Methods of treating lysosomal disorders

Abstract

To provide methods for treating a lysosomal transmembrane protein disease or disorder in a subject.SOLUTION: Provided herein are methods for treating a lysosomal transmembrane protein disease or disorder through ex vivo introduction of a nucleic acid molecule into hematopoietic stem and progenitor cells (HSPCs) followed by transplantation of the HSPCs into a subject in need of treatment. Also provided are vectors containing the nucleic acid molecule.SELECTED DRAWING: Figure 12D

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. patent application Ser. No. 62 / 471,741, filed Mar. 15, 2017, and U.S. patent application Ser. No. 62 / 507,713, filed May 17, 2017, the entire contents of each of which are incorporated herein by reference.

[0002] Grant information This invention was made with Government support under Grant Nos. DK090058 and HL107755 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0003] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy, created on March 15, 2018, is named 20378-201753_SL.txt and is 109 kilobytes in size.

[0004] FIELD OF THEINVENTION The present invention relates generally to lysosomal diseases associated with malfunctioning transmembrane lysosomal proteins, and more specifically to the treatment of such diseases by hematopoietic stem and progenitor cell (HSPC) gene therapy. [Background technology]

[0005] Background information Lysosomal membrane proteins act at several critical stages of the lysosomal life cycle, including acidification of the lumen, excretion of metabolic products, recruitment of molecular motors, and fusion with other organelles. Lysosomal storage diseases are a group of inherited metabolic disorders resulting from defects in lysosomal function. Lysosomes are enzyme-containing sacs inside the cell that digest large molecules and send their fragments to other parts of the cell for recycling. This process requires several essential enzymes. If one of these enzymes is defective (e.g., due to a mutation), large molecules accumulate inside the cell and eventually kill the cell.

[0006] Some of the approximately 50 known lysosomal storage diseases are caused by the malfunction of lysosomal membrane proteins. One such lysosomal membrane protein disease is cystinosis, which is characterized by abnormal accumulation of the amino acid cystine in all cells of the body, leading to multiple organ failure. Cystinosis is caused by mutations in the CTNS gene, which encodes cystinosin, a cystine transporter specific to the lysosomal membrane. The intracellular metabolism of cystine, as with all amino acids, requires that cystine be transported across the cell membrane. Normally, cystine is transported to the cytosol after endocytosed proteins are degraded to cystine in lysosomes. However, when the transport protein is defective, cystine accumulates in lysosomes. Because cystine is extremely insoluble, when its concentration in tissue lysosomes increases, it quickly exceeds its solubility, and crystalline precipitates form in almost all organs and tissues. Another example is Danon disease, which is caused by mutations in the LAMP-2 gene, a lysosomal transmembrane protein essential for autophagic flux.

[0007] To date, there are no known cures or preventative measures for such lysosomal diseases, and current therapies are aimed at treating the associated symptoms. Thus, there is a need in the art for alternative or improved methods for treating lysosomal diseases / disorders. Summary of the Invention

