Gene therapy for tuberous sclerosis

Abstract

The present invention provides compositions and methods for treating tuberous sclerosis complex (TSC). The present invention provides condensed tuberin (c-tuberin), c-tuberin nucleic acid, and recombinant adeno-associated virus (rAAV) carrying c-tuberin nucleic acid for treating patients with TSC. The c-tuberin contains a hamartin-binding domain and a GTPase-activating protein (GAP) domain, but lacks the Akt phosphorylation site Thr 1462.

Description

[Technical Field]

[0001] Statement Regarding Federally Sponsored Research This invention was made with government support under Grant No. TS120038 awarded by the Department of Defense. The government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 507,358, filed May 17, 2017, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on May 15, 2018, is entitled 51317-002WO2_Sequence_Listing_5.15.18_ST25 and is 55,672 bytes in size. [Background technology]

[0004] Background of the Invention Tuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor syndrome occurring in approximately 1 in 5,500 individuals. Patients inherit a mutation in one allele of either the TSC1 gene (encoding hamartin) or the TSC2 gene (encoding tuberin). These proteins cooperate to suppress the activity of the mammalian target of rapamycin complex 1 (mTORC1). Mutations in the corresponding normal allele during development or in a subset of somatic cells lead to cell enlargement and increased proliferation, resulting in the formation of benign tumors (e.g., hamartomas). Various tissues, including the brain, heart, kidney, skin, and lung, can be affected by such tumors. In the brain, these tumors can cause developmental delay, autism, epilepsy, and hydrocephalus. Life-threatening TSC manifestations include renal angiomyolipomas, which can cause internal bleeding, and lymphangioleiomyomatosis (LAM), which can compromise breathing. Although rapamycin and related drugs have been effective in reducing the size of some tumor lesions, they require continuous administration and have side effects, including impaired brain development and immunosuppression. In addition, some patients do not respond to these drugs, or respond initially but then become resistant. Therefore, there is a need in the art for improved treatment of TSC. Summary of the Invention

[0005] The present invention provides compositions and methods for treating tuberous sclerosis complex (TSC), which is caused by mutations in the TSC2 gene. The compositions and methods described herein relate to condensed tuberin (c-tuberin) and nucleic acid molecules encoding c-tuberin.

[0006] In a first aspect, the invention features cTuberin, which contains the hamartin binding domain and the GTPase-activating protein (GAP) domain but lacks the Akt phosphorylation site Thr 1462.

[0007] In some embodiments, the cTuberin has at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 1. In particular embodiments, the cTuberin is SEQ ID NO: 1.

[0008] In some embodiments, the hamartin binding region has at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:2. In particular embodiments, the hamartin binding region is SEQ ID NO:2.

[0009] In some embodiments, the GAP region has at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:3. In particular embodiments, the GAP region is SEQ ID NO:3.

[0010] The engineered cTuberin further lacks the Akt phosphorylation site of human tuberin (SEQ ID NO:10). In a particular embodiment, the cTuberin lacks amino acids 451-1514 of human tuberin (SEQ ID NO:10), which includes the Akt phosphorylation site at Thr 1462 of human tuberin.

[0011] In still other embodiments, the c-tuberin comprises a spacer between the hamartin binding region and the GAP region. In some embodiments, the spacer comprises at least SGGG. Exemplary spacers include: The file is TIFF0007725514000001.tif4128.

[0012] In still other embodiments, c-tuberin is produced using human tuberin isoforms as disclosed herein.

[0013] In a second aspect, the invention features a nucleic acid molecule encoding the c-tuberin of any of the preceding embodiments.

[0014] In other embodiments, the nucleic acid molecule is codon-optimized for expression in a human cell (eg, a brain cell, a heart cell, a kidney cell, a skin cell, or a lung cell).

[0015] In some embodiments, the nucleic acid molecule is operably linked to a regulatory control sequence. Exemplary regulatory control sequences include, but are not limited to, the human cytomegalovirus (CMV) promoter, chicken beta-actin (CBA) promoter, Rous sarcoma virus (RSV) LTR promoter / enhancer, SV40 promoter, dihydrofolate reductase promoter, phosphoglycerol kinase promoter, CMV immediate early gene enhancer / CBA promoter, synapsin promoter, or glial fibrillary acidic protein (GFAP) promoter. In one example, the regulatory control sequence comprises the CMV immediate early gene enhancer / CBA promoter and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

[0016] In other embodiments, the nucleic acid molecule has at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:5. In particular embodiments, the nucleic acid molecule is SEQ ID NO:5.

[0017] In some embodiments, the nucleic acid molecule is operably linked to an expression cassette.

[0018] In a third aspect, the invention features a cell or virus that includes a nucleic acid molecule of any of the preceding embodiments.

[0019] In a fourth aspect, the invention features a composition including a nucleic acid molecule of any of the preceding embodiments.

[0020] In a fifth aspect, the present invention features a recombinant adeno-associated virus (rAAV). Such rAAV comprises an AAV capsid and an AAV genome packaged therein, the AAV genome comprising a nucleic acid molecule capable of expressing c-tuberin. For example, the rAAV comprises an AAV capsid and an AAV genome packaged therein, the AAV genome comprising (a) an AAV 5' inverted terminal repeat (ITR) sequence; (b) a regulatory control sequence; (c) a nucleic acid molecule encoding c-tuberin; and (d) an AAV 3' ITR sequence. In another example, c-tuberin comprises a hamartin binding region and a GAP region, but lacks the Akt phosphorylation site Thr 1462.

[0021] In some embodiments, the AAV capsid is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid, or a variant of any one of these AAV capsids.

[0022] In other embodiments, the nucleic acid molecule is codon-optimized for expression in human cells. In further embodiments, the nucleic acid molecule has at least 90% sequence identity to SEQ ID NO:5. In particular embodiments, the nucleic acid molecule is SEQ ID NO:5.

[0023] In other embodiments, the nucleic acid is operably linked to a regulatory control sequence. Exemplary regulatory control sequences include, but are not limited to, a human cytomegalovirus (CMV) promoter, a chicken beta-actin (CBA) promoter, a Rous sarcoma virus (RSV) LTR promoter / enhancer, an SV40 promoter, a dihydrofolate reductase promoter, a phosphoglycerol kinase promoter, a CMV immediate early gene enhancer / CBA promoter, a synapsin promoter, or a glial fibrillary acidic protein (GFAP) promoter.

[0024] In some embodiments, the nucleic acid molecule comprises an inverted terminal repeat (ITR). In some embodiments, the nucleic acid molecule comprises a polyadenylation signal, such as a polyA tract.

[0025] In a sixth aspect, the invention features a composition including the rAAV of any one of the preceding embodiments and a pharmaceutically acceptable carrier.

[0026] In a seventh aspect, the invention features a method of treating a patient with tuberous sclerosis complex (TSC), the method comprising administering to the patient c-tuberin that includes the hamartin binding domain and the GAP domain but lacks the Akt phosphorylation site Thr 1462.

[0027] In some embodiments, a nucleic acid molecule encoding c-tuberin is administered to the patient.

[0028] In some embodiments, the patient is administered an rAAV of any of the preceding aspects.

[0029] In some embodiments, extracellular vesicles (EVs) comprising the nucleic acid molecule of any of the preceding aspects are administered to the patient.

[0030] In some embodiments, the patient has a renal angiomyolipoma. In some embodiments, the c-tuberin is administered intravascularly or into the renal artery or vein.

[0031] In other embodiments, the patient has lymphangioleiomyomatosis (LAM). In some embodiments, c-tuberin is administered intravascularly or pulmonary.

[0032] In yet other embodiments, the patient has a brain dysfunction. In some embodiments, the c-tuberin is administered intravascularly, intracerebrally, or intrathecally.

[0033] In some embodiments, cTuberin is administered to a renal angiomyolipoma, a LAM, or the brain.

[0034] In some embodiments, the rAAV is administered to brain cells, heart cells, kidney cells, skin cells, or lung cells. In still other embodiments, the rAAV is administered intravascularly, intravenously, intracerebrally, intracerebroventricularly, intrathecally, or to the skin.

[0035] In some embodiments, the patient is further administered a drug used in the treatment of TSC, which may be rapamycin or a rapamycin analog.

[0036] definition As used herein, "administering" or grammatical derivatives thereof refers to placing an agent disclosed herein into a subject by a method or route that at least partially localizes the agent at a desired site.

[0037] As used herein, "codon optimization" refers to modifying a nucleic acid sequence to alter individual nucleic acids without causing any changes to the encoded amino acids. Sequences modified in this manner are referred to herein as "codon-optimized." This process can be performed on any of the sequences described herein to improve expression or stability. Codon optimization can be performed, for example, as described in U.S. Pat. Nos. 7,561,972, 7,561,973, and 7,888,112, each of which is incorporated herein by reference in its entirety. The sequence surrounding the translation start site can be converted to a consensus Kozak sequence according to known methods. See, for example, Kozak et al., Nucleic Acids Res. 15(20): 8125-8148 (1987), incorporated herein by reference in its entirety.

[0038] As used herein, a sequence "encoding" a particular protein is a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences, is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo; however, one of skill in the art will readily appreciate that various polynucleotides (e.g., antisense RNA, siRNA, ribozymes, when the product is an RNA transcript) do not function in this manner. For protein products (i.e., not RNA products), the boundaries of the coding sequence are determined by a start codon at the 5' (i.e., amino) terminus and a translation stop codon at the 3' (i.e., carboxy) terminus. A gene can include, but is not limited to, cDNA derived from prokaryotic or eukaryotic mRNA, genomic DNA sequences derived from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence is typically located 3' of the gene sequence. Furthermore, a "gene" (i) includes a coding sequence beginning with a promoter region that contains multiple regulatory elements, possibly including enhancers, for directing transcription of the coding sequence; (ii) includes a coding sequence that begins with a transcription initiation site located upstream of the translation initiation site and ends with a transcription termination site that may be significantly downstream of a stop codon (usually a polyadenylation signal is associated with the transcription termination site and located upstream of the transcription termination site); and (iii) may include introns and other regulatory sequences that regulate expression and improve the stability of the RNA transcript. Furthermore, according to the present invention, a "gene" may refer to a sequence that encodes a protein.

[0039] As used herein, "expression" refers to the process by which a structural gene produces a polypeptide. This process involves transcription of the gene into mRNA and the translation of such mRNA into the polypeptide.

[0040] As used herein, an "expression vector" is a vector or vehicle that is similar to a cloning vector but can express a cloned gene after transformation into a host. The cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences, such as a promoter sequence. The expression control sequences vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host, and may further include transcription elements such as enhancer elements, termination sequences, tissue-specific elements, and / or translation start and stop sites.

[0041] As used herein, "nucleic acid" or "nucleic acid molecule," as generally understood and used herein, refers to a chain of nucleotides linked together by phosphodiester bonds to form a nucleic acid heteropolymer. Nucleic acid molecules can be double-stranded or single-stranded, and can be deoxyribonucleotide (DNA) molecules, such as cDNA or genomic DNA, or ribonucleotide (RNA) molecules. Thus, nucleic acid molecules can contain one or more exons, and may or may not contain introns, as appropriate.

[0042] As used herein, "operably linked" means that a first molecule is associated with a second molecule, with the molecules positioned such that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single, contiguous molecule, and may or may not be adjacent. For example, a promoter is operably linked to a transcribable polynucleotide molecule if it regulates the transcription of the transcribable polynucleotide molecule of interest in a cell. In addition, two portions of a transcriptional regulatory element are operably linked to each other if they are linked such that the transcriptional activation function of one portion is not adversely affected by the presence of the other portion. Two transcriptional regulatory elements may be operably linked to each other by a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or without any intervening nucleotides.

[0043] As used herein, the "percent identity" between two sequences is determined by the BLAST 2.0 algorithm described in Altschul et al., (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information.

[0044] As used herein, "pharmaceutically acceptable" means that such compound, material, composition, and / or dosage form is suitable for contact with the tissues of a subject, such as a mammal (e.g., a human) without undue toxicity, irritation, allergic response, and other significant complications, commensurate with a reasonable benefit / risk ratio.

[0045] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein and refer to a polymer of amino acids. Peptides are relatively short polypeptides, typically about 2 to 60 amino acids in length. As used herein, the terms "polypeptide sequence" or "amino acid sequence" can refer to the polypeptide material itself and / or the sequence information biochemically characterizing a polypeptide (i.e., the series of letters or three-letter codes used as abbreviations for amino acid names). Polypeptide sequences presented herein are presented in an N-terminal to C-terminal direction unless otherwise indicated.

[0046] As used herein, "regulatory control elements" or "regulatory control sequences" refer collectively to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which cooperate to provide for the replication, transcription, and translation of a coding sequence in a recipient cell. These control elements need not necessarily be present so long as the selected coding sequence is capable of being replicated, transcribed, and translated in an appropriate host cell.

[0047] As used herein, "recombinant virus" means a virus that has been genetically altered (eg, by the addition or insertion of a heterologous nucleic acid construct into the particle).

[0048] As used herein, the terms "subject" and "patient" are used interchangeably and refer to an organism undergoing treatment for a particular disease or condition described herein.

[0049] As used herein, the terms "treat," "treatment," "treating," or "amelioration," when used in reference to a disease, disorder, or medical condition, refer to therapeutic treatment for a condition, with the goal of reversing, alleviating, ameliorating, inhibiting, slowing, or halting the progression or severity of a symptom or condition. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease state is slowed or halted. That is, "treatment" goes beyond simply improving symptoms or markers to also include halting or at least slowing the progression or worsening of symptoms that would be expected in the absence of treatment. For example, in the case of renal angiomyolipoma, tumor size can be monitored by MRI, and cell size reduction due to replacement of tuberin function can be demonstrated according to standard procedures (e.g., those used to monitor TSC treatment with rapamycin).

[0050] Beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, reduction in the extent of the defect, stabilization (i.e., not worsening) of the tuberous sclerosis progression, delay or slowing of the aggressiveness or growth of the tumor or hamartoma, and improvement or alleviation of symptoms associated with such tumor or hamartoma. Treatment also includes reduced mortality or increased lifespan of a subject compared to one not receiving treatment.

[0051] As used herein, "vector" refers to any genetic element, e.g., plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., that is capable of replication and transfer of genetic sequences between cells when associated with the proper control elements. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0052] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art to which this disclosure belongs. The present invention is not limited to the particular methodology, protocols, and reagents, etc., described herein, which may vary accordingly. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0053] The inventions described herein offer numerous advantages. For example, the present specification describes compositions and methods useful for treating tuberous sclerosis complex (TSC) through gene therapy using recombinant adeno-associated viruses (RAVs). Previously, gene therapy could not cure TSC caused by TSC2 mutations due to the relatively small insert capacity of AAV vectors (4.7 kb compared to the 5.4 kb human tuberin cDNA). As described herein, the disclosed method for repairing TSC2 mutations utilizes c-tuberin, a condensed form of human tuberin. The c-tuberin cDNA described herein is approximately 2.3 kb and can be easily expressed by AAV vectors. In fact, AAV vectors have proven safe and beneficial in gene therapy. When delivered intravascularly, for example, this vector can reach multiple tissues with a single injection, and depending on the serotype, it can cross the blood-brain barrier. Long-term beneficial results are typically achieved with a single injection. Our compositions and methods enable the use of AAV vectors expressing c-tuberin to treat symptoms of tuberous sclerosis complex in patients with mutations in TSC2. Such vectors are useful not only for the treatment of renal angiomyolipoma, but also for LAM and brain dysfunction caused by TSC2.

[0054] Furthermore, the present invention provides an alternative method for treating TSC without necessarily requiring rapamycin or its analogs, which can cause toxicity and adverse events associated with overinhibition of mTORC1. Instead, the disclosed compositions and methods exploit the ability of tuberin to be active only when complexed with hamartin. Because hamartin levels are normal in TSC2 patients, overexpression of c-tuberin via vector delivery is expected to result in little to no toxicity. Furthermore, while rapamycin and related drugs can inhibit mTORC1 activity, c-tuberin may be more effective than previous therapies for tuberous sclerosis complex because it can inhibit both mTORC1 pathological effects and Rheb-dependent pathological effects that are independent of mTORC1.

