln His Thr Arg His Glu Ala 405 410 415 Gly Arg Ser Pro Val Asp Ser Leu Ser Ser Cys Ser Ser Ser Tyr Asp 420 425 430 Gly Ser Asp Arg Glu Asp His Arg Asn Glu Phe His Ser Pro Ile Gly 435 440 445 Leu Thr Arg Pro Ser Pro Asp Glu Arg Glu Ser Pro Ser Val Lys Arg 450 455 460 Met Arg Leu Ser Glu Gly Trp Ala Thr 465 470 <210> 31 <211> 353 <212> PRT <213> Unknown <220> <223> Human T-lymphotrophic virus <400> 31 Met Ala His Phe Pro Gly Phe Gly Gln Ser Leu Leu Phe Gly Tyr Pro 1 5 10 15 Val Tyr Val Phe Gly Asp Cys Val Gln Gly Asp Trp Cys Pro Ile Ser 20 25 30 Gly Gly Leu Cys Ser Ala Arg Leu His Arg His Ala Leu Leu Ala Thr 35 40 45 Cys Pro Glu His Gln Ile Thr Trp Asp Pro Ile Asp Gly Arg Val Ile 50 55 60 Gly Ser Ala Leu Gln Phe Leu Ile Pro Arg Leu Pro Ser Phe Pro Thr 65 70 75 80 Gln Arg Thr Ser Lys Thr Leu Lys Val Leu Thr Pro Pro Ile Thr His 85 90 95 Thr Thr Pro Asn Ile Pro Pro Ser Phe Leu Gln Ala Met Arg Lys Tyr 100 105 110 Ser Pro Phe Arg Asn Gly Tyr Met Glu Pro Thr Leu Gly Gln His Leu 115 120 125 Pro Thr Leu Ser Phe Pro Asp Pro Gly Leu Arg Pro Gln Asn Leu Tyr 130 135 140 Thr Leu Trp Gly Gly Ser Val Val Cys Met Tyr Leu Tyr Gln Leu Ser 145 150 155 160 Pro Pro Ile Thr Trp Pro Leu Leu Pro His Val Ile Phe Cys His Pro 165 170 175 Gly Gln Leu Gly Ala Phe Leu Thr Asn Val Pro Tyr Lys Arg Ile Glu 180 185 190 Glu Leu Leu Tyr Lys Ile Ser Leu Thr Thr Gly Ala Leu Ile Ile Leu 195 200 205 Pro Glu Asp Cys Leu Pro Thr Thr Leu Phe Gln Pro Ala Arg Ala Pro 210 215 220 Val Thr Leu Thr Ala Trp Gln Asn Gly Leu Leu Pro Phe His Ser Thr 225 230 235 240 Leu Thr Thr Pro Gly Leu Ile Trp Thr Phe Thr Asp Gly Thr Pro Met 245 250 255 Ile Ser Gly Pro Cys Pro Lys Asp Gly Gln Pro Ser Leu Val Leu Gln 260 265 270 Ser Ser Ser Phe Ile Phe His Lys Phe Gln Thr Lys Ala Tyr His Pro 275 280 285 Ser Phe Leu Leu Ser His Gly Leu Ile Gln Tyr Ser Ser Phe His Ser 290 295 300 Leu His Leu Leu Phe Glu Glu Tyr Thr Asn Ile Pro Ile Ser Leu Leu 305 310 315 320 Phe Asn Glu Lys Glu Ala Asp Asp Asn Asp His Glu Pro Gln Ile Ser 325 330 335 Pro Gly Gly Leu Glu Pro Pro Ser Glu Lys His Phe Arg Glu Thr Glu 340 345 350 Val <210> 32 <211> 505 <212> PRT <213> Homo sapiens <400> 32 Met Gly Glu Thr Leu Gly Asp Ser Pro Ile Asp Pro Glu Ser Asp Ser 1 5 10 15 Phe Thr Asp Thr Leu Ser Ala Asn Ile Ser Gln Glu Met Thr Met Val 20 25 30 Asp Thr Glu Met Pro Phe Trp Pro Thr Asn Phe Gly Ile Ser Ser Val 35 40 45 Asp Leu Ser Val Met Glu Asp His Ser His Ser Phe Asp Ile Lys Pro 50 55 60 Phe Thr Thr Val Asp Phe Ser Ser Ile Ser Thr Pro His Tyr Glu Asp 65 70 75 80 Ile Pro Phe Thr Arg Thr Asp Pro Val Val Ala Asp Tyr Lys Tyr Asp 85 90 95 Leu Lys Leu Gln Glu Tyr Gln Ser Ala Ile Lys Val Glu Pro Ala Ser 100 105 110 Pro Pro Tyr Tyr Ser Glu Lys Thr Gln Leu Tyr Asn Lys Pro His Glu 115 120 125 Glu Pro Ser Asn Ser Leu Met Ala Ile Glu Cys Arg Val Cys Gly Asp 130 135 140 Lys Ala Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys 145 