[0008] Thus, in one aspect, the present invention provides a method for treating a lysosomal transmembrane protein disease or disorder in a subject.The method includes introducing a corresponding functional human lysosomal transmembrane protein into the hematopoietic stem and progenitor cells (HSPCs) of the subject, and transplanting the HSPCs into the subject, thereby treating the lysosomal transmembrane protein disease or disorder. Thus, when the lysosomal transmembrane protein disease or disorder is cystinosis, the corresponding functional human lysosomal transmembrane protein is cystinosin (CTNS); when the lysosomal transmembrane protein disease or disorder is Salla disease or infantile sialic acid storage disorder, the corresponding functional human lysosomal transmembrane protein is sialin (SLC17A5); when the lysosomal transmembrane protein disease or disorder is cobalamin disease type F, the corresponding functional human lysosomal transmembrane protein is LMBD1; when the lysosomal transmembrane protein disease or disorder is late-onset childhood neuronal ceroid lipofuscinosis, the corresponding functional human lysosomal transmembrane protein is CLN7; when the lysosomal transmembrane protein disease or disorder is juvenile neuronal ceroid lipofuscinosis, the corresponding functional human lysosomal transmembrane protein is cystinosin (CTNS); the protein is Battenin (CLN3); if the lysosomal transmembrane protein disease or disorder is malignant infantile osteopetrosis, the corresponding functional human lysosomal transmembrane protein is ClC-7 or OSTM1; if the lysosomal transmembrane protein disease or disorder is mucolipidosis IV, the corresponding functional human lysosomal transmembrane protein is TRPML-1; if the lysosomal transmembrane protein disease or disorder is mucopolysaccharidosis type IIC, the corresponding functional human lysosomal transmembrane protein is HGSNAT; if the lysosomal transmembrane protein disease or disorder is Niemann-Pick disease type C, the corresponding functional human lysosomal transmembrane protein is NPC-1; if the lysosomal transmembrane protein disease or disorder is Danon disease, the corresponding functional human lysosomal transmembrane protein is LAMP2.

[0009] In various embodiments, the introducing step may include contacting the HSPC with a vector comprising a polynucleotide encoding a functional human lysosomal transmembrane protein and a functional promoter, and expressing the functional human lysosomal transmembrane protein. In various embodiments, the lysosomal transmembrane protein disease or disorder is cystinosis, and the functional human lysosomal transmembrane protein is CTNS. In various embodiments, the lysosomal transmembrane protein disease or disorder is Danon disease, and the functional human lysosomal transmembrane protein is LAMP2. The LAMP2 may be an isoform selected from the group consisting of LAMP-2A, LAMP-2B, and LAMP-2C. The subject may be a mammal, such as a human. In various embodiments, the vector is a viral vector selected from the group consisting of a lentiviral vector, an adenoviral vector, and an AAV vector. In various embodiments, the vector is a lentiviral vector. In various embodiments, the vector is an adenoviral vector. In various embodiments, the vector is an AAV vector. In various embodiments, the vector is a self-inactivating (SIN) lentiviral vector, such as pCCL-CTNS or pCCL-LAMP2. In various embodiments, the introducing step is carried out ex vivo. In various embodiments, the HSPC is isolated from the bone marrow of the subject.

[0010] In another aspect, the present invention provides an expression cassette comprising a promoter operably linked to a transgene encoding a functional human lysosomal transmembrane protein selected from the group consisting of CTNS, SLC17A5, LMBRD1, CLN7, CLN3, CLC-7, OSTM1, TRPML1, HGSNAT, NPC1, and LAMP2. Also provided is a vector, such as a self-inactivating (SIN) lentiviral vector, comprising a promoter operably linked to a polynucleotide encoding a functional human lysosomal transmembrane protein selected from the group consisting of CTNS, SLC17A5, LMBRD1, CLN7, CLN3, CLC-7, OSTM1, TRPML1, HGSNAT, NPC1, and LAMP2. In various embodiments, the functional human lysosomal transmembrane protein is CTNS. In various embodiments, the functional human lysosomal transmembrane protein is LAMP2.