[0055] [The present invention 1001] Condensed tuberin (c-tuberin), which contains the hamartin-binding domain and the GTPase-activating protein (GAP) domain but lacks the Akt phosphorylation site Thr 1462. [The present invention 1002] 1001. The cTuberin of claim 10, wherein said hamartin binding region has at least 90% sequence identity to SEQ ID NO:2. [The present invention 1003] 1002. The cTuberin of the present invention, wherein said hamartin binding domain is SEQ ID NO:2. [The present invention 1004] 1001. The cTuberin of the present invention, wherein said GAP region has at least 90% sequence identity to SEQ ID NO:3. [The present invention 1005] 1004. The cTuberin of the present invention, wherein said GAP region is SEQ ID NO:3. [The present invention 1006] 1001. A tuberin of the present invention, lacking amino acids 451 to 1514 of human tuberin (SEQ ID NO: 10). [The present invention 1007] 1001. The c-tuberin of the present invention, comprising a spacer between the hamartin binding domain and the GAP domain. [The present invention 1008] 1001. The tuberin of claim 10, wherein the spacer comprises at least SGGG. [The present invention 1009] 1008. The cTuberin of the present invention, wherein the spacer is SEQ ID NO:4. [The present invention 1010] 1001. A tuberin of the present invention, having at least 90% sequence identity to SEQ ID NO:1. [The present invention 1011] 1001. A tuberin of the present invention, which is SEQ ID NO:1. [The present invention 1012] A nucleic acid molecule encoding any one of c-tuberin according to the present invention 1001 to 1011. [The present invention 1013] 1012. A nucleic acid molecule of the invention that is codon-optimized for expression in human cells. [The present invention 1014] A nucleic acid molecule of the present invention 1013 operably linked to a regulatory control sequence. [The present invention 1015] The nucleic acid molecule of the present invention 1014, wherein the regulatory control sequence comprises a human cytomegalovirus (CMV) promoter, a chicken beta-actin (CBA) promoter, a Rous sarcoma virus (RSV) LTR promoter / enhancer, an SV40 promoter, a dihydrofolate reductase promoter, a phosphoglycerol kinase promoter, a CMV immediate early gene enhancer / CBA promoter, a synapsin promoter, or a glial fibrillary acidic protein (GFAP) promoter. [The present invention 1016] The nucleic acid molecule of the present invention 1015, wherein said regulatory control sequence comprises a CMV immediate early gene enhancer / CBA promoter and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). [The present invention 1017] The nucleic acid molecule of any one of 10 13 to 10 16, wherein the cell is a brain cell, a heart cell, a kidney cell, a skin cell, or a lung cell. [The present invention 1018] Any of the nucleic acid molecules of 1012 to 1017 of the present invention, having at least 90% sequence identity to SEQ ID NO:5. [The present invention 1019] The nucleic acid molecule of the present invention, which is SEQ ID NO:5. [The present invention 1020] 10. The nucleic acid molecule of any one of claims 1012 to 1019, operably linked to an expression cassette. [The present invention 1021] A cell or virus comprising any one of the nucleic acid molecules of the present invention 1012 to 1020. [The present invention 1022] A composition comprising any one of the nucleic acid molecules of the present invention 1012 to 1020. [The present invention 1023] A recombinant adeno-associated virus (rAAV), the rAAV comprises an AAV capsid and an AAV genome packaged therein; The AAV genome A nucleic acid molecule capable of expressing c-tuberin containing the hamartin binding domain and GAP domain but lacking the Akt phosphorylation site Thr 1462 The rAAV comprising: [The present invention 1024] The rAAV of the present invention, wherein the AAV capsid is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid, or a mutant of any one of these AAV capsids. [The present invention 1025] The rAAV of any one of claims 1023 to 1024, wherein the nucleic acid is operably linked to a regulatory control sequence. [The present invention 1026] The rAAV of any of claims 1023 to 1025, wherein the regulatory control sequence comprises a human cytomegalovirus (CMV) promoter, a chicken beta-actin (CBA) promoter, a Rous sarcoma virus (RSV) LTR promoter / enhancer, an SV40 promoter, a dihydrofolate reductase promoter, a phosphoglycerol kinase promoter, a CMV immediate early gene enhancer / CBA promoter, a synapsin promoter, or a glial fibrillary acidic protein (GFAP) promoter. [The present invention 1027] The rAAV of any one of claims 1023 to 1026, wherein the nucleic acid molecule comprises an ITR. [The present invention 1028] The rAAV of any one of 1023 to 1027, wherein the nucleic acid molecule contains polyA. [The present invention 1029] The rAAV of the present invention, wherein the nucleic acid molecule is SEQ ID NO:5. [The present invention 1030] A composition comprising any of the rAAVs of the present inventions 1023 to 1029 and a pharmaceutically acceptable carrier. [The present invention 1031] A method for treating a patient with tuberous sclerosis complex (TSC), comprising administering to the patient c-tuberin that contains the hamartin binding domain and GAP domain but lacks the Akt phosphorylation site Thr 1462. [The present invention 1032] 1032. The method of claim 1031, wherein a nucleic acid molecule encoding tuberin is administered to said patient. [The present invention 1033] The method of the present invention 1031, wherein the rAAV of any one of the present inventions 1022 to 1027 is administered to the patient. [The present invention 1034] The method of claim 1031, wherein extracellular vesicles (EVs) comprising the nucleic acid molecule of any one of claims 1012 to 1020 are administered to the patient. [This invention 1035] 1032. The method of claim 1031, wherein the patient has renal angiomyolipoma. [The present invention 1036] The method of the present invention 1035, wherein the c-tuberin is administered intravascularly. [This invention 1037] The method of claim 1036, wherein the c-tuberin is administered into the renal artery or renal vein. [The present invention 1038] The method of claim 1031, wherein said patient has lymphangioleiomyomatosis (LAM). [This invention 1039] The method of the present invention 1038, wherein the c-tuberin is administered intravascularly. [The present invention 1040] The method of claim 1038, wherein the c-tuberin is administered to the lungs. [This invention 1041] The method of claim 1031, wherein the patient has a brain dysfunction. [The present invention 1042] The method of the present invention 1041, wherein the c-tuberin is administered intravascularly. [This invention 1043] The method of claim 1041, wherein the c-tuberin is administered intracerebrally. [This invention 1044] 1041. The method of claim 1041, wherein the c-tuberin is administered intrathecally. [This invention 1045] The method of claim 1031, wherein the c-tuberin is administered to renal angiomyolipoma, LAM, or the brain. [The present invention 1046] The method of claim 1033, wherein the rAAV is administered to a brain cell, a heart cell, a kidney cell, a skin cell, or a lung cell. [This invention 1047] The method of claim 1033, wherein the rAAV is administered intravascularly, intravenously, intracerebrally, intracerebroventricularly, intrathecally, or skin. [This invention 1048] The method of claim 1031, wherein said patient further comprises administering rapamycin. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. [Brief explanation of the drawings]

[0056] [Figure 1A] c The amino acid sequence of tuberin (SEQ ID NO:1) is shown. [Figure 1B] c The nucleic acid sequence encoding tuberin (SEQ ID NO:5) is shown. [Figure 2A] Schematic diagram of the functional domains of the full-length human TSC1 and TSC2 proteins and the condensed tuberin (cTuberin) protein. Numbers above the arrows indicate amino acid residues. T2BD refers to the TSC2-binding domain; T1BD refers to the TSC1-binding domain; coil refers to the predicted coiled-coil domain; and GAP refers to the GTPase-activating protein, which is the domain of human tuberin that is homologous to that of Rap1 GAP. [Figure 2B] c Schematic diagram of the tuberin AAV vector. [Figure 2C] FIG. 1 is a schematic diagram of the AAV-CBA-cTuberin vector plasmid construct. [Figure 3A] 1 is a Western blot showing the expression level of c-tuberin in COS-7 cells transfected with an AAV-CBA-c-tuberin vector plasmid construct, revealing a band at approximately 85 kDa, the predicted molecular weight of c-tuberin. [Figure 3B] c Western blot showing the expression levels of pS6, S6, and GAPDH in cells transfected with various constructs. Expression of pS6 was elevated in cells lacking tuberin activity. [Figure 4A] Figure 11 is a graph showing the survival of Tsc2c / c mice injected with the AAV1-CBA-Cre vector at birth (postnatal day 0 (P0)), Tsc2c / c mice injected with AAV9-CBA-cTuberin at P21, and uninjected mice. The median survival time for mice injected with AAV1-CBA-Cre was 35 days, while the median survival time for mice injected with AAV9-CBA-cTuberin was >185 days and >175 days for uninjected mice. [Figure 4B]Figure 12 is a graph showing the survival of Tsc2c / c mice injected with the AAV1-CBA-Cre vector at P0 and mice injected with the AAV1-CBA-Cre vector at P0 followed by the AAV1-CBA-cTuberin vector at P21. Median survival was 26.5 days for uninjected mice and 35 days for AAV1-CBA-cTuberin-injected mice. [Figure 4C] Figure 12 is a graph showing the survival of Tsc2c / c mice injected with the AAV1-CBA-Cre vector at P3 and mice injected with the AAV1-CBA-Cre vector at P3 followed by the AAV1-CBA-cTuberin vector at P21. Median survival was 36 days for uninjected mice and 54 days for mice injected with AAV1-CBA-cTuberin. [Figure 4D] Figure 10 is a graph showing the survival of Tsc2c / c mice injected with the AAV1-CBA-Cre vector at P3 and mice injected with the AAV1-CBA-Cre vector at P3 followed by the AAV9-CBA-cTuberin vector at P21. Median survival was 32 days for uninjected mice and 45 days for mice injected with AAV9-CBA-cTuberin. [Figure 4E] This graph shows the survival of four groups of Tsc2c / c mice. Group 1 was injected with the AAV1-CBA-Cre vector only at P0; Group 2 was injected with the AAV1-CBA-Cre vector at P0 and then with AAV9-CBA-cTuberin at P3; Group 3 was injected with the AAV1-CBA-Cre vector only at P0 and treated with vigabatrin; and Group 4 was injected with the AAV1-CBA-Cre vector at P0 and then with AAV9-CBA-cTuberin at P3 and then treated with vigabatrin. The median survival time for uninjected, drug-free mice was 23 days, for uninjected, vigabatrin-treated mice was 27 days, for injected, drug-free mice was 35.5 days, and for injected, vigabatrin-treated mice was 40 days. [Figure 5]Figures 5A-5F show hematoxylin and eosin (H&E) staining or immunohistochemistry (IHC) staining for pS6 in the brains of mice treated according to the experimental design in Figure 4B. Figure 5A shows staining in a normal, uninjected mouse (control). Figures 5B-5E show staining in a mouse injected with the AAV1-CBA-Cre vector at P0, and Figure 5F shows staining in a mouse further treated with AAV1-CBA-cTuberin at P3. [Figure 6A] 1 shows the volume of lymphangioleiomyomatosis (LAM) tumors injected subcutaneously into NOD-SCID Il2R gamma (NSG) mice. [Figure 6B] 1 is a graph showing LAM tumor growth in uninjected mice and mice injected with AAV9-CBA-cTuberin at weeks 4 and 9. DETAILED DESCRIPTION OF THE INVENTION

[0057] Detailed Description of Embodiments of the Invention Systemic gene therapy as disclosed herein can be achieved in TSC patients by delivery (e.g., via the vasculature) of condensed tuberin (c-tuberin), such as that encoded by an AAV vector, which is useful for reducing the size of affected cells and reducing hamartomas in multiple tissues, including throughout the central nervous system, kidney, and lung. c-tuberin is useful, for example, for its ability to inhibit mTOR activity. Useful forms of c-tuberin can be identified according to any method, for example, by testing their ability to inhibit S6 phosphorylation in in vitro cells lacking tuberin, as described herein. The following describes the design of c-tuberins useful for treating TSC.

[0058] The following example describes the engineering of a condensed form of human tuberin, designated c-tuberin, that is compatible with AAV vectors. The size of the human tuberin cDNA is 5.4 kb, exceeding the transgene packaging capacity of AAV. To this end, we engineered a condensed form of tuberin by deleting the central portion of the human tuberin cDNA. Our c-tuberin retains the hamartin-binding domain at the N-terminus and the GAP domain at the C-terminus of human tuberin, but lacks the Akt phosphorylation site Thr1462, thereby reducing or eliminating Akt activation of mTORC1. The central region of the protein is replaced with a glycine-serine linker that confers conformational flexibility. This c-tuberin cDNA was then cloned into an AAV vector under the ubiquitous chicken beta-actin promoter. Transduction of mouse embryonic fibroblasts with this AAV-CBA-cTuberin construct resulted in a decrease in mTORC1 activation and therefore S6 kinase activity, a marker that defines the biological activity of cTuberin, as shown by Western blot analysis.

[0059] Ic tuberin cTuberin as described herein generally comprises a hamartin binding domain, a GTPase-activating protein (GAP) domain, and a spacer linking the hamartin binding domain to the GAP domain, and lacks the Akt phosphorylation site at Thr 1462 of human tuberin.

[0060] An exemplary c-tuberin useful for treating TSC has the amino acid sequence of SEQ ID NO: 1. In this c-tuberin (SEQ ID NO: 1), the amino acid sequence of the hamartin binding region is SEQ ID NO: 2. Also, in this c-tuberin (SEQ ID NO: 1), the amino acid sequence of the GAP region is SEQ ID NO: 3.

[0061] The hamartin-binding region and the GAP region of this c-tuberin (SEQ ID NO:1) are connected by a protein spacer sequence. In one example, the spacer sequence comprises a glycine-serine (SGGG) linker sequence, such as SEQ ID NO:4. In this case, a 16 aa linker connects the hamartin-binding region and the GAP region. Compared to human tuberin (SEQ ID NO:10), the c-tuberin protein lacks the Akt phosphorylation site Thr 1462 of human tuberin, which is one of several phosphorylation sites involved in the regulation of tuberin activity (Huang et al., Biochem. J. 412(2):179-190 2008).

[0062] In another example, cTuberin has at least 80% sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:1. In this c-tuberin, the hamartin binding region of the c-tuberin has at least 80% sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:2, and the GAP region of the c-tuberin has at least 80% sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:3. In some c-tuberins, the hamartin binding region and the GAP region of the c-tuberin are connected by a protein spacer sequence. In one example, the spacer sequence comprises a glycine-serine (SGGG) linker sequence, e.g., SEQ ID NO:4.

[0063] The c-tuberin of SEQ ID NO:1 described herein was generated using human tuberin, the amino acid and nucleic acid sequences of which can be found under NCBI accession number NP_000539.2 and GenBank accession number X75621.1, respectively. Other human tuberin isoforms can also be used to generate additional c-tuberins. Exemplary human tuberin isoforms useful for generating such molecules include tuberin isoform 4 (NCBI accession number NP_001070651.1), tuberin isoform 5 (NCBI accession number NP_001107854.1), tuberin isoform 6 (NCBI accession number NP_001305756.1), tuberin isoform 7 (NCBI accession number NP_001305758.1), tuberin isoform 8 (NCBI accession number NP_001305760.1), and tuberin isoform 9 (NCBI accession number NP_001305761.1). Examples of tuberin isoforms include, but are not limited to, tuberin isoform X1 (NCBI accession number XP_024306181.1), tuberin isoform X8 (NCBI accession number XP_005255586.2), tuberin isoform X9 (NCBI accession number XP_016879105.1), tuberin isoform X10 (NCBI accession number XP_005255588.2), tuberin isoform X11 (NCBI accession number XP_016879106.1), and tuberin isoform X12 (NCBI accession number XP_016879107.1). Such tuberins are useful for engineering any of the c-tuberins described herein.

[0064] For example, c-tuberin can be engineered using human tuberin isoform 4 (NCBI accession number NP_001070651.1), which is used to generate a hamartin binding region amino acid sequence having at least 80% sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:2, and a GAP region amino acid sequence having at least 80% sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:3, as described for c-tuberin (SEQ ID NO:1). Furthermore, the hamartin binding domain and the GAP domain can be connected by a protein spacer sequence. In one example, the spacer sequence comprises a glycine-serine (SGGG) linker sequence, such as SEQ ID NO: 4. cTuberin, engineered from human tuberin isoform 4, further lacks the Akt phosphorylation site.

[0065] II.c Tuberin nucleic acid molecule Furthermore, the exemplary c-tuberin of SEQ ID NO:1 is encoded by a nucleic acid molecule having the sequence of SEQ ID NO:5. In this c-tuberin nucleic acid molecule (SEQ ID NO:5), the hamartin binding region is encoded by SEQ ID NO:6. Also, in this c-tuberin nucleic acid molecule (SEQ ID NO:5), the GAP region is encoded by SEQ ID NO:7.

[0066] In this exemplary c-tuberin of SEQ ID NO:1, encoded by the nucleic acid molecule of SEQ ID NO:5, the hamartin binding region and the GAP region are linked by a protein spacer sequence, i.e., the glycine-serine linker of SEQ ID NO:4, which is encoded by SEQ ID NO:8.

[0067] In a further embodiment, the nucleic acid encoding ctuberin has at least 80% sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:5. In this c-tuberin, the hamartin binding region is encoded by a nucleic acid having at least 80% sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:6, and the GAP region is encoded by a nucleic acid having at least 80% sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:7. Furthermore, the nucleic acid encoding c-tuberin may include a sequence encoding a protein spacer, for example, SEQ ID NO:8.

[0068] The cTuberin nucleic acid molecule may be codon-optimized for expression in human cells. Furthermore, the cTuberin nucleic acid molecule may be operably linked to regulatory control sequences, such as, for example, the CMV immediate early gene enhancer / CBA promoter and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), or, but not limited to, the human cytomegalovirus (CMV) promoter, chicken beta-actin (CBA) promoter, Rous sarcoma virus (RSV) LTR promoter / enhancer, SV40 promoter, dihydrofolate reductase promoter, phosphoglycerol kinase promoter, CMV immediate early gene enhancer / CBA promoter, synapsin promoter, or glial fibrillary acidic protein (GFAP) promoter. The nucleic acid molecule may also be operably linked to an expression cassette.