150 155 160 Gly Phe Phe Arg Arg Thr Ile Arg Leu Lys Leu Ile Tyr Asp Arg Cys 165 170 175 Asp Leu Asn Cys Arg Ile His Lys Ser Arg Asn Lys Cys Gln Tyr 180 185 190 Cys Arg Phe Gln Lys Cys Leu Ala Val Gly Met Ser His Asn Ala Ile 195 200 205 Arg Phe Gly Arg Met Pro Gln Ala Glu Lys Glu Lys Leu Leu Ala Glu 210 215 220 Ser Ile Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala Asp Leu Arg 225 230 235 240 Ala Leu Ala Lys His Leu Tyr Asp Ser Tyr Ile Lys Ser Phe Pro Leu 245 250 255 Thr Lys Ala Lys Ala Arg Ala Ile Leu Thr Gly Lys Thr Thr Asp Lys 260 265 270 Ser Pro Phe Val Ile Tyr Asp Met Asn Ser Leu Met Met Gly Glu Asp 275 280 285 Lys Ile Lys Phe Lys His Ile Thr Pro Leu Gln Glu Gln Ser Lys Glu 290,295,300 Val Ala Ile Arg Ile Phe Gln Gly Cys Gln Phe Arg Ser Val Glu Ala 305 310 315 320 Val Gln Glu Ile Thr Glu Tyr Ala Lys Ser Ile Pro Gly Phe Val Asn 325 330 335 Leu Asp Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val His Glu 340 345 350 Ile Ile Tyr Thr Met Leu Ala Ser Leu Met Asn Lys Asp Gly Val Leu 355 360 365 Ile Ser Glu Gly Gln Gly Phe Met Thr Arg Glu Phe Leu Lys Ser Leu 370 375 380 Arg Lys Pro Phe Gly Asp Phe Met Glu Pro Lys Phe Glu Phe Ala Val 385 390 395 400 Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Ile Phe Ile 405 410 415 Ala Val Ile Ile Leu Ser Gly Asp Arg Pro Gly Leu Leu Asn Val Lys 420 425 430 Pro Ile Glu Asp Ile Gln Asp Asn Leu Leu Gln Ala Leu Glu Leu Gln 435 440 445 Leu Lys Leu Asn His Pro Glu Ser Ser Gln Leu Phe Ala Lys Leu Leu 450 455 460 Gln Lys Met Thr Asp Leu Arg Gln Ile Val Thr Glu His Val Gln Leu 465 470 475 480 Leu Gln Val Ile Lys Lys Thr Glu Thr Asp Met Ser Leu His Pro Leu 485 490 495 Leu Gln Glu Ile Tyr Lys Asp Leu Tyr 500 505 <210> 33 <211> 366 <212> PRT <213> Homo sapiens <400> 33 Met Asp Ser Asp Asp Glu Met Val Glu Glu Ala Val Glu Gly His Leu 1 5 10 15 Asp Asp Asp Gly Leu Pro His Gly Phe Cys Thr Val Thr Tyr Ser Ser 20 25 30 Thr Asp Arg Phe Glu Gly Asn Phe Val His Gly Glu Lys Asn Gly Arg 35 40 45 Gly Lys Phe Phe Phe Phe Asp Gly Ser Thr Leu Glu Gly Tyr Tyr Val 50 55 60 Asp Asp Ala Leu Gln Gly Gln Gly Val Tyr Thr Tyr Glu Asp Gly Gly 65 70 75 80 Val Leu Gln Gly Thr Tyr Val Asp Gly Glu Leu Asn Gly Pro Ala Gln 85 90 95 Glu Tyr Asp Thr Asp Gly Arg Leu Ile Phe Lys Gly Gln Tyr Lys Asp 100 105 110 Asn Ile Arg His Gly Val Cys Trp Ile Tyr Tyr Pro Asp Gly Gly Ser 115 120 125 Leu Val Gly Glu Val Asn Glu Asp Gly Glu Met Thr Gly Glu Lys Ile 130 135 140 Ala Tyr Val Tyr Pro Asp Glu Arg Thr Ala Leu Tyr Gly Lys Phe Ile 145 150 155 160 Asp Gly Glu Met Ile Glu Gly Lys Leu Ala Thr Leu Met Ser Thr Glu 165 170 175 Glu Gly Arg Pro His Phe Glu Leu Met Pro Gly Asn Ser Val Tyr His 180 185 190 Phe Asp Lys Ser Thr Ser Ser Cys Ile Ser Thr Asn Ala Leu Leu Pro 195 200 205 Asp Pro Tyr Glu Ser Glu Arg Val Tyr Val Ala Glu Ser Leu Ile Ser 210 215 220 Ser Ala Gly Glu Gly Leu Phe Ser Lys Val Ala Val Gly Pro Asn Thr 225 230 235 