[0011] In another aspect, the present invention provides a method for treating or ameliorating a lysosomal protein disease or disorder in a subject, comprising isolating hematopoietic stem cells and HSPC cells from the bone marrow of a subject, introducing a functional human lysosomal transmembrane gene into the HSPC, the gene encoding a protein corresponding to the lysosomal protein disease or disorder, and transplanting the HSPC back into the subject, thereby treating or ameliorating the lysosomal protein disease or disorder. Thus, if the lysosomal transmembrane protein disease or disorder is cystinosis, the functional human lysosomal transmembrane gene is CTNS; if the lysosomal transmembrane protein disease or disorder is Salla disease or infantile sialic acid storage disorder, the functional human lysosomal transmembrane gene is SLC17A5; if the lysosomal transmembrane protein disease or disorder is cobalaminopathy type F, the functional human lysosomal transmembrane gene is LMBRD1; if the lysosomal transmembrane protein disease or disorder is late-onset childhood neuronal ceroid lipofuscinosis, the functional human lysosomal transmembrane gene is MFSD8; if the lysosomal transmembrane protein disease or disorder is juvenile neuronal ceroid lipofuscinosis, the functional human lysosomal transmembrane gene is MFSD8. if the lysosomal transmembrane protein disease or disorder is malignant infantile osteopetrosis, the functional human lysosomal transmembrane gene is CLCN7 or OSTM1; if the lysosomal transmembrane protein disease or disorder is mucolipidosis IV, the functional human lysosomal transmembrane gene is MCOLN1; if the lysosomal transmembrane protein disease or disorder is mucopolysaccharidosis IIC, the functional human lysosomal transmembrane gene is HGSNAT; if the lysosomal transmembrane protein disease or disorder is Niemann-Pick disease type C, the functional human lysosomal transmembrane gene is NPC1; if the lysosomal transmembrane protein disease or disorder is Danon disease, the functional human lysosomal transmembrane gene is LAMP2.

[0012] In various embodiments, the HSPCs are CD34+ cells. In various embodiments, the lysosomal protein disease or disorder is cystinosis and the functional human lysosomal transmembrane gene is CTNS. In various embodiments, the lysosomal protein disease or disorder is Danon disease and the functional human lysosomal transmembrane gene is LAMP2. In various embodiments, the step of introducing the functional human CTNS gene into the HSPCs comprises using a vector, such as a viral vector. In various embodiments, the vector is a viral vector selected from the group consisting of a lentiviral vector, an adenoviral vector, and an AAV vector. In various embodiments, the level of cystine in the subject's eye, skin, white blood cells, parenchymal tissue, or gastrointestinal tract is reduced after treatment. In various embodiments, the dosage is about 1.0×10 6 ~5.0×10 6 Cells / kg, e.g. 2.5 x 10 6 cells / kg and administered as a single dose.

[0013] The subject may have received cysteamine therapy, such as oral cysteamine therapy, prior to treatment. The dose administration may be intravenous. In various embodiments, cystine or cystine crystals in the eye, skin, white blood cells, parenchymal tissue, and / or gastrointestinal tract are measured prior to and / or after treatment. In various embodiments, cystine or cystine crystals in the eye are measured prior to and / or after treatment. In various embodiments, cystine crystals are measured using in vivo confocal microscopy. In various embodiments, cystine levels may be measured prior to, during, and / or after treatment. In various embodiments, cystine levels are measured using a biological sample, such as blood, rectal biopsy, or buccal mucosa. In various embodiments, cystine levels are measured from a rectal biopsy.

[0014] In another aspect, the present invention provides a method for treating or ameliorating lysosomal protein disease or disorder in a subject.The method includes using gene editing to create a functional human lysosomal transmembrane gene in a subject.Therefore, when the lysosomal transmembrane protein disease or disorder is cystinosis, the functional human lysosomal transmembrane gene is CTNS; when the lysosomal transmembrane protein disease or disorder is Salla disease or infantile sialic acid storage disorder, the functional human lysosomal transmembrane gene is SLC17A5; when the lysosomal transmembrane protein disease or disorder is cobalamin disease type F, the functional human lysosomal transmembrane gene is LMBRD1; when the lysosomal transmembrane protein disease or disorder is late-onset childhood neuronal ceroid lipofuscinosis, the functional human lysosomal transmembrane gene is MFSD8; when the lysosomal transmembrane protein disease or disorder is juvenile neuronal ceroid lipofuscinosis, the functional human lysosomal transmembrane gene is MFSD8. if the lysosomal transmembrane protein disease or disorder is malignant infantile osteopetrosis, the functional human lysosomal transmembrane gene is CLCN7 or OSTM1; if the lysosomal transmembrane protein disease or disorder is mucolipidosis IV, the functional human lysosomal transmembrane gene is MCOLN1; if the lysosomal transmembrane protein disease or disorder is mucopolysaccharidosis IIC, the functional human lysosomal transmembrane gene is HGSNAT; if the lysosomal transmembrane protein disease or disorder is Niemann-Pick disease type C, the functional human lysosomal transmembrane gene is NPC1; if the lysosomal transmembrane protein disease or disorder is Danon disease, the functional human lysosomal transmembrane gene is LAMP2.