[0069] For example, the cTuberin nucleic acid molecule (SEQ ID NO:5) can be inserted under the CBA promoter with a Kozak sequence, followed by the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and the SV40 and bovine growth hormone polyadenylation signals. The vector is inserted into an AAV2-LTR backbone and flanked by AAV2 ITR sequences.

[0070] Additionally, the cTuberin nucleic acid molecule (SEQ ID NO:5) may be incorporated into a vector plasmid genome. An exemplary vector plasmid genome comprising the cTuberin nucleic acid molecule (SEQ ID NO:5) has the sequence of SEQ ID NO:11.

[0071] III. Recombinant AAV molecules Any suitable nucleic acid vector can be used in conjunction with the compositions and methods of the present invention to design and assemble the components of a ctuberin-encoding nucleic acid molecule and a recombinant adeno-associated virus (AAV). The rAAV vectors useful in the compositions and methods described herein are recombinant nucleic acid constructs containing (1) a heterologous sequence to be expressed (e.g., a ctuberin-encoding nucleic acid molecule) and (2) viral sequences that facilitate integration and expression of the heterologous gene. The viral sequences may include AAV sequences required in cis for DNA replication and packaging into virions (e.g., functional ITRs). Such rAAV vectors may also contain marker or reporter genes. Useful rAAV vectors lack all or part of one or more AAV WT genes but retain functional flanking ITR sequences. The AAV ITRs may be of any serotype appropriate for the particular application. Methods for using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279-291 (2000) and Monahan et al., Gene Therapy. 7:24-30 (2000), the disclosures of each of which, when relevant to AAV vectors for gene delivery, are incorporated herein by reference.

[0072] In one embodiment, the vector is a recombinant AAV that carries a c-tuberin nucleic acid molecule and is driven by a promoter that expresses the c-tuberin molecule in selected cells of the subject.Methods for assembling recombinant vectors are known in the art.See, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989;Kay, MA et al., Nat. Med. 7(1):33-40 (2001); and Walther W. and Stein U., Drugs 2000, 60(2):249-71.

[0073] In certain embodiments described herein, c-tuberin nucleic acid molecules are delivered to selected cells in need of treatment, such as brain cells, heart cells, kidney cells, skin cells, or lung cells, using AAV vectors according to standard methods known in the art. More than 30 natural AAV serotypes are available. Many natural variants of AAV capsids exist, allowing AAVs with properties particularly suited to selected cells to be identified and used. By manipulating the AAV virus using conventional molecular biology techniques, these particles can be optimized for cell-specific delivery of c-tuberin nucleic acid molecule sequences, minimized immunogenicity, tailored stability and particle lifespan, efficient degradation, and precise intracellular delivery (e.g., into the nucleus).

[0074] Expression of the ctuberin nucleic acid molecules described herein can be achieved in selected cells through delivery by recombinantly engineered or engineered AAVs containing sequences encoding the desired ctuberin nucleic acid molecules. The use of AAVs is a common method of exogenous DNA delivery because they are relatively non-toxic, provide efficient gene transfer, and can be easily optimized for specific purposes. Among the well-characterized serotypes of AAV isolated from humans or non-human primates, human serotype 2 has been widely used for efficient gene transfer experiments in various target tissues and animal models. Other AAV serotypes include, but are not limited to, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or hybrid serotypes thereof. Unless otherwise specified, AAV ITRs and other selected AAV components described herein can be readily selected from any AAV serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, hybrid serotypes thereof, or other known and unknown AAV serotypes. In one embodiment, the ITRs are derived from AAV2. Such ITRs or other AAV components can be readily isolated from AAV serotypes using techniques available to those of skill in the art. Such AAVs can be isolated or obtained from academic, commercial, or public sources (e.g., American Type Culture Collection, Manassas, VA). Alternatively, AAV sequences can be obtained by synthetic or other suitable means by reference to published sequences such as those provided in the literature or databases such as GenBank, PubMed, etc.

[0075] Desirable AAV fragments for assembly into vectors include cap proteins including vp1, vp2, vp3, and hypervariable regions, rep proteins including rep78, rep68, rep52, and rep40, and sequences encoding these proteins. These fragments can be readily utilized in a variety of vector systems and host cells. Such fragments can be used alone, in combination with other AAV serotype sequences or fragments, or in combination with elements from other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, but are not limited to, AAVs containing non-native capsid proteins. Selected AAV sequences (e.g., fragments of the vp1 capsid protein) can be used in combination with heterologous sequences that can be obtained from a selected different AAV serotype, non-contiguous portions of the same AAV serotype, non-AAV viral sources, or non-viral sources to generate such artificial capsids by any suitable technique. Artificial AAV serotypes can be, but are not limited to, pseudotyped AAV, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids. Pseudotype vectors that utilize the capsid of one AAV with ITRs from an AAV with different capsid proteins are useful in the compositions and methods described herein.

[0076] In one example, the AAV comprises a capsid sequence derived from AAV1. In another embodiment, the AAV comprises a capsid sequence derived from AAV9. The use of AAV1 and AAV9 has been previously described in Broekman et al., Neuroscience. 138:501-510, 2006, which is incorporated herein by reference.

[0077] In another example, vectors useful in the compositions and methods described herein contain sequences encoding a selected AAV serotype capsid, e.g., AAV1 or AAV9 capsid, or fragments thereof. Other useful vectors contain sequences encoding a selected AAV serotype rep protein, e.g., AAV1 or AAV9 rep protein, or fragments thereof. Such vectors may optionally contain both AAV cap and rep proteins. In vectors providing both AAV rep and cap, the AAV rep and AAV cap sequences can both be from a single serotype source, e.g., AAV1 or AAV9.

[0078] Alternatively, a vector may be used in which the rep sequence is derived from a different AAV serotype than that which provides the cap sequence. In one embodiment, the rep sequence and the cap sequence are expressed from separate sources (e.g., separate vectors, or the host cell and the vector). In another embodiment, the rep sequences are fused in-frame to the cap sequence of a different AAV serotype to form a chimeric AAV vector as described in U.S. Patent No. 7,282,199, which is incorporated herein by reference.

[0079] Suitable recombinant AAV (rAAV) is produced by culturing host cells that contain a nucleic acid sequence encoding an AAV serotype capsid protein or fragment thereof as defined herein; a functional rep gene; a minigene composed, for example, of AAV ITRs and c-tuberin nucleic acid sequences; and sufficient helper functions to allow packaging of the minigene into an AAV capsid protein. Components required for culturing the host cell to package the AAV minigene into an AAV capsid may be provided to the host cell in trans. Alternatively, one or more required components (e.g., minigene, rep sequence, cap sequence, and / or helper functions) may be provided by a stable host cell engineered to contain one or more required components using methods known to those skilled in the art.

[0080] In one example, the AAV contains a promoter (or a functional fragment of a promoter). The promoter for use in the rAAV can be selected from among many constitutive or inducible promoters known in the art, which can express a selected transgene in the desired target cell. In one embodiment, the promoter is cell-specific. The term "cell-specific" means that the particular promoter selected for the recombinant vector is capable of directing expression of the selected transgene in a specific cell type. In one embodiment, the promoter is specific for expression of the transgene in brain cells, heart cells, kidney cells, skin cells, or lung cells.

[0081] In another embodiment, the promoter is the native promoter of the target gene to be expressed. Useful promoters include, but are not limited to, the human cytomegalovirus (CMV) promoter, chicken beta-actin (CBA) promoter, Rous sarcoma virus (RSV) LTR promoter / enhancer, SV40 promoter, dihydrofolate reductase promoter, phosphoglycerol kinase promoter, CMV immediate early (IE) enhancer / CBA promoter, synapsin promoter, and glial fibrillary acidic protein (GFAP) promoter.

[0082] Other conventional regulatory sequences for inclusion in minigenes or rAAVs are known in the art, and one of skill in the art can select from among these and other expression control sequences without departing from the scope described herein.

[0083] The AAV minigene can comprise the c-tuberin nucleic acid molecule and its regulatory sequences described herein, as well as the 5' and 3' AAV ITRs. In one embodiment, the ITRs of AAV serotype 2 are used; however, ITRs from other suitable serotypes may also be selected. In some embodiments, the minigene is packaged into capsid proteins and delivered to a selected host cell.

[0084] The minigene, rep sequence, cap sequence, and helper functions required for rAAV production can be delivered to packaging host cells in the form of any genetic element that introduces the sequences carried by the rAAV. The selected genetic element can be delivered by any suitable method, including those described herein. The methods used to construct any of the embodiments described herein are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for producing rAAV virions are well known, and the selection of a suitable method does not limit the present invention. See, e.g., K. Fisher et al., J. Virol., 1993 70:520-532 and U.S. Patent No. 5,478,745, each of which is incorporated herein by reference.

[0085] In another example, the c-tuberin minigene is prepared in a proviral plasmid, such as that disclosed in International Publication No. 2012 / 158757, which is incorporated herein by reference. Such a proviral plasmid contains a modular recombinant AAV genome containing functional junctions, including a wild-type 5' AAV2 ITR sequence flanked by unique restriction sites that allow for easy removal or replacement of the ITRs; a promoter containing a cytomegalovirus (CMV)-chicken beta-actin sequence upstream of a cytomegalovirus sequence, or a cell-specific promoter / enhancer, where the promoter is flanked by unique restriction sites that allow for easy removal or replacement of the entire promoter sequence, and the upstream sequence is flanked by unique restriction sites that allow for easy removal or replacement of only the upstream CMV or enhancer sequence from the promoter sequence. The c-tuberin nucleic acid molecule described herein can be inserted into the site of a multiple cloning polylinker, where the c-tuberin nucleic acid molecule is operably linked to and under the regulatory control of the promoter. Also part of this plasmid are a bovine growth hormone polyadenylation sequence flanked by unique restriction sites that allow for easy removal or replacement of the polyA sequence, and a wild-type 3' AAV2 ITR sequence flanked by unique restriction sites that allow for easy removal or replacement of the 3' ITR. The plasmid backbone contains the elements necessary for replication in bacterial cells and is itself flanked by transcription terminator / insulator sequences.

[0086] In yet another embodiment, the proviral plasmid contains a modular recombinant AAV genome comprising functional links: (i) wild-type 5' AAV2 ITR sequences flanked by unique restriction sites allowing for facile removal or replacement of the ITRs; (ii) (A) a CMV immediate-early enhancer sequence upstream of the CMV-chicken beta-actin sequence; or (B) a promoter comprising a cell-specific promoter / enhancer, including, for example, the human cytomegalovirus (CMV) promoter, chicken beta-actin (CBA) promoter, Rous sarcoma virus (RSV) LTR promoter / enhancer, SV40 promoter, dihydrofolate reductase promoter, phosphoglycerol kinase promoter, CMV immediate-early gene enhancer / CBA promoter, synapsin promoter, or glial fibrillary acidic protein (GFAP) promoter. The promoter is flanked by unique restriction sites that allow for easy removal or replacement of the entire promoter sequence, and the upstream sequence is flanked by unique restriction sites that allow for easy removal or replacement of only the upstream CMV or enhancer sequence from the promoter sequence. Also included in this proviral plasmid are a multicloning polylinker sequence that allows for the insertion of a c-tuberin nucleic acid sequence (the c-tuberin nucleic acid molecule is operably linked to and under the regulatory control of the promoter), including any of those described herein; a bovine growth hormone polyadenylation sequence flanked by unique restriction sites that allow for easy removal or replacement of the polyA sequence; and a wild-type 3' AAV2 ITR sequence flanked by unique restriction sites that allow for easy removal or replacement of the 3' ITR. The proviral plasmid also includes a plasmid backbone that contains elements necessary for replication in bacterial cells and further contains a kanamycin resistance gene, and the plasmid backbone is flanked by transcription terminator / insulator sequences. The proviral plasmids described herein may also contain a non-coding 5.1 kb stuffer sequence of lambda phage in the plasmid backbone to increase the backbone length and prevent reverse packaging of non-functional AAV genomes.

[0087] In some embodiments, the proviral plasmid contains multiple copies of the c-tuberin nucleic acid molecule. Thus, for example, a c-tuberin nucleic acid molecule that is less than half the upper packaging limit of AAV may be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times on a single proviral plasmid.

[0088] In yet another aspect, the promoter of the proviral plasmid is modified to reduce the size of the promoter, allowing for the insertion of larger c-tuberin nucleic acid molecule sequences into the rAAV. In one embodiment, the CMV / CBA hybrid promoter, which normally contains a total of about 1,000 base pairs of non-coding exons and introns, is replaced with a 130 base pair chimeric intron, as known in the art.

[0089] These proviral plasmids are then used in existing conventional packaging techniques to generate recombinant viruses expressing the c-tuberin molecule transgene carried by the proviral plasmid. Suitable producer cell lines can be readily selected by those skilled in the art. Suitable host cells can be selected from any biological organism, including, for example, prokaryotic (e.g., bacterial) cells and eukaryotic cells, including insect cells, yeast cells, and mammalian cells. Briefly, the proviral plasmid can be transfected into the selected packaging cells to allow transient expression. Alternatively, a minigene or gene expression cassette with flanking ITRs can be stably integrated into the host cell genome, either chromosomally or episomally. Suitable transfection techniques for delivering recombinant AAV genomes into host cells are known and readily available. Typically, the proviral plasmid is cultured in host cells that express cap and / or rep proteins. In the host cells, a minigene consisting of a c-tuberin nucleic acid molecule with flanking AAV ITRs is rescued and packaged into capsid or envelope proteins to form infectious viral particles. Thus, recombinant AAV infectious particles are produced by culturing packaging cells carrying the proviral plasmid in the presence of sufficient viral sequences to allow packaging of the viral genome of the gene expression cassette into an infectious AAV envelope or capsid.

[0090] IV. Extracellular vesicles Extracellular vesicles (EVs) are useful in the methods and compositions described herein.For example, EVs containing any c-tuberin described herein can be administered to subjects according to standard methods.Furthermore, for example, EVs containing any nucleic acid molecule encoding c-tuberin can also be administered to subjects.

[0091] Extracellular vesicles (EVs) are fragments of plasma membrane, ranging from 20 nm to 10 μm, released from nearly all cell types, including, but not limited to, exosomes, microvesicles, microparticles, circulating microvesicles, shed microvesicles, nanovesicles, nanoparticles, apoptotic bodies, and membrane vesicles. Microvesicles act as intercellular signaling mediators and can transport mRNA, miRNA, and proteins between cells. As will be apparent to those skilled in the art, there are a variety of EV isolation and purification protocols based on filtration, differential centrifugation, ultracentrifugation, vesicle flotation on density gradients (sucrose, OptiPrep™), and immunoaffinity capture using antibodies against membrane proteins. Exemplary information for isolating extracellular vesicles can be found in Simpson RJ, Mathivanan S (2012) Extracellular Microvesicles: The Need for Internationally Recognized Nomenclature and Stringent Purification Criteria. J Proteomics Bioinform 5: ii-ii; van der Pol et al., Classification, functions, and clinical relevance of extracellular vesicles, Pharmacol Rev. 2012 July;64(3):676-705; Raposo and Stoorvogel, Extracellular vesicles: exosomes, microvesicles, and friends, J Cell Biol. 2013 February 18;200(4):373-83; and Witwer et al., Standardization of sample collection, isolation, and analysis methods in extracellular vesicle research, J Extracell Vesicles. 2013 May 27;2, which are incorporated herein by reference in their entireties.See also Sarkar el al., 2009, Taylor and Gercel-Taylor, 2008, and Balaj et al., 2011, which are incorporated by reference in their entireties.

[0092] Typically, any of the c-tuberins described herein are loaded into EVs according to standard procedures. For example, EVs can be loaded with c-tuberin of SEQ ID NO:1.

[0093] Similarly, EVs can be loaded with any of the c-tuberin-encoding nucleic acid molecules described herein. The nucleic acid molecule can be integrated into the AAV genome. Furthermore, the nucleic acid molecule can be operably linked to regulatory control sequences, including, for example, the human cytomegalovirus (CMV) promoter, chicken beta-actin (CBA) promoter, Rous sarcoma virus (RSV) LTR promoter / enhancer, SV40 promoter, dihydrofolate reductase promoter, phosphoglycerol kinase promoter, CMV immediate early gene enhancer / CBA promoter, synapsin promoter, or glial fibrillary acidic protein (GFAP) promoter. In one example, the regulatory control sequence includes the CMV immediate early gene enhancer / CBA promoter and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). As a further example, the nucleic acid molecule can include ITRs. In another example, the nucleic acid molecule includes poly(A).

[0094] Any of the EVs described herein may also be included in a composition comprising a pharmaceutically acceptable carrier.

[0095] V. Pharmaceutical Compositions and Kits Provided herein are pharmaceutical compositions comprising a ctuberin nucleic acid molecule, an EV comprising a ctuberin nucleic acid molecule (e.g., rAAV) described herein, or an rAAV comprising a ctuberin nucleic acid molecule as described herein. Such pharmaceutical compositions comprise either a ctuberin nucleic acid molecule or ctuberin described herein.