240 Val Met Ser Phe Tyr Asn Gly Val Arg Ile Thr His Gln Glu Val Asp 245 250 255 Ser Arg Asp Trp Ala Leu Asn Gly Asn Thr Leu Ser Leu Asp Glu Glu 260 265 270 Thr Val Ile Asp Val Pro Glu Pro Tyr Asn His Val Ser Lys Tyr Cys 275 280 285 Ala Ser Leu Gly His Lys Ala Asn His Ser Phe Thr Pro Asn Cys Ile 290 295 300 Tyr Asp Met Phe Val His Pro Arg Phe Gly Pro Ile Lys Cys Ile Arg 305 310 315 320 Thr Leu Arg Ala Val Glu Ala Asp Glu Glu Leu Thr Val Ala Tyr Gly 325 330 335 Tyr Asp His Ser Pro Pro Gly Lys Ser Gly Pro Glu Ala Pro Glu Trp 340 345 350 Tyr Gln Val Glu Leu Lys Ala Phe Gln Ala Thr Gln Gln Lys 355 360 365 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 34 gtcaaagggg catatggaag g 21 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 35 gggaagaaag ccccacttgg 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 36 gcccagtcgc gtgggggggg 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 37 ggagcgcgag tgtcactcgg 20 <210> 38 <211> 21 <212> DNA <213> Mus musculus <400> 38 gctcactgta ggacccgagc c 21 <210> 39 <211> 21 <212> DNA <213> Mus musculus <400> 39 gacgcggcgc tcattggcca a 21 <210> 40 <211> 21 <212> DNA <213> Mus musculus <400> 40 cgagccgcga gcccagtcgc g 21 <210> 41 <211> 21 <212> DNA <213> Mus musculus <400> 41 tccccccccc cccccacgcg a 21 <210> 42 <211> 21 <212> DNA <213> Mus musculus <400> 42 gtcactcacc ccgattggcc a 21 <210> 43 <211> 21 <212> DNA <213> Mus musculus <400> 43 cgcgagccca gtcgcgtggg g 21 <210> 44 <211> 21 <212> DNA <213> Mus musculus <400> 44 gttggcttat ccaaacatct c 21 <210> 45 <211> 21 <212> DNA <213> Mus musculus <400> 45 atgttaagca agggtaatag a 21 <210> 46 <211> 21 <212> DNA <213> Mus musculus <400> 46 ctgtgaaagg aatacaattc a 21 <210> 47 <211> 21 <212> DNA <213> Mus musculus <400> 47 gccaattctt ggcaaccgag c 21 <210> 48 <211> 21 <212> DNA <213> Mus musculus <400> 48 gaattggcca aagggagggg t 21 <210> 49 <211> 21 <212> DNA <213> Mus musculus <400> 49 aattagcaga cagcttggta c 21 <210> 50 <211> 21 <212> DNA <213> Mus musculus <400> 50 ctggctgatt cccgaggatt t 21 <210> 51 <211> 21 <212> DNA <213> Mus musculus <400> 51 cactgaatac ggattggtca g 21 <210> 52 <211> 21 <212> DNA <213> Mus musculus <400> 52 gatgtctcag aaccactgaa t 21 <210> 53 <211> 21 <212> DNA <213> Mus musculus <400> 53 aaccactgaa tacggattgg t 21 <210> 54 <211> 21 <212> DNA <213> Mus musculus <400> 54 accaatccgt attcagtggt t 21 <210> 55 <211> 21 <212> DNA <213> Homo sapiens <400> 55 ggcgcggggc ggacggggcg a 21 <210> 56 <211> 21 <212> DNA <213> Homo sapiens <400> 56 gcgccccggg aacgcgtggg g 21 <210> 57 <211> 21 <212> DNA <213> Homo sapiens <400> 57 cgccccgcgc cgcgcgggga g 21 <210> 58 <211> 21 <212> DNA <213> Homo sapiens <400> 58 tccgccccgc gccgcgcggg g 21 <210> 59 <211> 21 <212> DNA <213> Homo sapiens <400> 59 ggaacgcgtg gggcggagct t 21 <210> 60 <211> 21 <212> DNA <213> Homo sapiens <400> 60 gccccgcgcc gcgcggggag g 21 <210> 61 <211> 21 <212> DNA <213> Homo sapiens <400> 61 tgcgccccgg gaacgcgtgg g 21 <210> 62 <211> 21 <212> DNA <213> Homo sapiens <400> 62 gaacgcgtgg ggcggagctt c 21 <210> 63 <211> 21 <212> DNA <213> Homo sapiens <400> 63 gcggcgcggg gcggacgggg c 21 <210> 64 <211> 21 <212> DNA <213> Homo sapiens <400> 64 cccgtccgcc ccgcgccgcg c 21 <210> 65 <211> 21 <212> DNA <213> Homo sapiens <400> 65 ggcccactcg ccgccaatca g 21 <210> 66 <211> 21 <212> DNA <213> Homo sapiens <400> 66 ggaagccgcc ggggccgcct a 21 <210> 67 <211> 21 <212> DNA <213> Homo sapiens <400> 67 tgattggcgg cgagtgggcc a 21 <210> 68 <211> 21 <212> DNA <213> Homo sapiens <400> 68 gccgccaatc agcggaagcc g 21 <210> 69 <211> 21 <212> DNA <213> Homo sapiens <400> 69 ggcggcttcc gctgattggc g 21 <210> 70 <211> 21 <212> DNA <213> Homo sapiens <400> 70 ccgccaatca gcggaagccg c 21 <210> 71 <211> 21 <212> DNA <213> Homo sapiens <400> 71 agccgccggg gccgcctaga g 21 <210> 72 <211> 21 <212> DNA <213> Homo sapiens <400> 72 gcttccgctg attggcggcg a 21 <210> 73 <211> 21 <212> DNA <213> Homo sapiens <400> 73 cggcgagtgg gccaatgggt g 21 <210> 74 <211> 21 <212> DNA <213> Homo sapiens <400> 74 ccaatgggtg cggggcggtg g 21 <210> 75 <211> 21 <212> DNA <213> Mus musculus <400> 75 ggctgccggg gccgcctaaa g 21 <210> 76 <211> 21 <212> DNA <213> Mus musculus <400> 76 ggaggctgcc ggggccgcct a 21 <210> 77 <211> 21 <212> DNA <213> Mus musculus <400> 77 gccgccaatc agcggaggct g 21 <210> 78 <211> 21 <212> DNA <213> Mus musculus <400> 78 ccgccaatca gcggaggctg c 21 <210> 79 <211> 21 <212> DNA <213> Mus musculus <400> 79 tggccggtgc gccgccaatc a 21 <210> 80 <211> 21 <212> DNA <213> Mus musculus <400> 80 ggccggtgcg ccgccaatca g 21 <210> 81 <211> 21 <212> DNA <213> Mus musculus <400> 81 cggcgcaccg gccaataagt g 21 <210> 82 <211> 21 <212> DNA <213> Mus musculus <400> 82 ataagtgtgg ggcggtgggc g 21 <210> 83 <211> 21 <212> DNA <213> Mus musculus <400> 83 ccaataagtg tggggcggtg g 21 <210> 84 <211> 21 <212> DNA <213> Mus musculus <400> 84 caataagtgt ggggcggtgg g 21 <210> 85 <211> 20 <212> DNA <213> Mus musculus <400> 85 cctttctatg acctagtcgg 20 <210> 86 <211> 20 <212> DNA <213> Mus musculus <400> 86 cagaatcagt aacgcactgt 20 <210> 87 <211> 20 <212> DNA <213> Mus musculus <400> 87 gaaaccagga gagataaccc 20 <210> 88 <211> 20 <212> DNA <213> Mus musculus <400> 88 ggaccccaga tattctggaa 20 <210> 89 <211> 20 <212> DNA <213> Mus musculus <400> 89 ttattgttga cttaacgaag 20 <210> 90 <211> 20 <212> DNA <213> Mus musculus <400> 90 aaaaagaagc aatagctaa 20 <210> 91 <211> 20 <212> DNA <213> Mus musculus <400> 91 agaatcagta acgcactgta 20 <210> 92 <211> 27 <212> DNA <213> Mus musculus <400> 92 tgttggttta ttggacccca gatattc 27 <210> 93 <211> 27 <212> DNA <213> Mus musculus <400> 93 tgttggagaa attaactta gtgcata 27 <210> 94 <211> 27 <212> DNA <213> Mus musculus <400> 94 tgttggtata actgccacta gagggct 27 <210> 95 <211> 19 <212> DNA <213> Homo sapiens <400> 95 aggagccggg acccaccgg 19