[0015] In another aspect, the present invention provides a method for treating or ameliorating a lysosomal protein disease or disorder in a subject, comprising contacting a cell from the subject expressing a defective lysosomal transmembrane protein with a vector encoding a gene editing system that, when transfected into the cell, removes a trinucleotide expansion mutation in a gene encoding an endogenous lysosomal transmembrane protein, thereby treating the lysosomal protein disease or disorder. Thus, if the lysosomal transmembrane protein disease or disorder is cystinosis, the lysosomal transmembrane protein is cystinosin (CTNS); if the lysosomal transmembrane protein disease or disorder is Salla disease or infantile sialic acid storage disorder, the lysosomal transmembrane protein is sialin (SLC17A5); if the lysosomal transmembrane protein disease or disorder is cobalaminopathy type F, the lysosomal transmembrane protein is LMBD1; if the lysosomal transmembrane protein disease or disorder is late-onset childhood neuronal ceroid lipofuscinosis, the lysosomal transmembrane protein is CLN7; if the lysosomal transmembrane protein disease or disorder is juvenile neuronal ceroid lipofuscinosis, the lysosomal transmembrane protein is cystinosin (CTNS); the protein is Battenin (CLN3); if the lysosomal transmembrane protein disease or disorder is malignant infantile osteopetrosis, the lysosomal transmembrane protein is ClC-7 or OSTM1; if the lysosomal transmembrane protein disease or disorder is mucolipidosis IV, the lysosomal transmembrane protein is TRPML-1; if the lysosomal transmembrane protein disease or disorder is mucopolysaccharidosis type IIC, the lysosomal transmembrane protein is HGSNAT; if the lysosomal transmembrane protein disease or disorder is Niemann-Pick disease type C, the lysosomal transmembrane protein is NPC-1; if the lysosomal transmembrane protein disease or disorder is Danon disease, the lysosomal transmembrane protein is LAMP2.