[0096] The pharmaceutical compositions described herein can be assessed for contamination by conventional methods and then formulated into pharmaceutical compositions intended for a suitable route of administration. Additional compositions containing c-tuberin nucleic acid molecules, EVs containing c-tuberin nucleic acid molecules, or rAAVs containing c-tuberin nucleic acid molecules may be similarly formulated with a suitable carrier. Such formulations include the use of pharmaceutically and / or physiologically acceptable vehicles or carriers specifically intended for administration to target cells. In one embodiment, carriers suitable for administration to target cells include buffered saline, isotonic sodium chloride solution, or other buffers to maintain pH at appropriate physiological levels, such as HEPES, as well as, optionally, other agents, pharmaceuticals, stabilizers, buffers, carriers, adjuvants, diluents, etc.

[0097] Typically, the carrier is an injectable solution.Exemplary physiologically acceptable carriers include sterile pyrogen-free water and sterile pyrogen-free phosphate buffered saline.In one embodiment, the carrier is an isotonic sodium chloride solution.In another example, the carrier is a balanced salt solution.Other carriers include Tween.When the virus is stored for a long period of time, it may be frozen in the presence of glycerol or Tween 20.

[0098] Compositions containing the c-tuberin nucleic acid molecules described herein may also contain a surfactant. The inclusion of a useful surfactant, such as Pluronic F68 (Poloxamer 188, also known as LUTROL® F68), can prevent AAV from adhering to inert surfaces, thereby ensuring delivery of the desired dose. For example, one exemplary composition designed for the treatment of a disease or disorder caused by a TSC2 mutation described herein, such as tuberous sclerosis, comprises a recombinant adeno-associated vector carrying a nucleic acid sequence encoding a c-tuberin described herein under the control of a regulatory sequence that expresses the c-tuberin nucleic acid molecule in brain, heart, kidney, skin, or lung cells of a mammalian subject, and a pharmaceutically acceptable carrier. The carrier is an isotonic sodium chloride solution and includes the surfactant Pluronic F68. In one embodiment, the c-tuberin nucleic acid molecule is any of those described herein.

[0099] In yet another exemplary embodiment, a composition comprises a recombinant AAV1 or AAV9 pseudotyped adeno-associated virus carrying an alternative c-tuberin nucleic acid molecule, where the nucleic acid sequence is under the control of a promoter that directs expression of the c-tuberin nucleic acid molecule in brain, heart, kidney, skin, or lung cells, wherein the composition is formulated with a carrier and additional components suitable for cutaneous administration or intravascular, intraventricular, intracranial, or intrathecal injection. In yet another embodiment, compositions or components for making or assembling this composition, including carriers, rAAV particles, surfactants, and / or components for producing rAAV, as well as laboratory hardware suitable for preparing the composition, can be incorporated into a kit.

[0100] Further provided herein are kits comprising a first pharmaceutical composition comprising a c-tuberin nucleic acid molecule and a second pharmaceutical composition comprising a drug used in the treatment of tuberous sclerosis, e.g., rapamycin and its analogs. In some embodiments, the kit includes instructions for mixing the two pharmaceutical compositions prior to administration.

[0101] VI. Method The above compositions are useful in methods for treating diseases or disorders caused by mutations in TSC2 by replacing the normal allele of TSC2. Such methods include contacting a target TSC2 gene with a c-tuberin nucleic acid molecule described herein under conditions that deliver the c-tuberin nucleic acid molecule to selected cells and correct TSC2 expression in the target cells. Thus, the methods and compositions are used to treat diseases or disorders caused by specific mutations and / or mutations in TSC2 associated with gene expression.

[0102] In some embodiments, the c-tuberin nucleic acid molecule, the EV comprising the c-tuberin nucleic acid molecule, or the rAAV comprising the c-tuberin nucleic acid molecule is administered to brain cells, heart cells, kidney cells, skin cells, or lung cells. In some embodiments, the c-tuberin nucleic acid molecule, the EV comprising the c-tuberin nucleic acid molecule, or the rAAV comprising the c-tuberin nucleic acid molecule is administered to the affected subject percutaneously or by intravascular, intraventricular, intracranial, or intrathecal injection.

[0103] In some embodiments, the method comprises administering a c-tuberin nucleic acid molecule, an EV comprising a c-tuberin nucleic acid molecule, or an rAAV comprising a c-tuberin nucleic acid molecule to treat a subject with a disorder associated with a TSC2 mutation, such as tuberous sclerosis complex. Such selection can be based on the subject's genotype. In some embodiments, the disorder associated with TSC2 can be an autosomal dominant disorder. In some cases, the subject is homozygous or compound heterozygous for a TSC2 mutation. Methods for screening and identifying specific mutations in TSC2 are known in the art.

[0104] Dosage and Combination Therapy In this specification, standard administration method is used.In addition, the administration for treating renal angiomyolipoma, lymphangioleiomyomatosis (LAM) and brain dysfunction, and the administration to brain cells, heart cells, kidney cells, skin cells or lung cells are described below.The administration method for administration by percutaneous, intravascular, intracerebral, intraventricular or intrathecal injection is also described.

[0105] The effective concentration of recombinant adeno-associated virus carrying the c-tuberin nucleic acid molecule described herein is about 10 9 ~10 15 The effective concentration is in the range of genome copies (gc) / kg body weight of the subject (gc / kg). For example, the effective concentration is 10 9 ~10 15 gc / kg range, e.g., 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , and 10 15 In another example, the effective concentration is 10 10 ~10 13 gc / kg range, e.g., 10 10 , 10 11 , 10 12 , and 10 13 gc / kg. Further dosages within the above ranges or in other units may be selected by the attending physician, taking into consideration the physical condition of the subject being treated, including the age of the subject; the composition being administered, and the particular disorder; and, if progressive, the target cells in which the disorder is manifested and the extent to which it occurs.

[0106] renal angiomyolipoma For example, rAAV carrying a c-tuberin nucleic acid molecule can be used to treat patients with renal angiomyolipoma. The rAAV can be administered to patients by any method, such as by intravascular injection into the renal artery or renal vein. An effective dose of rAAV for treating renal angiomyolipoma by intravascular injection is 10 9 ~10 15 In one embodiment, the amount of rAAV administered to a patient is about 10 9 In a further embodiment, the amount of rAAV administered to the patient is about 10 10 In a further embodiment, the amount of rAAV administered to the patient is about 10 11 In a further embodiment, the amount of rAAV administered to the patient is about 10 12 In a further embodiment, the amount of rAAV administered to the patient is about 1013 In a further embodiment, the amount of rAAV administered to the patient is about 10 14 In a further embodiment, the amount of rAAV administered to the patient is about 10 15 gc / kg.

[0107] LAM rAAV carrying a c-tuberin nucleic acid molecule can also be used to treat patients with lymphangioleiomyomatosis (LAM). The rAAV can be administered to patients by any method, such as intravascular injection. An effective dose of rAAV for the treatment of LAM by intravascular injection is 10 9 ~10 15 In one embodiment, the amount of rAAV administered to a patient is about 10 9 In a further embodiment, the amount of rAAV administered to the patient is about 10 10 In a further embodiment, the amount of rAAV administered to the patient is about 10 11 In a further embodiment, the amount of rAAV administered to the patient is about 10 12 In a further embodiment, the amount of rAAV administered to the patient is about 10 13 In a further embodiment, the amount of rAAV administered to the patient is about 10 14 In a further embodiment, the amount of rAAV administered to the patient is about 10 15 Additionally, rAAV can be administered to patients with LAM via the nasal route or other medically approved pulmonary routes.

[0108] Brain dysfunction In another example, rAAV carrying c-tuberin nucleic acid molecules can be used in patients with brain dysfunction. rAAV can be administered to patients according to any method (e.g., intravascular, intraventricular, intracranial, or intrathecal injection).

[0109] For the treatment of brain dysfunction by intravascular injection, the effective dose of rAAV is 10 9 ~1015 In one embodiment, the amount of rAAV administered to a patient is about 10 9 In a further embodiment, the amount of rAAV administered to the patient is about 10 10 In a further embodiment, the amount of rAAV administered to the patient is about 10 11 In a further embodiment, the amount of rAAV administered to the patient is about 10 12 In a further embodiment, the amount of rAAV administered to the patient is about 10 13 In a further embodiment, the amount of rAAV administered to the patient is about 10 14 In a further embodiment, the amount of rAAV administered to the patient is about 10 15 gc / kg.

[0110] Furthermore, for the treatment of brain dysfunction by intraventricular injection, the effective dose of rAAV is 10 10 ~10 13 In one embodiment, the amount of rAAV administered to a patient is about 10 10 In a further embodiment, the amount of rAAV administered to the patient is about 10 11 In a further embodiment, the amount of rAAV administered to the patient is about 10 12 In a further embodiment, the amount of rAAV administered to the patient is about 10 13 gc / kg.

[0111] Furthermore, for the treatment of brain dysfunction by intracranial injection, the effective dose of rAAV is 10 10 ~10 13 In one embodiment, the amount of rAAV administered to a patient is about 10 10 In a further embodiment, the amount of rAAV administered to the patient is about 10 11 In a further embodiment, the amount of rAAV administered to the patient is about 10 12 In a further embodiment, the amount of rAAV administered to the patient is about 10 13 gc / kg.

[0112] Furthermore, for the treatment of brain dysfunction by intrathecal injection, the effective dose of rAAV is 10 10 ~10 13 In one embodiment, the amount of rAAV administered to a patient is about 10 10 In a further embodiment, the amount of rAAV administered to the patient is about 10 11 In a further embodiment, the amount of rAAV administered to the patient is about 10 12 In a further embodiment, the amount of rAAV administered to the patient is about 10 13 gc / kg.

[0113] Delivery Depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method, the composition can be delivered in a volume of about 50 μL to about 1 mL, including all numbers within that range. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 70 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 250 μL. In another embodiment, the volume is about 300 μL. In another embodiment, the volume is about 350 μL. In another embodiment, the volume is about 400 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 750 μL. In another embodiment, the volume is about 850 μL. In another embodiment, the volume is about 1,000 μL.

[0114] In one embodiment, the volume and concentration of rAAV composition are selected so that only a specific anatomical region with target cells is affected.In another embodiment, the volume and / or concentration of rAAV composition are increased to reach a wider area of target organ, such as the brain, heart, kidney, skin or lung.Similarly, when administered to other organs, the dosage is adjusted accordingly.

[0115] This document provides a method for treating tuberous sclerosis in patients.In some embodiments, the present invention provides a method for treating renal angiomyolipoma, LAM or brain dysfunction for subjects.For each of the methods described, this treatment can be used to prevent further damage or to restore the tissue with mild or progressive disease.As used herein, the term "restore" refers to preventing the progression of disease, preventing the spread of damage to non-damaged cells, or improving the damage of damaged cells.

[0116] Thus, in one embodiment, the composition is administered before the onset of disease. In another embodiment, the composition is administered before the onset of symptoms. In another embodiment, the composition is administered after the onset of symptoms. In yet another embodiment, the composition is administered when less than 90% of the target cells are functioning or surviving, e.g., compared to a reference tissue. In yet another embodiment, the composition is administered when more than 10% of the target cells are functioning or surviving, e.g., compared to a reference tissue. In yet another embodiment, the composition is administered when more than 20% of the target cells are functioning or surviving. In yet another embodiment, the composition is administered when more than 30% of the target cells are functioning or surviving.

[0117] In yet another embodiment, any of the above methods is performed in combination with another treatment, i.e., a secondary treatment. The treatment can be any currently known or as yet unknown treatment that helps prevent, inhibit, or ameliorate such mutations or defects, or any effects associated therewith. The secondary treatment can be administered before, simultaneously with, or after administration of a c-tuberin nucleic acid molecule or an rAAV carrying such a c-tuberin nucleic acid molecule. In one embodiment, the secondary treatment includes treatment of seizures in a subject, including, for example, administration of an anticonvulsant drug. In a further embodiment, the secondary treatment includes administration of rapamycin. In a further embodiment, the secondary treatment includes co-administration of rapamycin. The administration or co-administration of rapamycin can be for a subject with tuberous sclerosis. Furthermore, the administration or co-administration of rapamycin can be for a subject with renal angiomyolipoma, LAM, or brain dysfunction. In some embodiments, the administration or co-administration of rapamycin can be during infantile spasms in early childhood. In a further embodiment, administration or co-administration of rapamycin can occur after detection of subependymal overgrowth, e.g., by MRI, or at any time thereafter due to symptoms caused by overgrowth due to loss of somatic tuberin function.

[0118] When used in these methods, the amount and viral titer of each injection is determined individually, and dosage, administration, and regimen can be determined by the attending physician in view of the teachings of the present disclosure. [Example]

[0119] The following are examples of methods and compositions of the present invention. It will be understood that various other embodiments can be practiced in view of the general description provided above.

[0120] Example 1. Cell culture Tsc2 null mouse embryonic fibroblasts (MEFs) (Huang et al., Biochem. J. 412(2):179-190 2008) and immortalized TRI102 human angiomyolipoma cells (Hong et al., Mol. Cell. 30:701-711, 2008; Yu et al., Am. J. Physiol. Lung Cell. Mol. Physiol. 286:L694-L700, 2004) were grown in Dulbecco's modified Eagle's medium (DMEM) (Cellgro®, Manassas, VA) growth medium supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich®, St. Louis, MO) and 1% penicillin / streptomycin (Cellgro®), and cells were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Lentiviral vectors were generated using CSCW-IG (Sena-Esteves et al., J. Virol. Methods. 122(2):131-139, 2004), a self-inactivating lentiviral vector with a CMV promoter controlling the expression of both the transgene and GFP cDNA, separated by an IRES element. cDNAs encoding Fluc (pGL3-basic; Promega®, Madison, WI) and monomeric red fluorescent protein (mCherry) (Rizzo et al., 2004) were amplified by PCR. The Fluc sequence was inserted directly into the Nhe I site downstream of the CMV promoter, and the mCherry sequence was inserted into the Bsa I and Sal I sites in place of the GFP cDNA, generating pCSCW-Fluc-IRES-mCherry. The lentiviral vector was administered at a concentration of 10 per ml. 8 ~10 10Routine titers of transducing units (tu) were generated as described (Sena-Esteves et al., J. Virol. Methods. 122(2):131-139, 2004). To obtain stable expression of Fluc and mCherry in lymphangioleiomyomatosis (LAM) cells, we infected them with CSCW-Fluc-IRES-mCherry lentivirus at a multiplicity of infection (MOI) of 100, resulting in over 90% susceptibility (the cell line designated TSC2-LAM-FC). COS-7 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin and transfected with a plasmid vector containing c-tuberin cDNA (pAAV-CBA-c-tuberin) using Lipofectamine™ 3000 (Thermo Fisher Scientific®, Waltham, MA).

[0121] Example 2. AAV Vector Design and Packaging The AAV vector plasmid AAV-CBA-Cre-BGHpA was derived as described by Prabhakar et al., PLoS One. 8(5):e64224, 2013. These AAV vectors contain AAV2 ITR elements, and gene expression is controlled by a hybrid promoter (chicken β-actin (CBA)) consisting of the CMV immediate-early gene enhancer fused to the β-actin promoter (Gray et al., Hum. Gene Ther. 22:1143-1153, 2011). The AAV vector plasmid AAV-CBA-cTuberin was derived from the plasmid pAAV-CBA-W (CSCW-IG) (Sena-Esteves et al., J. Virol. Methods. 122(2):131-139, 2004). This vector contains a CBA promoter driving c-tuberin, followed by the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and polyadenylation (polyA) signal sequences from SV40 and bovine growth hormone (BGH) (Figure 2C). The condensed tuberin (c-tuberin) construct contains ACC (Kozak sequence)::amino acids 1-450 of human tuberin::gly / ser linker::amino acids 1515-1807 of human tuberin::cmyc tag. The 2,307 bp cDNA sequence encodes an 85 kDa protein (Figure 1A).

[0122] AAV1 and AAV9 serotype vectors were generated by transient cotransfection of 293T cells with the vector plasmid (AAV-CBA-ctuberin-cmyc), the adenovirus helper plasmid pFΔ6, and a plasmid encoding the AAV9 (pXR9) or AAV1 (pXR1) capsid gene via calcium phosphate precipitation, as previously described in Broekman et al., Neuroscience. 138:501-510, 2006. The identity of all PCR-amplified sequences was confirmed by sequencing. Briefly, AAV vectors were purified by iodixanol density gradient centrifugation. Virus-containing fractions were concentrated using Amicon® Ultra 100 kDa MWCO centrifugal devices (EMD Millipore®, Billerica, MA), and titers (genome copies (gc) / ml) were determined by real-time PCR amplification using primers and a probe specific for the bovine growth hormone polyadenylation signal.