Claims

1. 1. A method of treating a haploinsufficiency disease in a mammalian subject, comprising: (i) (a) a targeting region that specifically hybridizes to a promoter or enhancer region operably linked to a wild-type copy of a haploinsufficient gene under conditions present in the nucleus of said cell; and (b) a CRISPR nuclease binding region that specifically binds to a CRISPR nuclease under conditions present in the nucleus of the cell, or a region that specifically binds to the CRISPR nuclease binding region a guide RNA comprising (ii) CRISPR nuclease contacting the substrate with a composition comprising By the contacting step, a complex containing the CRISPR nuclease bound to the guide RNA is formed, and the targeting region of the guide RNA in the complex hybridizes to the promoter or enhancer; the complex comprises a catalytically inactive CRISPR nuclease and a transcription activation domain; and the complex activates transcription of a wild-type copy of the haploinsufficient gene in an amount and for a sufficient duration to treat the haploinsufficiency disease in the subject. The method.

2. 10. The method of claim 1, wherein the contacting step comprises contacting the cell with an episomal vector encoding a guide RNA or a CRISPR nuclease.

3. 3. The method of claim 1 or 2, wherein the contacting step comprises contacting the cell with an episomal vector encoding a guide RNA and a CRISPR nuclease.

4. 3. The method of claim 1 or 2, wherein the contacting step comprises contacting the cell with an episomal vector encoding a guide RNA and a second episomal vector encoding a CRISPR nuclease.

5. The method of any one of the preceding claims, wherein the episomal vector is non-integrating.

6. The method of any one of the preceding claims, wherein the episomal vector is non-replicating.

7. The method of any one of the preceding claims, wherein the episomal vector is an adeno-associated virus (AAV) vector.

8. 10. The method of claim 1, wherein the episomal vector independently comprises a first end and a second end, and the first end and the second end each independently comprise an AAV inverted terminal repeat sequence.

9. The method of any one of the preceding claims, wherein the CRISPR nuclease comprises (i) a nuclease domain modified to eliminate nuclease activity and nicking activity, and (ii) a transcription activation domain.