[0123] Example 3. Western Blot Briefly, cultured cells were lysed in lysis buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 2 mM EDTA, 2.5% sodium dodecyl sulfate, 2% CHAPS, 2.5 mM sucrose, 10% glycerol, 10 mM sodium fluoride, 2 mM sodium vanadate, 1 mM PMSF, 10 mM sodium pyrophosphate, protease inhibitor cocktail). After sonication and incubation at 8°C for 10 min, samples were centrifuged at 14,000 g for 30 min at 8°C. Equal amounts of protein, determined by a detergent-compatible protein assay kit (Bio-Rad®, Hercules, CA), were boiled for 5 min in Laemmli sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose membranes (Bio-Rad®). Equal protein loading was confirmed by Ponceau S staining. The membranes were blocked with 2% blocking reagent (GE Healthcare, Pittsburgh, PA) for 1 hour at room temperature and then incubated with primary antibodies overnight at 4°C. Primary antibodies used were anti-tuberin / TSC2 (#3612), anti-phospho-S6 (#2211), anti-S6 (#2212) (Cell Signaling Technology®), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#2275-PC) (Trevigen®, Gaithersburg, MD). Secondary antibodies used were anti-rabbit or anti-mouse IgG antibodies conjugated with horseradish peroxidase. Antigen-antibody complexes were detected using enhanced chemiluminescence reagent, Lumigen® ECL Ultra (TMA-6) (Lumigen®, Southfield, MI).

[0124] Example 4. Animals and intracerebroventricular (ICV) injection The experimental research protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at Massachusetts General Hospital (MGH) in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. c / cThis was performed in floxed mice (Onda et al., J. Clin. Invest. 104(6):687-695, 1999). In response to Cre recombinase, Tsc2 c / c The allele is converted to a null allele, and the lacZ allele expresses β-galactosidase. These mice remain normal and healthy throughout life.

[0125] For vector injections, neonates were hypothermically anesthetized on the day of birth (postnatal day 0 (P0)) or P3. 1 or 2 μl of the viral vector AAV1-CBA-Cre was injected into each lateral ventricle using a glass micropipette (tip diameter 70-100 mm) with a Narishige® IM300 microinjector (Narishige International, East Meadow, NY) at a rate of 2.4 psi / sec. The mice were then placed on a heating pad and allowed to regain normal color and full activity, as is typical of neonatal mice, before being returned to their mothers.

[0126] Example 5. Retro-orbital (RO) injection Three-week-old (P21) mice were anesthetized by isoflurane inhalation (3.5% isoflurane in an induction chamber, followed by 2-3% isoflurane and 1-2 L / min oxygen for the duration of the experiment). AAV vectors were injected retroorbitally into the vasculature directly behind one eye using a 0.3 ml insulin syringe over a period of less than 2 minutes in a 70 μl volume (10 μl AAV1- or AAV9-CBA-cTuberin + 60 μl saline solution) or not (Yardeni et al., Lab. Anim. (NY). 40(5):155-160, 2011).

[0127] Example 6. Subcutaneous lymphangioleiomyomatosis (LAM) model Three million human TSC2-null immortalized angiomyolipoma cells expressing Fluc were suspended in 50 μl of reduced serum medium (Opti-MEM®, Gibco®), mixed with 50 μl of Matrigel® (BD Matrigel™ Matrix HC) (BD Biosciences, Bedford, MA), and subcutaneously implanted into the backs of NOD-SCID Il2R gamma (NSG™) mice. Four weeks later, mice were intraperitoneally injected with the Fluc substrate D-luciferin (LUCNA-1G) (Gold Biotechnology®, St. Louis, MO). Five minutes later, signals were acquired using a high-efficiency IVIS® Spectrum (Caliper Life Sciences, Hopkinton, MA) equipped with an XGI-8 gas anesthesia system (Caliper Life Sciences).

[0128] Example 7. Histology and Immunohistochemistry (IHC) Standard histology of mouse brains was performed as described in Prabhakar et al., PLoS One. 8(5):e64224, 2013. Five-micrometer sections were stained with hematoxylin and eosin (H&E) or used for IHC as described (ibid.) using an antibody against pS6 (#2211, Cell Signaling) with the secondary antibody described (ibid.).

[0129] Example 8. Statistical Analysis All survival curve analyses (chi-square tests) were performed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA). The indicated P values indicate statistical significance.

[0130] Example 9. Expression of cTuberin in COS-7 cells transfected with AAV-CBA-cTuberin vector plasmid Following the procedure of Example 1, COS-7 cells were transfected with the AAV-CBA-cTuberin vector plasmid of Example 2. After 24 hours, cTuberin expression was detected by Western blotting as described in the previous example using an anti-tuberin / TSC2 antibody. As shown in Figure 3A, cTuberin expression was observed at the expected molecular weight (MW) of 85 kDa.

[0131] Example 10. pS6 kinase activity in COS-7 cells transfected with various AAV constructs To test the activity of c-tuberin, COS-7 cells were transfected with GFP, pAAV-CBA-cTSC2, TSC2-FLAG, pAAV-CBA-cTSC2+TSC1-FLAG, TSC1-FLAG+TSC2-FLAG, and TSC1-FLAG vectors. Expression levels of phosphorylated S6 (pS6), S6, and GAPDH were detected by Western blotting. Although pS6 kinase levels are normally elevated in the absence of tuberin activity, cells transfected with the AAV-CBA-c-tuberin plasmid showed lower pS6 levels, indicating reduced pS6 kinase activity. This is shown in Figure 3B, which shows elevated pS6 levels in columns 1 (GFP (control)) and 7 (control, no plasmid) compared to the other cells.

[0132] Example 11. Survival of mice injected with AAV1-CBA-Cre vector at P0, mice injected with AAV9-CBA-cTuberin vector at P21, and uninjected mice Efficacy of AAV-CBA-cTuberin vectors was confirmed by Tsc2 c / cThe study was conducted in mice. Intracerebroventricular (ICV) and retroorbital (RO) injections were performed as described in the previous examples. AAV1-CBA-Cre and AAV9-CBA-cTuberin vectors were generated as described above. Mice were injected ICV with AAV1-CBA-Cre at P0 (N=16), and RO with AAV9-CBA-cTuberin at P21 (N=7) or not injected at all (N=6). The titers of AAV1-CBA-Cre and AAV9-CBA-cTuberin injections were 9.1 x 10 12 gc / ml and 4.5 × 10 12 The median survival time was 35 days for AAV1-CBA-Cre mice, over 175 days for uninjected mice, and over 185 days for AAV9-CBA-cTuberin mice. Differences between groups were statistically significant, with P<0.0001 (log-rank) or P<0.0001 (Gehan-Breslow-Wilcoxon). Survival curves are shown in Figure 4A.

[0133] Example 12. Survival and histology of mice injected with AAV1-CBA-Cre vector at P0 plus AAV1-CBA-cTuberin vector at P21 The efficacy of gene therapy using the AAV1-CBA-cTuberin vector was tested in mice lacking tuberin. The AAV1-CBA-cTuberin vector was generated as described in the previous example. At P0, all mice were injected ICV with AAV1-CBA-Cre. At P21, one group of mice was injected RO with AAV1-CBA-cTuberin (N=7), and the second group was not (N=10). The titers of the AAV1-CBA-Cre and AAV1-CBA-cTuberin injections were 5.1 x 10, respectively. 13 gc / ml and 3 × 10 11 The median survival time for uninjected mice was 26.5 days, whereas mice injected with AAV1-CBA-cTuberin survived a median of 35 days. The difference between the two groups was statistically significant, P = 0.0001 (log-rank) or P = 0.0004 (Gehan-Breslow-Wilcoxon). The survival curves are shown in Figure 4B.

[0134] Additionally, the brains of mice lacking tuberin and mice treated with AAV-CBA-cTuberin were examined using H&E staining or IHC for pS6, performed as described in Example 7. Following the same design as the previous experiment, AAV1-CBA-Cre was administered at P0 and AAV1-CBA-cTuberin was administered at P21 to Tsc2 c / c Mice were injected with AAV1-CBA-cTuberin and sacrificed at P27. The results are shown in Figures 5A-5F. Figure 5A shows staining of an uninjected normal brain (control). Figures 5B-5E show staining of a mouse injected with only AAV1-CBA-Cre at P0, demonstrating proliferation of ependymal cells (Figure 5B), expansion of hippocampal pyramidal cells (Figure 5C), subependymal nodules (Figure 5D), and numerous subependymal nodules and proliferations (Figure 5E). Finally, Figure 5F shows minimal nodules, inflammation, and edema in the subependymal region in a mouse treated with AAV1-CBA-cTuberin at P21.

[0135] Example 13. Survival of mice injected with AAV1-CBA-Cre vector at P3 plus AAV1- or AAV9-CBA-cTuberin vector at P21 The efficacy of gene therapy using the AAV1-CBA-cTuberin vector or the AAV9-CBA-cTuberin vector was further tested in the following two experiments. First injecting AAV1-CBA-Cre into mice at P3, when the permeability of the cerebrospinal fluid (CSF) barrier is slightly lower than at P0, should result in less loss of tuberin in the brain.

[0136] AAV1-CBA-cTuberin At P3, AAV1-CBA-Cre transfected all Tsc2 c / c Mice were injected ICV. At P21, one group of mice was injected RO with AAV1-CBA-cTuberin (N=9) and the second group was not (N=7). The titers of AAV1-CBA-Cre and AAV1-CBA-cTuberin injections were 5.1 × 10, respectively. 13 gc / ml and 3 × 10 11The median survival time for uninjected mice was 36 days, while mice injected with AAV1-CBA-cTuberin survived a median of 54 days. The difference between the two groups was statistically significant, P < 0.0001 (log-rank) or P = 0.0004 (Gehan-Breslow-Wilcoxon). Survival curves are shown in Figure 4C.

[0137] AAV9-CBA-cTuberin At P3, AAV1-CBA-Cre transfected all Tsc2 c / c Mice were injected ICV. At P21, one group of mice was injected RO with AAV9-CBA-cTuberin (N=11) and the second group was not (N=9). The titers of AAV1-CBA-Cre and AAV9-CBA-cTuberin injections were 5.1 x 10, respectively. 13 gc / ml and 4.5 × 10 12 The median survival time for uninjected mice was 32 days, while mice injected with AAV9-CBA-cTuberin survived a median of 45 days. The difference between the two groups was statistically significant, P < 0.0006 (log-rank) or P = 0.0014 (Gehan-Breslow-Wilcoxon). The survival curves are shown in Figure 4D.

[0138] Example 14. Survival of mice injected with AAV9-CBA-cTuberin vector and vigabatrin Therapeutic vectors may also reduce seizures, which, if not reduced, can lead to early death, often preceding hydrocephalus caused by subependymal nodules (SEN). The efficacy of AAV9-CBA-cTuberin in combination with vigabatrin, which was shown to be effective in preventing seizures, was further tested in Tsc1-floxed / GFAP-Cre mice (Zhang et al., PLoS One. 8(2):e57445, 2013).

[0139] At P0, all mice were injected ICV with AAV1-CBA-Cre. At P3, one group of mice was injected RO with AAV9-CBA-cTuberin, and the second group was not. Of the injected mice, one group was treated with vigabatrin (50 mg / kg) (N = 7), and the second group was not treated (N = 10). Of the uninjected mice, one group was treated with vigabatrin (200 mg / kg) (N = 7), and the second group was not treated (N = 8). The titers of AAV1-CBA-Cre and AAV9-CBA-cTuberin were 5.1 × 10, respectively. 13 gc / ml and 4.5 × 10 12 The vigabatrin dose delivered was 50 mg / kg in mice injected with AAV9-CBA-cTuberin and 200 mg / kg in uninjected mice. Median survival times were 23 days for uninjected, drug-free mice, 27 days for uninjected, vigabatrin-treated mice, 35.5 days for injected, drug-free mice, and 40 days for injected, vigabatrin-treated mice. The difference between the two AAV9-CBA-cTuberin-injected groups was statistically significant, P<0.0001 (log-rank) or P<0.0001 (Gehan-Breslow-Wilcoxon).

[0140] Example 15. Efficacy of AAV9-CBA-cTuberin against LAM tumors in vivo The efficacy of AAV9-CBA-cTuberin against lymphangioleiomyomatosis (LAM) tumors subcutaneously injected into immunodeficient NSG mice, generated as described in Example 4, was also tested in vivo. A Fluc-expressing LAM tumor is shown in Figure 6A. Tumor volume was monitored by bioluminescence at weeks 1, 4, 6, 9, and 14. Tumors were injected with the AAV9-CBA-cTuberin vector (N=7) or not (N=5) at weeks 4 and 9. The titer of the AAV9-CBA-cTuberin vector was 4.3 x 10 10 As shown in Figure 6B, the size of the cTuberin vector-injected tumors did not increase until week 14, while the volume of the uninjected tumors continued to expand.

[0141] Other Aspects While the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entireties.