10. 10. The method of claim 1, wherein the modification comprises mutations at positions corresponding to D10 and H840 of S. pyogenes Cas9.

11. The method of any one of the preceding claims, wherein the CRISPR nuclease comprises D10A,H840A Streptococcus pyogenes dCas9.

12. 10. The method of any one of the preceding claims, wherein the guide RNA comprises a dead guide sequence.

13. 10. The method of any one of the preceding claims, wherein the guide RNA comprises a transcription activation binding domain, and the transcription activation binding domain specifically binds to a composition comprising one or more transcription activation domains.

14. The method of any one of the preceding claims, wherein the complex comprising the CRISPR nuclease bound to the guide RNA further comprises a transcription activation domain selected from the group consisting of HSF1, VP16, VP64, p65, MyoD1, RTA, SET7 / 9, VPR, histone acetyltransferase p300, a hydroxylase catalytic domain of a TET family protein (e.g., a TET1 hydroxylase catalytic domain), LSD1, CIB1, AD2, CR3, EKLF1, GATA4, PRVIE, p53, SP1, MEF2C, TAX, and PPARγ.

15. 3. The method of any one of the preceding claims, wherein the CRISPR nuclease is a CRISPR nuclease-VP64 fusion polypeptide.

16. 10. The method of any one of the preceding claims, wherein the guide RNA comprises a scaffold region.

17. 17. The method of claim 16, wherein the scaffold region comprises an ms2, f6, PP7, com, or L7a ligand sequence.

18. 18. The method of claim 17, wherein the scaffold region of the guide RNA in the complex is bound to a transcription activation domain fused to an MCP polypeptide, a COM polypeptide, a PCP polypeptide, or an L7a polypeptide.

19. Haploinsufficient genes include SIM1, leptin, leptin receptor, MC4R, SCN2A, SETD5, PAX6, PKD1, MC3R, POMC, STAT3, STAT5, SOCS3, GHR, NPY, NPY1R, NPY2R, NPY5R, PYY, AMPK (PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3), OXT, JAK2, SHP2, NOS3, NROB2, BRS3, CARTPT, FABP4, HTR2C, IL6, and NHLH 2, NMU, NPB, NPBWRI, PNPLA2, UCP3, ADIPOQ, APOA5, ARNT2, ASIP, C1QTNF2, C3AR1, CCK, CPT1B, CSF2, DGAT1, DGAT2, GHRL, GHSR, HSD11B1, HTR7, INSIG1, INSIG2, LIPC, NMURI, NMUR2, NPBWR2, NTS, PPARGC1A, PPY, RETN, SIRT1, TGFBR2, WDTC1, or FOXO1.

20. The targeting region of the guide RNA is 10. The method of claim 9, wherein the nucleic acid sequence is encoded by or specifically hybridizes to:

21. The targeting region of the guide RNA is 20. The method of any one of claims 1 to 19, wherein the nucleic acid sequence is encoded by or specifically hybridizes to:

22. The targeting region of the guide RNA is 20. The method of any one of claims 1 to 19, wherein the nucleic acid sequence is encoded by or specifically hybridizes to:

23. The targeting region of the guide RNA is 20. The method of any one of claims 1 to 19, wherein the nucleic acid sequence is encoded by or specifically hybridizes to:

24. The targeting region of the guide RNA is 20. The method of any one of claims 1 to 19, wherein the nucleic acid sequence is encoded by or specifically hybridizes to:

25. The targeting region of the guide RNA is 20. The method of any one of claims 1 to 19, wherein the nucleic acid sequence is encoded by or specifically hybridizes to:

26. The targeting region of the guide RNA is 20. The method of any one of claims 1 to 19, wherein the nucleic acid sequence is encoded by or specifically hybridizes to:

27. The targeting region of the guide RNA is 20. The method of any one of claims 1 to 19, wherein the nucleic acid sequence is encoded by or specifically hybridizes to:

28. The targeting region of the guide RNA is 20. The method of any one of claims 1 to 19, wherein the nucleic acid sequence is encoded by or specifically hybridizes to:

29. The targeting region of the guide RNA is 20. The method of any one of claims 1 to 19, wherein the nucleic acid sequence is encoded by or specifically hybridizes to:

30. The targeting region of the guide RNA is 20. The method of any one of claims 1 to 19, wherein the nucleic acid sequence is encoded by or specifically hybridizes to:

31. The targeting region of the guide RNA is 20. The method of any one of claims 1 to 19, wherein the nucleic acid sequence is encoded by or specifically hybridizes to:

32. The method of any one of the preceding claims, wherein the cells are non-dividing cells.

33. The method of any one of the preceding claims, wherein the cell is a neuron.

34. The method of any one of the preceding claims, wherein the cell is a hypothalamic cell.

35. 10. The method of any one of the preceding claims, wherein the contacting step comprises injection of a nucleic acid encoding a guide RNA and / or a CRISPR nuclease into a region of the brain that includes the hypothalamus.

36. 10. The method of any one of the preceding claims, wherein the contacting step comprises injecting an adeno-associated viral vector comprising a nucleic acid encoding a guide RNA and / or a CRISPR nuclease into a region of the brain comprising the hypothalamus.

37. 10. The method of any one of the preceding claims, wherein the haploinsufficiency disease is selected from the group consisting of obesity, autism, epilepsy, intellectual disability, aniridia, and polycystic kidney disease.

38. The method of any of the preceding claims, wherein the haploinsufficiency disease is selected from Table 1.

39. 38. The method of claim 37, wherein the haploinsufficiency disease is obesity.

40. (I.) A genome comprising at least one functional copy of a target gene, wherein the functional copy does not produce sufficient corresponding gene product to result in a wild-type phenotype in an organism in the absence of transcriptional activation by a heterologous complex; and (II.) (a) (i.) a targeting region that specifically hybridizes, under conditions present in the nucleus of a cell, to a promoter or enhancer region operably linked to a functional copy of a target gene; (ii.) a CRISPR nuclease binding domain that specifically binds to a CRISPR nuclease under conditions present in the nucleus of a cell; and a guide RNA, and (b) CRISPR nuclease A heterologous complex comprising 1. An isolated mammalian host cell comprising: A guide RNA of a heterologous complex comprising a CRISPR nuclease bound to the guide RNA is hybridized to the promoter or enhancer; the CRISPR nuclease is catalytically inactive, and the complex activates transcription of a functional copy of the target gene in a sufficient amount and for a sufficient duration to produce a wild-type phenotype when the host cell is present in an organism; The isolated mammalian host cell.

41. 41. The isolated mammalian host cell of claim 40, wherein said genome comprises only one functional copy of the target gene.

42. 42. The isolated mammalian host cell of claim 41, wherein the only functional copy of the target gene comprises a haploinsufficient gene.

43. Haploinsufficient genes include SIM1, leptin, leptin receptor, MC4R, SCN2A, SETD5, PAX6, PKD1, MC3R, POMC, STAT3, STAT5, SOCS3, GHR, NPY, NPY1R, NPY2R, NPY5R, PYY, AMPK (PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3), OXT, JAK2, SHP2, NOS3, NROB2, BRS3, CARTPT, FABP4, HTR2C, IL6, NHLH2, 43. The isolated mammalian host cell of claim 42, wherein the host cell is NMU, NPB, NPBWRI, PNPLA2, UCP3, ADIPOQ, APOA5, ARNT2, ASIP, C1QTNF2, C3AR1, CCK, CPT1B, CSF2, DGAT1, DGAT2, GHRL, GHSR, HSD11B1, HTR7, INSIG1, INSIG2, LIPC, NMURI, NMUR2, NPBWR2, NTS, PPARGC1A, PPY, RETN, SIRT1, TGFBR2, WDTC1, or FOXO1.

44. 44. The isolated mammalian host cell of claim 43, wherein said haploinsufficient gene treats a haploinsufficiency disease selected from Table 1.

45. 45. The isolated mammalian host cell of claim 44, wherein the haploinsufficiency disease is selected from obesity, autism, epilepsy, intellectual disability, aniridia, and polycystic kidney disease.

46. 46. The isolated mammalian host cell of claim 45, wherein the haploinsufficiency disease is obesity.

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