[0142] Sequence information SEQUENCE LISTING <110> The General Hospital Corporation <120> GENE THERAPY FOR TUBEROUS SCLEROSIS <150> US 62 / 507,358 <151> 2017-05-17 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 758 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 1 Met Ala Lys Pro Thr Ser Lys Asp Ser Gly Leu Lys Glu Lys Phe Lys 1 5 10 15 Ile Leu Leu Gly Leu Gly Thr Pro Arg Pro Asn Pro Arg Ser Ala Glu 20 25 30 Gly Lys Gln Thr Glu Phe Ile Ile Thr Ala Glu Ile Leu Arg Glu Leu 35 40 45 Ser Met Glu Cys Gly Leu Asn Asn Arg Ile Arg Met Ile Gly Gln Ile 50 55 60 Cys Glu Val Ala Lys Thr Lys Lys Phe Glu Glu His Ala Val Glu Ala 65 70 75 80 Leu Trp Lys Ala Val Ala Asp Leu Leu Gln Pro Glu Arg Pro Leu Glu 85 90 95 Ala Arg His Ala Val Leu Ala Leu Leu Lys Ala Ile Val Gln Gly Gln 100 105 110 Gly Glu Arg Leu Gly Val Leu Arg Ala Leu Phe Phe Lys Val Ile Lys 115 120 125 Asp Tyr Pro Ser Asn Glu Asp Leu His Glu Arg Leu Glu Val Phe Lys 130 135 140 Ala Leu Thr Asp Asn Gly Arg His Ile Thr Tyr Leu Glu Glu Glu Leu 145 150 155 160 Ala Asp Phe Val Leu Gln Trp Met Asp Val Gly Leu Ser Ser Glu Phe 165 170 175 Leu Leu Val Leu Val Asn Leu Val Lys Phe Asn Ser Cys Tyr Leu Asp 180 185 190 Glu Tyr Ile Ala Arg Met Val Gln Met Ile Cys Leu Leu Cys Val Arg 195 200 205 Thr Ala Ser Ser Val Asp Ile Glu Val Ser Leu Gln Val Leu Asp Ala 210 215 220 Val Val Cys Tyr Asn Cys Leu Pro Ala Glu Ser Leu Pro Leu Phe Ile 225 230 235 240 Val Thr Leu Cys Arg Thr Ile Asn Val Lys Glu Leu Cys Glu Pro Cys 245 250 255 Trp Lys Leu Met Arg Asn Leu Leu Gly Thr His Leu Gly His Ser Ala 260 265 270 Ile Tyr Asn Met Cys His Leu Met Glu Asp Arg Ala Tyr Met Glu Asp 275 280 285 Ala Pro Leu Leu Arg Gly Ala Val Phe Phe Val Gly Met Ala Leu Trp 290 295 300 Gly Ala His Arg Leu Tyr Ser Leu Arg Asn Ser Pro Thr Ser Val Leu 305 310 315 320 Pro Ser Phe Tyr Gln Ala Met Ala Cys Pro Asn Glu Val Val Ser Tyr 325 330 335 Glu Ile Val Leu Ser Ile Thr Arg Leu Ile Lys Lys Tyr Arg Lys Glu 340 345 350 Leu Gln Val Val Ala Trp Asp Ile Leu Leu Asn Ile Ile Glu Arg Leu 355 360 365 Leu Gln Gln Leu Gln Thr Leu Asp Ser Pro Glu Leu Arg Thr Ile Val 370 375 380 His Asp Leu Leu Thr Thr Val Glu Glu Leu Cys Asp Gln Asn Glu Phe 385 390 395 400 His Gly Ser Gln Glu Arg Tyr Phe Glu Leu Val Glu Arg Cys Ala Asp 405 410 415 Gln Arg Pro Glu Ser Ser Leu Leu Asn Leu Ile Ser Tyr Arg Ala Gln 420 425 430 Ser Ile His Pro Ala Lys Asp Gly Trp Ile Gln Asn Leu Gln Ala Leu 435 440 445 Met Glu Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 450 455 460 Gly Gly Lys Pro Ile Leu Leu Pro Asn Glu Ser Gln Ser Phe Glu Arg 465 470 475 480 Ser Val Gln Leu Leu Asp Gln Ile Pro Ser Tyr Asp Thr His Lys Ile 485 490 495 Ala Val Leu Tyr Val Gly Glu Gly Gln Ser Asn Ser Glu Leu Ala Ile 500 505 510 Leu Ser Asn Glu His Gly Ser Tyr Arg Tyr Thr Glu Phe Leu Thr Gly 515 520 525 Leu Gly Arg Leu Ile Glu Leu Lys Asp Cys Gln Pro Asp Lys Val Tyr 530 535 540 Leu Gly Gly Leu Asp Val Cys Gly Glu Asp Gly Gln Phe Thr Tyr Cys 545 550 555 560 Trp His Asp Asp Ile Met Gln Ala Val Phe His Ile Ala Thr Leu Met 565 570 575 Pro Thr Lys Asp Val Asp Lys His Arg Cys Asp Lys Lys Arg His Leu 580 585 590 Gly Asn Asp Phe Val Ser Ile Val Tyr Asn Asp Ser Gly Glu Asp Phe 595 600 605 Lys Leu Gly Thr Ile Lys Gly Gln Phe Asn Phe Val His Val Ile Val 610 615 620 Thr Pro Leu Asp Tyr Glu Cys Asn Leu Val Ser Leu Gln Cys Arg Lys 625 630 635 640 Asp Met Glu Gly Leu Val Asp Thr Ser Val Ala Lys Ile Val Ser Asp 645 650 655 Arg Asn Leu Pro Phe Val Ala Arg Gln Met Ala Leu His Ala Asn Met 660 665 670 Ala Ser Gln Val His His Ser Arg Ser Asn Pro Thr Asp Ile Tyr Pro 675 680 685 Ser Lys Trp Ile Ala Arg Leu Arg His Ile Lys Arg Leu Arg Gln Arg 690 695 700 Ile Cys Glu Glu Ala Ala Tyr Ser Asn Pro Ser Leu Pro Leu Val His 705 710 715 720 Pro Pro Ser His Ser Lys Ala Pro Ala Gln Thr Pro Ala Glu Pro Thr 725 730 735 Pro Gly Tyr Glu Val Gly Gln Arg Lys Arg Leu Ile Ser Ser Val Glu 740 745 750 Asp Phe Thr Glu Phe Val 755 <210> 2 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 2 Met Ala Lys Pro Thr Ser Lys Asp Ser Gly Leu Lys Glu Lys Phe Lys 1 5 10 15 Ile Leu Leu Gly Leu Gly Thr Pro Arg Pro Asn Pro Arg Ser Ala Glu 20 25 30 Gly Lys Gln Thr Glu Phe Ile Ile Thr Ala Glu Ile Leu Arg Glu Leu 35 40 45 Ser Met Glu Cys Gly Leu Asn Asn Arg Ile Arg Met Ile Gly Gln Ile 50 55 60 Cys Glu Val Ala Lys Thr Lys Lys Phe Glu Glu His Ala Val Glu Ala 65 70 75 80 Leu Trp Lys Ala Val Ala Asp Leu Leu Gln Pro Glu Arg Pro Leu Glu 85 90 95 Ala Arg His Ala Val Leu Ala Leu Leu Lys Ala Ile Val Gln Gly Gln 100 105 110 Gly Glu Arg Leu Gly Val Leu Arg Ala Leu Phe Phe Lys Val Ile Lys 115 120 125 Asp Tyr Pro Ser Asn Glu Asp Leu His Glu Arg Leu Glu Val Phe Lys 130 135 140 Ala Leu Thr Asp Asn Gly Arg His Ile Thr Tyr Leu Glu Glu Glu Leu 145 150 155 160 Ala Asp Phe Val Leu Gln Trp Met Asp Val Gly Leu Ser Ser Glu Phe 165 170 175 Leu Leu Val Leu Val Asn Leu Val Lys Phe Asn Ser Cys Tyr Leu Asp 180 185 190 Glu Tyr Ile Ala Arg Met Val Gln Met Ile Cys Leu Leu Cys Val Arg 195 200 205 Thr Ala Ser Ser Val Asp Ile Glu Val Ser Leu Gln Val Leu Asp Ala 210 215 220 Val Val Cys Tyr Asn Cys Leu Pro Ala Glu Ser Leu Pro Leu Phe Ile 225 230 235 240 Val Thr Leu Cys Arg Thr Ile Asn Val Lys Glu Leu Cys Glu Pro Cys 245 250 255 Trp Lys Leu Met Arg Asn Leu Leu Gly Thr His Leu Gly His Ser Ala 260 265 270 Ile Tyr Asn Met Cys His Leu Met Glu Asp Arg Ala Tyr Met Glu Asp 275 280 285 Ala Pro Leu Leu Arg Gly Ala Val Phe Phe Val Gly Met Ala Leu Trp 290 295 300 Gly Ala His Arg Leu Tyr Ser Leu Arg Asn Ser Pro Thr Ser Val Leu 305 310 315 320 Pro Ser Phe Tyr Gln Ala Met Ala Cys Pro Asn Glu Val Val Ser Tyr 325 330 335 Glu Ile Val Leu Ser Ile Thr Arg Leu Ile Lys Lys Tyr Arg Lys Glu 340 345 350 Leu Gln Val Val Ala Trp Asp Ile Leu Leu Asn Ile Ile Glu Arg Leu 355 360 365 Leu Gln Gln Leu Gln Thr Leu Asp Ser Pro Glu Leu Arg Thr Ile Val 370 375 380 His Asp Leu Leu Thr Thr Val Glu Glu Leu Cys Asp Gln Asn Glu Phe 385 390 395 400 His Gly Ser Gln Glu Arg Tyr Phe Glu Leu Val Glu Arg Cys Ala Asp 405 410 415 Gln Arg Pro Glu Ser Ser Leu Leu Asn Leu Ile Ser Tyr Arg Ala Gln 420 425 430 Ser Ile His Pro Ala Lys Asp Gly Trp Ile Gln Asn Leu Gln Ala Leu 435 440 445 Met Glu 450 <210> 3 <211> 292 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 3 Lys Pro Ile Leu Leu Pro Asn Glu Ser Gln Ser Phe Glu Arg Ser Val 1 5 10 15 Gln Leu Leu Asp Gln Ile Pro Ser Tyr Asp Thr His Lys Ile Ala Val 20 25 30 Leu Tyr Val Gly Glu Gly Gln Ser Asn Ser Glu Leu Ala Ile Leu Ser 35 40 45 Asn Glu His Gly Ser Tyr Arg Tyr Thr Glu Phe Leu Thr Gly Leu Gly 50 55 60 Arg Leu Ile Glu Leu Lys Asp Cys Gln Pro Asp Lys Val Tyr Leu Gly 65 70 75 80 Gly Leu Asp Val Cys Gly Glu Asp Gly Gln Phe Thr Tyr Cys Trp His 85 90 95 Asp Asp Ile Met Gln Ala Val Phe His Ile Ala Thr Leu Met Pro Thr 100 105 110 Lys Asp Val Asp Lys His Arg Cys Asp Lys Lys Arg His Leu Gly Asn 115 120 125 Asp Phe Val Ser Ile Val Tyr Asn Asp Ser Gly Glu Asp Phe Lys Leu 130 135 140 Gly Thr Ile Lys Gly Gln Phe Asn Phe Val His Val Ile Val Thr Pro 145 150 155 160 Leu Asp Tyr Glu Cys Asn Leu Val Ser Leu Gln Cys Arg Lys Asp Met 165 170 175 Glu Gly Leu Val Asp Thr Ser Val Ala Lys Ile Val Ser Asp Arg Asn 180 185 190 Leu Pro Phe Val Ala Arg Gln Met Ala Leu His Ala Asn Met Ala Ser 195 200 205 Gln Val His His Ser Arg Ser Asn Pro Thr Asp Ile Tyr Pro Ser Lys 210 215 220 Trp Ile Ala Arg Leu Arg His Ile Lys Arg Leu Arg Gln Arg Ile Cys 225 230 235 240 Glu Glu Ala Ala Tyr Ser Asn Pro Ser Leu Pro Leu Val His Pro Pro 245 250 255 Ser His Ser Lys Ala Pro Ala Gln Thr Pro Ala Glu Pro Thr Pro Gly 260 265 270 Tyr Glu Val Gly Gln Arg Lys Arg Leu Ile Ser Ser Val Glu Asp Phe 275 280 285 Thr Glu Phe Val 290 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 4 Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 <210> 5 <211> 2306 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 5 gctagcacta gtaccatggc gaaaccgacc agcaaagata gcggcctgaa agaaaaattt 60 aaaattctgc tgggcctggg caccccgcgc ccgaacccgc gcagcgcgga aggcaaacag 120 accgaattta ttattaccgc ggaaattctg cgcgaactga gcatggaatg cggcctgaac 180 aaccgcattc gcatgattgg ccagatttgc gaagtggcga aaaccaaaaa atttgaagaa 240 catgcggtgg aagcgctgtg gaaagcggtg gcggatctgc tgcagccgga acgcccgctg 300 gaagcgcgcc atgcggtgct ggcgctgctg aaagcgattg tgcagggcca gggcgaacgc 360 ctgggcgtgc tgcgcgcgct gttttttaaa gtgattaaag attatccgag caacgaagat 420 ctgcatgaac gcctggaagt gtttaaagcg ctgaccgata acggccgcca tattacctat 480 ctggaagaag aactggcgga ttttgtgctg cagtggatgg atgtgggcct gagcagcgaa 540 tttctgctgg tgctggtgaa cctggtgaaa tttaacagct gctatctgga tgaatatatt 600 gcgcgcatgg tgcagatgat ttgcctgctg tgcgtgcgca ccgcgagcag cgtggatatt 660 gaagtgagcc tgcaggtgct ggatgcggtg gtgtgctata actgcctgcc ggcggaaagc 720 ctgccgctgt ttattgtgac cctgtgccgc accattaacg tgaaagaact gtgcgaaccg 780 tgctggaaac tgatgcgcaa cctgctgggc acccatctgg gccatagcgc gatttataac 840 atgtgccatc tgatggaaga tcgcgcgtat atggaagatg cgccgctgct gcgcggcgcg 900 gtgttttttg tgggcatggc gctgtggggc gcgcatcgcc tgtatagcct gcgcaacagc 960 ccgaccagcg tgctgccgag cttttatcag gcgatggcgt gcccgaacga agtggtgagc 1020 tatgaaattg tgctgagcat tacccgcctg attaaaaaat atcgcaaaga actgcaggtg 1080 gtggcgtggg atattctgct gaacattatt gaacgcctgc tgcagcagct gcagaccctg 1140 gatagcccgg aactgcgcac cattgtgcat gatctgctga ccaccgtgga agaactgtgc 1200 gatcagaacg aatttcatgg cagccaggaa cgctattttg aactggtgga acgctgcgcg 1260 gatcagcgcc cggaaagcag cctgctgaac ctgattagct atcgcgcgca gagcattcat 1320 ccggcgaaag atggctggat tcagaacctg caggcgctga tggaatctgg tgggggtagc 1380 ggaggagggt cagggggcgg cagtggaggc ggaaaaccga ttctgctgcc gaacgaaagc 1440 cagagctttg aacgcagcgt gcagctgctg gatcagattc cgagctatga tacccataaa 1500 attgcggtgc tgtatgtggg cgaaggccag agcaacagcg aactggcgat tctgagcaac 1560 gaacatggca gctatcgcta taccgaattt ctgaccggcc tgggccgcct gattgaactg 1620 aaagattgcc agccggataa agtgtatctg ggcggcctgg atgtgtgcgg cgaagatggc 1680 cagtttacct attgctggca tgatgatatt atgcaggcgg tgtttcatat tgcgaccctg 1740 atgccgacca aagatgtgga taaacatcgc tgcgataaaa aacgccatct gggcaacgat 1800 tttgtgagca ttgtgtataa cgatagcggc gaagatttta aactgggcac cattaaaggc 1860 cagtttaact ttgtgcatgt gattgtgacc ccgctggatt atgaatgcaa cctggtgagc 1920 ctgcagtgcc gcaaagatat ggaaggcctg gtggatacca gcgtggcgaa aattgtgagc 1980 gatcgcaacc tgccgtttgt ggcgcgccag atggcgctgc atgcgaacat ggcgagccag 2040 gtgcatcata gccgcagcaa cccgaccgat atttatccga gcaaatggat tgcgcgcctg 2100 cgccatatta aacgcctgcg ccagcgcatt tgcgaagaag cggcgtatag caacccgagc 2160 ctgccgctgg tgcatccgcc gagccatagc aaagcgccgg cgcagacccc ggcggaaccg 2220 accccgggct atgaagtggg ccagcgcaaa cgcctgatta gcagcgtgga agattttacc 2280 gaatttgtgt aggcggccgc ctcgag 2306 <210> 6 <211> 1353 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 6 accatggcga aaccgaccag caaagatagc ggcctgaaag aaaaatttaa aattctgctg 60 ggcctgggca ccccgcgccc gaacccgcgc agcgcggaag gcaaacagac cgaatttatt 120 attaccgcgg aaattctgcg cgaactgagc atggaatgcg gcctgaacaa ccgcattcgc 180 atgattggcc agatttgcga agtggcgaaa accaaaaaat ttgaagaaca tgcggtggaa 240 gcgctgtgga aagcggtggc ggatctgctg cagccggaac gcccgctgga agcgcgccat 300 gcggtgctgg cgctgctgaa agcgattgtg cagggccagg gcgaacgcct gggcgtgctg 360 cgcgcgctgt tttttaaagt gattaaagat tatccgagca acgaagatct gcatgaacgc 420 ctggaagtgt ttaaagcgct gaccgataac ggccgccata ttacctatct ggaagaagaa 480 ctggcggatt ttgtgctgca gtggatggat gtgggcctga gcagcgaatt tctgctggtg 540 ctggtgaacc tggtgaaatt taacagctgc tatctggatg aatatattgc gcgcatggtg 600 cagatgattt gcctgctgtg cgtgcgcacc gcgagcagcg tggatattga agtgagcctg 660 caggtgctgg atgcggtggt gtgctataac tgcctgccgg cggaaagcct gccgctgttt 720 attgtgaccc tgtgccgcac cattaacgtg aaagaactgt gcgaaccgtg ctggaaactg 780 atgcgcaacc tgctgggcac ccatctgggc catagcgcga tttaataacat gtgccatctg 840 atggaagatc gcgcgtatat ggaagatgcg ccgctgctgc gcggcgcggt gttttttgtg 900 ggcatggcgc tgtggggcgc gcatcgcctg tatagcctgc gcaacagccc gaccagcgtg 960 ctgccgagct tttatcaggc gatggcgtgc ccgaacgaag tggtgagcta tgaaattgtg 1020 ctgagcatta cccgcctgat taaaaaatat cgcaaagaac tgcaggtggt ggcgtgggat 1080 attctgctga acattattga acgcctgctg cagcagctgc agaccctgga tagcccggaa 1140 ctgcgcacca ttgtgcatga tctgctgacc accgtggaag aactgtgcga tcagaacgaa 1200 tttcatggca gccaggaacg ctattttgaa ctggtggaac gctgcgcgga tcagcgcccg 1260 gaaagcagcc tgctgaacct gattagctat cgcgcgcaga gcattcatcc ggcgaaagat 1320 ggctggattc agaacctgca ggcgctgatg gaa 1353 <210> 7 <211> 876 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 7 aaaccgattc tgctgccgaa cgaaagccag agctttgaac gcagcgtgca gctgctggat 60 cagattccga gctatgatac ccataaaatt gcggtgctgt atgtgggcga aggccagagc 120 aacagcgaac tggcgattct gagcaacgaa catggcagct atcgctatac cgaatttctg 180 accggcctgg gccgcctgat tgaactgaaa gattgccagc cggataaagt gtatctgggc 240 ggcctggatg tgtgcggcga agatggccag tttacctatt gctggcatga tgatattatg 300 caggcggtgt ttcatattgc gaccctgatg ccgaccaaag atgtggataa acatcgctgc 360 gataaaaaac gccatctggg caacgatttt gtgagcattg tgtataacga tagcggcgaa 420 gattttaaac tgggcaccat taaaggccag tttaactttg tgcatgtgat tgtgaccccg 480 ctggattatg aatgcaacct ggtgagcctg cagtgccgca aagatatgga aggcctggtg 540 gataccagcg tggcgaaaat tgtgagcgat cgcaacctgc cgtttgtggc gcgccagatg 600 gcgctgcatg cgaacatggc gagccaggtg catcatagcc gcagcaaccc gaccgatatt 660 tatccgagca aatggattgc gcgcctgcgc catattaaac gcctgcgcca gcgcatttgc 720 gaagaagcgg cgtatatagcaa cccgagcctg ccgctggtgc atccgccgag ccatagcaaa 780 gcgccggcgc agaccccggc ggaaccgacc ccgggctatg aagtgggcca gcgcaaacgc 840 ctgattagca gcgtggaaga ttttaccgaa tttgtg 876 <210> 8 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 8 tctggtgggg gtagcggagg agggtcaggg ggcggcagtg gaggcgga 48 <210> 9 <211> 7540 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 9 ggccgctcta gaagataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat 60 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca 120 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc 180 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc 240 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt 300 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg 360 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc 420 ctttccatgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta 480 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg 540 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc 600 cccgcatcgg actagagaga tccagacatg ataagataca ttgatgagtt tggacaaacc 660 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 720 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg 780 tttcaggttc agggggaggt gtgggaggtt ttttagtcga ctagagctcg ctgatcagcc 840 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg 900 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat 960 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag 1020 gattgggaag acaatagcag gcatgctggg gagagatcta ggaaccccta gtgatggagt 1080 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc 1140 gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg 1200 ccatgcagcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt 1260 gcgtagcctg aatggcgaat ggcgcgacgc gccctgtagc ggcgcattaa gcgcggcggg 1320 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 1380 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 1440 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 1500 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 1560 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 1620 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 1680 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat 1740 ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc 1800 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca 1860 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 1920 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga 1980 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 2040 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 2100 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 2160 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 2220 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 2280 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 2340 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 2400 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 2460 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 2520 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 2580 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 2640 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 2700 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg tagccctcc 2940 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 3480 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 3540 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 3600 cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 3660 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 3720 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 3780 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 3840 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 3900 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 3960 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 4020 attcattaat gcagctggggc tgcagggggg gggggggggg ggtgggggggg gggggggggg 4080 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc 4140 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gagaggagt 4200 ggccaactcc atcactaggg gttcctagat ctgaattcgg taccctagtt attaatagta 4260 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac 4320 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac 4380 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggactattt 4440 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat 4500 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga 4560 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag 4620 ccccacgtc tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt 4680 atttatttt taattatttt gtgcagcgat gggggcgggg gggggggggg ggcgcgcgcc 4740 aggcggggcg gggcggggcg aggggcgggg cggggcgagg cggagaggtg cggcggcgc 4800 caatcagagc ggcgcgctcc gaaagttttcc tttatggcg aggcggcggc ggcggcggcc 4860 ctataaaaaag cgaagcgcgc ggcgggcggg agtcgctgcg acgctgcctt cgccccgtgc 4920 cccgctccgc cgccgccctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac 4980 aggtgagcgg gcgggacggc ccttctcctc cgggctttaa ttagcgcttg gtttaatgac 5040 ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg gctccgggag ctagagcctc 5100 tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg tgctggttat 5160 tgtgctgtct catcattttg gcaaagaatt cctcgaagat ccgaaggggt tcaagcttaa 5220 aaactagtac catggcaaaa cctacatcaa aagatagcgg actgaaagag aagttcaaaa 5280 tcctgctggg actggggaca ccacgcccta atccacggtc cgccgagggc aagcagaccg 5340 agttcatcat cacagccgag atcctgcgcg agctgtctat ggagtgcggc ctgaacaatc 5400 ggatcagaat gatcggccag atttgcgagg tggccaagac caagaagttt gaggagcacg 5460 cagtggaggc cctgtggaag gcagtggccg atctgctgca gcctgagaga ccactggagg 5520 caaggcacgc cgtgctggcc ctgctgaagg ccatcgtgca gggacaggga gagcgcctgg 5580 gcgtgctgcg ggccctgttc tttaaagtga tcaaggacta ccctagcaac gaggatctgc 5640 acgagagact ggaggtgttc aaggccctga ccgataatgg caggcacatc acatatctgg 5700 aggaggagct ggccgacttt gtgctgcagt ggatggatgt gggcctgagc tccgagttcc 5760 tgctggtgct ggtgaacctg gtgaagttta attcctgcta cctggacgag tatatcgccc 5820 gcatggtgca gatgatctgc ctgctgtgcg tgcggaccgc ctctagcgtg gacatcgagg 5880 tgtctctgca ggtgctggat gccgtggtgt gctacaactg tctgcccgcc gagagcctgc 5940 ctctgttcat cgtgaccctg tgcagaacaa tcaatgtgaa ggagctgtgc gagccttgtt 6000 ggaagctgat gaggaacctg ctgggcacac acctgggaca cagcgccatc tacaatatgt 6060 gccacctgat ggaggaccgc gcctatatgg aggatgcacc actgctgagg ggagccgtgt 6120 tctttgtggg aatggcactg tggggagcac acagactgta ctccctgagg aactctccaa 6180 ccagcgtgct gccctctttt taccaggcta tggcctgtcc aaatgaggtg gtgtcttatg 6240 agatcgtgct gagcatcaca cgcctgatca agaagtatcg gaaggagctg caggtggtgg 6300 cctgggacat cctgctgaac atcatcgagc gcctgctgca gcagctgcag accctggaca 6360 gcccagagct gaggacaatc gtgcacgatc tgctgaccac agtggaggag ctgtgcgacc 6420 agaatgagtt ccacggctcc caggagcggt actttgagct ggtggagcgg tgcgcagatc 6480 agaggccaga gtcctctctg ctgaacctga tctcctatcg ggcccagtct atccaccctg 6540 ccaaggacgg ctggattcag aatctgcagg ccctgatgga gagcggagga ggctccggag 6600 gaggctctgg aggcggcagc ggcggcggca agccaatcct gctgcccaac gagagccaga 6660 gcttcgagcg gagcgtgcag ctgctggacc agatccccag ctacgatacc cacaagatcg 6720 ccgtgctgta tgtgggcgag ggccagtcta acagcgagct ggccatcctg agcaatgagc 6780 acggctccta cagatatacc gagtttctga caggcctggg caggctgatc gagctgaagg 6840 actgccagcc cgataaggtg tacctgggag gcctggacgt gtgcggagag gatggccagt 6900 tcacctattg ttggcacgac gatatcatgc aggccgtgtt tcacatcgcc accctgatgc 6960 ctacaaagga cgtggataag cacagatgtg acaagaagag gcacctgggc aacgatttcg 7020 tgtccatcgt gtacaatgac tctggcgagg acttcaagct gggcaccatc aagggccagt 7080 tcaactttgt gcacgtgatc gtgacaccac tggattatga gtgcaatctg gtgagcctgc 7140 agtgtagaaa ggacatggag ggcctggtgg ataccagcgt ggccaagatc gtgtccgaca gaaacctgcc cttcgtggcc aggcagatgg ccctgcacgc caacatggcc agccaggtgc 7260 accactccag gtctaatcct acagacatct acccatcca gtggatcgcc aggctgcgcc acatcaagcg gctgagacag aggatctgcg aggaggccgc ctattccaat ccctctctgc ctctggtgca cccacctagc cactccaagg cacctgcaca gaccccagca gacccaacac caggatacga agtggacag aggaagcggc tgatctcctc cgtggagat ttcaccgaat ttgtggagca gaagctgatt agcgaagaag acctgtaagc <210> 10 <211> 1807 <212> PRT <213> Homo sapiens <400> 10 Met Ala Lys Pro Thr Ser Lys Asp Ser Gly Leu Lys Glu Lys Phe Lys 1 5 10 15 Ile Leu Leu Gly Leu Gly Thr Pro Arg Pro Asn Pro Arg Ser Ala Glu 20 25 30 Gly Lys Gln Thr Glu Phe Ile Ile Thr Ala Glu Ile Leu Arg Glu Leu 35 40 45 Ser Met Glu Cys Gly Leu Asn Asn Arg Ile Arg Met Ile Gly Gln Ile 50 55 60 Cys Glu Val Ala Lys Thr Lys Lys Phe Glu Glu His Ala Val Glu Ala 65 70 75 80 Leu Trp Lys Ala Val Ala Asp Leu Leu Gln Pro Glu Arg Pro Leu Glu 85 90 95 Ala Arg His Ala Val Leu Ala Leu Leu Lys Ala Ile Val Gln Gly Gln 100 105 110 Gly Glu Arg Leu Gly Val Leu Arg Ala Leu Phe Phe Lys Val Ile Lys 115 120 125 Asp Tyr Pro Ser Asn Glu Asp Leu His Glu Arg Leu Glu Val Phe Lys 130 135 140 Ala Leu Thr Asp Asn Gly Arg His Ile Thr Tyr Leu Glu Glu Glu Leu 145 150 155 160 Ala Asp Phe Val Leu Gln Trp Met Asp Val Gly Leu Ser Ser Glu Phe 165 170 175 Leu Leu Val Leu Val Asn Leu Val Lys Phe Asn Ser Cys Tyr Leu Asp 180 185 190 Glu Tyr Ile Ala Arg Met Val Gln Met Ile Cys Leu Leu Cys Val Arg 195 200 205 Thr Ala Ser Ser Val Asp Ile Glu Val Ser Leu Gln Val Leu Asp Ala 210 215 220 Val Val Cys Tyr Asn Cys Leu Pro Ala Glu Ser Leu Pro Leu Phe Ile 225 230 235 240 Val Thr Leu Cys Arg Thr Ile Asn Val Lys Glu Leu Cys Glu Pro Cys 245 250 255 Trp Lys Leu Met Arg Asn Leu Leu Gly Thr His Leu Gly His Ser Ala 260 265 270 Ile Tyr Asn Met Cys His Leu Met Glu Asp Arg Ala Tyr Met Glu Asp 275 280 285 Ala Pro Leu Leu Arg Gly Ala Val Phe Phe Val Gly Met Ala Leu Trp 290 295 300 Gly Ala His Arg Leu Tyr Ser Leu Arg Asn Ser Pro Thr Ser Val Leu 305 310 315 320 Pro Ser Phe Tyr Gln Ala Met Ala Cys Pro Asn Glu Val Val Ser Tyr 325 330 335 Glu Ile Val Leu Ser Ile Thr Arg Leu Ile Lys Lys Tyr Arg Lys Glu 340 345 350 Leu Gln Val Val Ala Trp Asp Ile Leu Leu Asn Ile Ile Glu Arg Leu 355 360 365 Leu Gln Gln Leu Gln Thr Leu Asp Ser Pro Glu Leu Arg Thr Ile Val 370 375 380 His Asp Leu Leu Thr Thr Val Glu Glu Leu Cys Asp Gln Asn Glu Phe 385 390 395 400 His Gly Ser Gln Glu Arg Tyr Phe Glu Leu Val Glu Arg Cys Ala Asp 405 410 415 Gln Arg Pro Glu Ser Ser Leu Leu Asn Leu Ile Ser Tyr Arg Ala Gln 420 425 430 Ser Ile His Pro Ala Lys Asp Gly Trp Ile Gln Asn Leu Gln Ala Leu 435 440 445 Met Glu Arg Phe Phe Arg Ser Glu Ser Arg Gly Ala Val Arg Ile Lys 450 455 460 Val Leu Asp Val Leu Ser Phe Val Leu Leu Ile Asn Arg Gln Phe Tyr 465 470 475 480 Glu Glu Glu Leu Ile Asn Ser Val Val Ile Ser Gln Leu Ser His Ile 485 490 495 Pro Glu Asp Lys Asp His Gln Val Arg Lys Leu Ala Thr Gln Leu Leu 500 505 510 Val Asp Leu Ala Glu Gly Cys His Thr His His Phe Asn Ser Leu Leu 515 520 525 Asp Ile Ile Glu Lys Val Met Ala Arg Ser Leu Ser Pro Pro Pro Glu 530 535 540 Leu Glu Glu Arg Asp Val Ala Ala Tyr Ser Ala Ser Leu Glu Asp Val 545 550 555 560 Lys Thr Ala Val Leu Gly Leu Leu Val Ile Leu Gln Thr Lys Leu Tyr 565 570 575 Thr Leu Pro Ala Ser His Ala Thr Arg Val Tyr Glu Met Leu Val Ser 580 585 590 His Ile Gln Leu His Tyr Lys His Ser Tyr Thr Leu Pro Ile Ala Ser 595 600 605 Ser Ile Arg Leu Gln Ala Phe Asp Phe Leu Leu Leu Leu Arg Ala Asp 610 615 620 Ser Leu His Arg Leu Gly Leu Pro Asn Lys Asp Gly Val Val Arg Phe 625 630 635 640 Ser Pro Tyr Cys Val Cys Asp Tyr Met Glu Pro Glu Arg Gly Ser Glu 645 650 655 Lys Lys Thr Ser Gly Pro Leu Ser Pro Pro Thr Gly Pro Pro Gly Pro 660 665 670 Ala Pro Ala Gly Pro Ala Val Arg Leu Gly Ser Val Pro Tyr Ser Leu 675 680 685 Leu Phe Arg Val Leu Leu Gln Cys Leu Lys Gln Glu Ser Asp Trp Lys 690 695 700 Val Leu Lys Leu Val Leu Gly Arg Leu Pro Glu Ser Leu Arg Tyr Lys 705 710 715 720 Val Leu Ile Phe Thr Ser Pro Cys Ser Val Asp Gln Leu Cys Ser Ala 725 730 735 Leu Cys Ser Met Leu Ser Gly Pro Lys Thr Leu Glu Arg Leu Arg Gly 740 745 750 Ala Pro Glu Gly Phe Ser Arg Thr Asp Leu His Leu Ala Val Val Pro 755 760 765 Val Leu Thr Ala Leu Ile Ser Tyr His Asn Tyr Leu Asp Lys Thr Lys 770 775 780 Gln Arg Glu Met Val Tyr Cys Leu Glu Gln Gly Leu Ile His Arg Cys 785 790 795 800 Ala Ser Gln Cys Val Val Ala Leu Ser Ile Cys Ser Val Glu Met Pro 805 810 815 Asp Ile Ile Ile Lys Ala Leu Pro Val Leu Val Val Lys Leu Thr His 820 825 830 Ile Ser Ala Thr Ala Ser Met Ala Val Pro Leu Leu Glu Phe Leu Ser 835 840 845 Thr Leu Ala Arg Leu Pro His Leu Tyr Arg Asn Phe Ala Ala Glu Gln 850 855 860 Tyr Ala Ser Val Phe Ala Ile Ser Leu Pro Tyr Thr Asn Pro Ser Lys 865 870 875 880 Phe Asn Gln Tyr Ile Val Cys Leu Ala His His Val Ile Ala Met Trp 885 890 895 Phe Ile Arg Cys Arg Leu Pro Phe Arg Lys Asp Phe Val Pro Phe Ile 900 905 910 Thr Lys Gly Leu Arg Ser Asn Val Leu Leu Ser Phe Asp Asp Thr Pro 915 920 925 Glu Lys Asp Ser Phe Arg Ala Arg Ser Thr Ser Leu Asn Glu Arg Pro 930 935 940 Lys Ser Leu Arg Ile Ala Arg Pro Pro Lys Gln Gly Leu Asn Asn Ser 945 950 955 960 Pro Pro Val Lys Glu Phe Lys Glu Ser Ser Ala Ala Glu Ala Phe Arg 965 970 975 Cys Arg Ser Ile Ser Val Ser Glu His Val Val Arg Ser Arg Ile Gln 980 985 990 Thr Ser Leu Thr Ser Ala Ser Leu Gly Ser Ala Asp Glu Asn Ser Val 995 1000 1005 Ala Gln Ala Asp Asp Ser Leu Lys Asn Leu His Leu Glu Leu Thr 1010 1015 1020 Glu Thr Cys Leu Asp Met Met Ala Arg Tyr Val Phe Ser Asn Phe 1025 1030 1035 Thr Ala Val Pro Lys Arg Ser Pro Val Gly Glu Phe Leu Leu Ala 1040 1045 1050 Gly Gly Arg Thr Lys Thr Trp Leu Val Gly Asn Lys Leu Val Thr 1055 1060 1065 Val Thr Thr Ser Val Gly Thr Gly Thr Arg Ser Leu Leu Gly Leu 1070 1075 1080 Asp Ser Gly Glu Leu Gln Ser Gly Pro Glu Ser Ser Ser Ser Pro 1085 1090 1095 Gly Val His Val Arg Gln Thr Lys Glu Ala Pro Ala Lys Leu Glu 1100 1105 1110 Ser Gln Ala Gly Gln Gln Val Ser Arg Gly Ala Arg Asp Arg Val 1115 1120 1125 Arg Ser Met Ser Gly Gly His Gly Leu Arg Val Gly Ala Leu Asp 1130 1135 1140 Val Pro Ala Ser Gln Phe Leu Gly Ser Ala Thr Ser Pro Gly Pro 1145 1150 1155 Arg Thr Ala Pro Ala Ala Lys Pro Glu Lys Ala Ser Ala Gly Thr 1160 1165 1170 Arg Val Pro Val Gln Glu Lys Thr Asn Leu Ala Ala Tyr Val Pro 1175 1180 1185 Leu Leu Thr Gln Gly Trp Ala Glu Ile Leu Val Arg Arg Pro Thr 1190 1195 1200 Gly Asn Thr Ser Trp Leu Met Ser Leu Glu Asn Pro Leu Ser Pro 1205 1210 1215 Phe Ser Ser Asp Ile Asn Asn Met Pro Leu Gln Glu Leu Ser Asn 1220 1225 1230 Ala Leu Met Ala Ala Glu Arg Phe Lys Glu His Arg Asp Thr Ala 1235 1240 1245 Leu Tyr Lys Ser Leu Ser Val Pro Ala Ala Ser Thr Ala Lys Pro 1250 1255 1260 Pro Pro Leu Pro Arg Ser Asn Thr Val Ala Ser Phe Ser Ser Leu 1265 1270 1275 Tyr Gln Ser Ser Cys Gln Gly Gln Leu His Arg Ser Val Ser Trp 1280 1285 1290 Ala Asp Ser Ala Val Val Met Glu Glu Gly Ser Pro Gly Glu Val 1295 1300 1305 Pro Val Leu Val Glu Pro Pro Gly Leu Glu Asp Val Glu Ala Ala 1310 1315 1320 Leu Gly Met Asp Arg Arg Thr Asp Ala Tyr Ser Arg Ser Ser Ser 1325 1330 1335 Val Ser Ser Gln Glu Glu Lys Ser Leu His Ala Glu Glu Leu Val 1340 1345 1350 Gly Arg Gly Ile Pro Ile Glu Arg Val Val Ser Ser Glu Gly Gly 1355 1360 1365 Arg Pro Ser Val Asp Leu Ser Phe Gln Pro Ser Gln Pro Leu Ser 1370 1375 1380 Lys Ser Ser Ser Ser Pro Glu Leu Gln Thr Leu Gln Asp Ile Leu 1385 1390 1395 Gly Asp Pro Gly Asp Lys Ala Asp Val Gly Arg Leu Ser Pro Glu 1400 1405 1410 Val Lys Ala Arg Ser Gln Ser Gly Thr Leu Asp Gly Glu Ser Ala 1415 1420 1425 Ala Trp Ser Ala Ser Gly Glu Asp Ser Arg Gly Gln Pro Glu Gly 1430 1435 1440 Pro Leu Pro Ser Ser Ser Pro Arg Ser Pro Ser Gly Leu Arg Pro 1445 1450 1455 Arg Gly Tyr Thr Ile Ser Asp Ser Ala Pro Ser Arg Arg Gly Lys 1460 1465 1470 Arg Val Glu Arg Asp Ala Leu Lys Ser Arg Ala Thr Ala Ser Asn 1475 1480 1485 Ala Glu Lys Val Pro Gly Ile Asn Pro Ser Phe Val Phe Leu Gln 1490 1495 1500 Leu Tyr His Ser Pro Phe Phe Gly Asp Glu Ser Asn Lys Pro Ile 1505 1510 1515 Leu Leu Pro Asn Glu Ser Gln Ser Phe Glu Arg Ser Val Gln Leu 1520 1525 1530 Leu Asp Gln Ile Pro Ser Tyr Asp Thr His Lys Ile Ala Val Leu 1535 1540 1545 Tyr Val Gly Glu Gly Gln Ser Asn Ser Glu Leu Ala Ile Leu Ser 1550 1555 1560 Asn Glu His Gly Ser Tyr Arg Tyr Thr Glu Phe Leu Thr Gly Leu 1565 1570 1575 Gly Arg Leu Ile Glu Leu Lys Asp Cys Gln Pro Asp Lys Val Tyr 1580 1585 1590 Leu Gly Gly Leu Asp Val Cys Gly Glu Asp Gly Gln Phe Thr Tyr 1595 1600 1605 Cys Trp His Asp Asp Ile Met Gln Ala Val Phe His Ile Ala Thr 1610 1615 1620 Leu Met Pro Thr Lys Asp Val Asp Lys His Arg Cys Asp Lys Lys 1625 1630 1635 Arg His Leu Gly Asn Asp Phe Val Ser Ile Val Tyr Asn Asp Ser 1640 1645 1650 Gly Glu Asp Phe Lys Leu Gly Thr Ile Lys Gly Gln Phe Asn Phe 1655 1660 1665 Val His Val Ile Val Thr Pro Leu Asp Tyr Glu Cys Asn Leu Val 1670 1675 1680 Ser Leu Gln Cys Arg Lys Asp Met Glu Gly Leu Val Asp Thr Ser 1685 1690 1695 Val Ala Lys Ile Val Ser Asp Arg Asn Leu Pro Phe Val Ala Arg 1700 1705 1710 Gln Met Ala Leu His Ala Asn Met Ala Ser Gln Val His His Ser 1715 1720 1725 Arg Ser Asn Pro Thr Asp Ile Tyr Pro Ser Lys Trp Ile Ala Arg 1730 1735 1740 Leu Arg His Ile Lys Arg Leu Arg Gln Arg Ile Cys Glu Glu Ala 1745 1750 1755 Ala Tyr Ser Asn Pro Ser Leu Pro Leu Val His Pro Pro Ser His 1760 1765 1770 Ser Lys Ala Pro Ala Gln Thr Pro Ala Glu Pro Thr Pro Gly Tyr 1775 1780 1785 Glu Val Gly Gln Arg Lys Arg Leu Ile Ser Ser Val Glu Asp Phe 1790 1795 1800 Thr Glu Phe Val 1805 <210> 11 <211> 768 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 11 Met Ala Lys Pro Thr Ser Lys Asp Ser Gly Leu Lys Glu Lys Phe Lys 1 5 10 15 Ile Leu Leu Gly Leu Gly Thr Pro Arg Pro Asn Pro Arg Ser Ala Glu 20 25 30 Gly Lys Gln Thr Glu Phe Ile Ile Thr Ala Glu Ile Leu Arg Glu Leu 35 40 45 Ser Met Glu Cys Gly Leu Asn Asn Arg Ile Arg Met Ile Gly Gln Ile 50 55 60 Cys Glu Val Ala Lys Thr Lys Lys Phe Glu Glu His Ala Val Glu Ala 65 70 75 80 Leu Trp Lys Ala Val Ala Asp Leu Leu Gln Pro Glu Arg Pro Leu Glu 85 90 95 Ala Arg His Ala Val Leu Ala Leu Leu Lys Ala Ile Val Gln Gly Gln 100 105 110 Gly Glu Arg Leu Gly Val Leu Arg Ala Leu Phe Phe Lys Val Ile Lys 115 120 125 Asp Tyr Pro Ser Asn Glu Asp Leu His Glu Arg Leu Glu Val Phe Lys 130 135 140 Ala Leu Thr Asp Asn Gly Arg His Ile Thr Tyr Leu Glu Glu Glu Leu 145 150 155 160 Ala Asp Phe Val Leu Gln Trp Met Asp Val Gly Leu Ser Ser Glu Phe 165 170 175 Leu Leu Val Leu Val Asn Leu Val Lys Phe Asn Ser Cys Tyr Leu Asp 180 185 190 Glu Tyr Ile Ala Arg Met Val Gln Met Ile Cys Leu Leu Cys Val Arg 195 200 205 Thr Ala Ser Ser Val Asp Ile Glu Val Ser Leu Gln Val Leu Asp Ala 210 215 220 Val Val Cys Tyr Asn Cys Leu Pro Ala Glu Ser Leu Pro Leu Phe Ile 225 230 235 240 Val Thr Leu Cys Arg Thr Ile Asn Val Lys Glu Leu Cys Glu Pro Cys 245 250 255 Trp Lys Leu Met Arg Asn Leu Leu Gly Thr His Leu Gly His Ser Ala 260 265 270 Ile Tyr Asn Met Cys His Leu Met Glu Asp Arg Ala Tyr Met Glu Asp 275 280 285 Ala Pro Leu Leu Arg Gly Ala Val Phe Phe Val Gly Met Ala Leu Trp 290 295 300 Gly Ala His Arg Leu Tyr Ser Leu Arg Asn Ser Pro Thr Ser Val Leu 305 310 315 320 Pro Ser Phe Tyr Gln Ala Met Ala Cys Pro Asn Glu Val Val Ser Tyr 325 330 335 Glu Ile Val Leu Ser Ile Thr Arg Leu Ile Lys Lys Tyr Arg Lys Glu 340 345 350 Leu Gln Val Val Ala Trp Asp Ile Leu Leu Asn Ile Ile Glu Arg Leu 355 360 365 Leu Gln Gln Leu Gln Thr Leu Asp Ser Pro Glu Leu Arg Thr Ile Val 370 375 380 His Asp Leu Leu Thr Thr Val Glu Glu Leu Cys Asp Gln Asn Glu Phe 385 390 395 400 His Gly Ser Gln Glu Arg Tyr Phe Glu Leu Val Glu Arg Cys Ala Asp 405 410 415 Gln Arg Pro Glu Ser Ser Leu Leu Asn Leu Ile Ser Tyr Arg Ala Gln 420 425 430 Ser Ile His Pro Ala Lys Asp Gly Trp Ile Gln Asn Leu Gln Ala Leu 435 440 445 Met Glu Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 450 455 460 Gly Gly Lys Pro Ile Leu Leu Pro Asn Glu Ser Gln Ser Phe Glu Arg 465 470 475 480 Ser Val Gln Leu Leu Asp Gln Ile Pro Ser Tyr Asp Thr His Lys Ile 485 490 495 Ala Val Leu Tyr Val Gly Glu Gly Gln Ser Asn Ser Glu Leu Ala Ile 500 505 510 Leu Ser Asn Glu His Gly Ser Tyr Arg Tyr Thr Glu Phe Leu Thr Gly 515 520 525 Leu Gly Arg Leu Ile Glu Leu Lys Asp Cys Gln Pro Asp Lys Val Tyr 530 535 540 Leu Gly Gly Leu Asp Val Cys Gly Glu Asp Gly Gln Phe Thr Tyr Cys 545 550 555 560 Trp His Asp Asp Ile Met Gln Ala Val Phe His Ile Ala Thr Leu Met 565 570 575 Pro Thr Lys Asp Val Asp Lys His Arg Cys Asp Lys Lys Arg His Leu 580 585 590 Gly Asn Asp Phe Val Ser Ile Val Tyr Asn Asp Ser Gly Glu Asp Phe 595 600 605 Lys Leu Gly Thr Ile Lys Gly Gln Phe Asn Phe Val His Val Ile Val 610 615 620 Thr Pro Leu Asp Tyr Glu Cys Asn Leu Val Ser Leu Gln Cys Arg Lys 625 630 635 640 Asp Met Glu Gly Leu Val Asp Thr Ser Val Ala Lys Ile Val Ser Asp 645 650 655 Arg Asn Leu Pro Phe Val Ala Arg Gln Met Ala Leu His Ala Asn Met 660 665 670 Ala Ser Gln Val His His Ser Arg Ser Asn Pro Thr Asp Ile Tyr Pro 675 680 685 Ser Lys Trp Ile Ala Arg Leu Arg His Ile Lys Arg Leu Arg Gln Arg 690 695 700 Ile Cys Glu Glu Ala Ala Tyr Ser Asn Pro Ser Leu Pro Leu Val His 705 710 715 720 Pro Pro Ser His Ser Lys Ala Pro Ala Gln Thr Pro Ala Glu Pro Thr 725 730 735 Pro Gly Tyr Glu Val Gly Gln Arg Lys Arg Leu Ile Ser Ser Val Glu 740 745 750 Asp Phe Thr Glu Phe Val Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 755 760 765 <210> 12 <211> 2336 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 12 gctagcacta gtaccatggc gaaaccgacc agcaaagata gcggcctgaa agaaaaattt 60 aaaattctgc tgggcctggg caccccgcgc ccgaacccgc gcagcgcgga aggcaaacag 120 accgaattta ttattaccgc ggaaattctg cgcgaactga gcatggaatg cggcctgaac 180 aaccgcattc gcatgattgg ccagatttgc gaagtggcga aaaccaaaaa atttgaagaa 240 catgcggtgg aagcgctgtg gaaagcggtg gcggatctgc tgcagccgga acgcccgctg 300 gaagcgcgcc atgcggtgct ggcgctgctg aaagcgattg tgcagggcca gggcgaacgc 360 ctgggcgtgc tgcgcgcgct gttttttaaa gtgattaaag attatccgag caacgaagat 420 ctgcatgaac gcctggaagt gtttaaagcg ctgaccgata acggccgcca tattacctat 480 ctggaagaag aactggcgga ttttgtgctg cagtggatgg atgtgggcct gagcagcgaa 540 tttctgctgg tgctggtgaa cctggtgaaa tttaacagct gctatctgga tgaatatatt 600 gcgcgcatgg tgcagatgat ttgcctgctg tgcgtgcgca ccgcgagcag cgtggatatt 660 gaagtgagcc tgcaggtgct ggatgcggtg gtgtgctata actgcctgcc ggcggaaagc 720 ctgccgctgt ttattgtgac cctgtgccgc accattaacg tgaaagaact gtgcgaaccg 780 tgctggaaac tgatgcgcaa cctgctgggc acccatctgg gccatagcgc gatttataac 840 atgtgccatc tgatggaaga tcgcgcgtat atggaagatg cgccgctgct gcgcggcgcg 900 gtgttttttg tgggcatggc gctgtggggc gcgcatcgcc tgtatagcct gcgcaacagc 960 ccgaccagcg tgctgccgag cttttatcag gcgatggcgt gcccgaacga agtggtgagc 1020 tatgaaattg tgctgagcat tacccgcctg attaaaaaat atcgcaaaga actgcaggtg 1080 gtggcgtggg atattctgct gaacattatt gaacgcctgc tgcagcagct gcagaccctg 1140 gatagcccgg aactgcgcac cattgtgcat gatctgctga ccaccgtgga agaactgtgc 1200 gatcagaacg aatttcatgg cagccaggaa cgctattttg aactggtgga acgctgcgcg 1260 gatcagcgcc cggaaagcag cctgctgaac ctgattagct atcgcgcgca gagcattcat 1320 ccggcgaaag atggctggat tcagaacctg caggcgctga tggaatctgg tgggggtagc 1380 ggaggagggt cagggggcgg cagtggaggc ggaaaaccga ttctgctgcc gaacgaaagc 1440 cagagctttg aacgcagcgt gcagctgctg gatcagattc cgagctatga tacccataaa 1500 attgcggtgc tgtatgtggg cgaaggccag agcaacagcg aactggcgat tctgagcaac 1560 gaacatggca gctatcgcta taccgaattt ctgaccggcc tgggccgcct gattgaactg 1620 aaagattgcc agccggataa agtgtatctg ggcggcctgg atgtgtgcgg cgaagatggc 1680 cagtttacct attgctggca tgatgatatt atgcaggcgg tgtttcatat tgcgaccctg 1740 atgccgacca aagatgtgga taaacatcgc tgcgataaaa aacgccatct gggcaacgat 1800 tttgtgagca ttgtgtataa cgatagcggc gaagatttta aactgggcac cattaaaggc 1860 cagtttaact ttgtgcatgt gattgtgacc ccgctggatt atgaatgcaa cctggtgagc 1920 ctgcagtgcc gcaaagatat ggaaggcctg gtggatacca gcgtggcgaa aattgtgagc 1980 gatcgcaacc tgccgtttgt ggcgcgccag atggcgctgc atgcgaacat ggcgagccag 2040 gtgcatcata gccgcagcaa cccgaccgat atttatccga gcaaatggat tgcgcgcctg 2100 cgccatatta aacgcctgcg ccagcgcatt tgcgaagaag cggcgtatag caacccgagc 2160 ctgccgctgg tgcatccgcc gagccatagc aaagcgccgg cgcagacccc ggcggaaccg 2220 accccgggct atgaagtggg ccagcgcaaa cgcctgatta gcagcgtgga agattttacc 2280 gaatttgtgg aacaaaaact catctcagaa gaggatctgt aggcggccgc ctcgag 2336

Claims

1. A recombinant adeno-associated virus (rAAV), comprising: the rAAV comprises an AAV capsid and an AAV genome packaged therein; The AAV genome A nucleic acid molecule capable of expressing c-tuberin, which contains the hamartin binding domain and the GAP domain but lacks the Akt phosphorylation site Thr 1462, and has at least 90% sequence identity to SEQ ID NO:

1. The rAAV comprising:

2. The rAAV of claim 1, wherein the AAV capsid is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid.

3. The rAAV of any one of claims 1 to 2, wherein the nucleic acid is operably linked to a regulatory control sequence.

4. The rAAV of any one of claims 1 to 3, wherein the regulatory control sequence comprises a human cytomegalovirus (CMV) promoter, a chicken beta-actin (CBA) promoter, a Rous sarcoma virus (RSV) LTR promoter / enhancer, an SV40 promoter, a dihydrofolate reductase promoter, a phosphoglycerol kinase promoter, a CMV immediate early gene enhancer / CBA promoter, a synapsin promoter, or a glial fibrillary acidic protein (GFAP) promoter.

5. The rAAV of any one of claims 1 to 4, wherein the nucleic acid molecule comprises an ITR.

6. The rAAV of any one of claims 1 to 5, wherein the nucleic acid molecule comprises polyA.

7. A composition comprising the rAAV of any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

8. A pharmaceutical for treating patients with tuberous sclerosis complex (TSC), comprising the rAAV of any one of claims 1 to 5.

9. 9. The pharmaceutical composition according to claim 8, characterized in that it is administered to brain cells, heart cells, kidney cells, skin cells, or lung cells.

10. 9. The pharmaceutical composition according to claim 8, which is administered intravascularly, intravenously, intracerebrally, intraventricularly, intrathecally, or to the skin.

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