[0016] In various embodiments, the gene editing system is selected from the group consisting of CRISPR / Cas, zinc finger nuclease, engineered meganuclease, ARCUS, and transcription activator-like effector nuclease. In various embodiments, the contacting step comprises administering an effective amount of the vector to the subject. In various embodiments, the contacting step comprises obtaining a cell sample from the subject, transfecting the cell sample with the gene editing system, and then transplanting the transfected cell into the subject. In various embodiments, the cell sample is selected from the group consisting of blood cells and HSPCs. [The present invention 1001] 1. A method of treating a lysosomal transmembrane protein disease or disorder in a subject, comprising: introducing a corresponding functional human lysosomal transmembrane protein into hematopoietic stem and progenitor cells (HSPCs) of the subject; and Transplanting the HSPCs into the subject. thereby treating a lysosomal transmembrane protein disease or disorder. [The present invention 1002] (a) When the lysosomal transmembrane protein disease or disorder is cystinosis, the corresponding functional human lysosomal transmembrane protein is cystinosine (CTNS); (b) when the lysosomal transmembrane protein disease or disorder is Salla disease or infantile sialic acid storage disorder, the corresponding functional human lysosomal transmembrane protein is sialin (SLC17A5); (c) when the lysosomal transmembrane protein disease or disorder is cobalaminopathy type F, the corresponding functional human lysosomal transmembrane protein is LMBD1; (d) when the lysosomal transmembrane protein disease or disorder is late-onset childhood neuronal ceroid lipofuscinosis, the corresponding functional human lysosomal transmembrane protein is CLN7; (e) when the lysosomal transmembrane protein disease or disorder is juvenile neuronal ceroid lipofuscinosis, the corresponding functional human lysosomal transmembrane protein is battenin (CLN3); (f) when the lysosomal transmembrane protein disease or disorder is malignant infantile osteopetrosis, the corresponding functional human lysosomal transmembrane protein is ClC-7 or OSTM1; (g) when the lysosomal transmembrane protein disease or disorder is mucolipidosis IV, the corresponding functional human lysosomal transmembrane protein is TRPML-1; (h) when the lysosomal transmembrane protein disease or disorder is mucopolysaccharidosis type IIC, the corresponding functional human lysosomal transmembrane protein is HGSNAT; (i) when the lysosomal transmembrane protein disease or disorder is Niemann-Pick disease type C, the corresponding functional human lysosomal transmembrane protein is NPC-1; and (j) when the lysosomal transmembrane protein disease or disorder is Danon disease, the corresponding functional human lysosomal transmembrane protein is LAMP2; The method of the present invention 1001. [The present invention 1003] The method of the present invention 1001, wherein the introducing step comprises contacting HSPCs with a vector comprising a polynucleotide encoding a functional human lysosomal transmembrane protein and a functional promoter, and expressing the functional human lysosomal transmembrane protein. [The present invention 1004] 1001. The method of claim 1001, wherein the lysosomal transmembrane protein disease or disorder is cystinosis and the functional human lysosomal transmembrane protein is CTNS. [The present invention 1005] 1001. The method of claim 1001, wherein the lysosomal transmembrane protein disease or disorder is Danon disease and the functional human lysosomal transmembrane protein is LAMP2. [The present invention 1006] 1005. The method of the present invention, wherein LAMP2 is an isoform selected from the group consisting of LAMP-2A, LAMP-2B, and LAMP-2C. [The present invention 1007] The method of claim 1001, wherein the subject is a mammal. [The present invention 1008] The method of claim 1007, wherein the subject is a human. [The present invention 1009] The method of claim 10, wherein the vector is a viral vector selected from the group consisting of a lentiviral vector, an adenoviral vector, and an AAV vector. [The present invention 1010] 1009. The method of claim 10, wherein the vector is a self-inactivating (SIN) lentiviral vector. [The present invention 1011] The method of claim 1004, wherein the vector is a self-inactivating (SIN) lentiviral vector. [The present invention 1012] The method of claim 1011, wherein the vector is pCCL-CTNS. [The present invention 1013] 1005. The method of claim 10, wherein the vector is a self-inactivating (SIN) lentiviral vector. [The present invention 1014] The method of claim 1013, wherein the vector is pCCL-LAMP2. [The present invention 1015] The method of any one of claims 10 to 13, wherein the introducing step is carried out ex vivo. [The present invention 1016] The method of claim 1001, wherein the HSPCs are isolated from the subject's bone marrow. [The present invention 1017] A vector comprising a promoter operably linked to a polynucleotide encoding a functional human lysosomal transmembrane protein selected from the group consisting of CTNS, SLC17A5, LMBRD1, CLN7, CLN3, CLC-7, OSTM1, TRPML1, HGSNAT, NPC1, and LAMP2. [The present invention 1018] The vector of the present invention, which is a viral vector selected from the group consisting of a lentiviral vector, an adenoviral vector, and an AAV vector. [The present invention 1019] The vector of the present invention 1017, which is a self-inactivating (SIN) lentiviral vector. [The present invention 1020] The vector of the present invention is pCCL-CTNS. [The present invention 1021] An isolated mammalian host cell comprising an expression vector according to any one of claims 1017 to 1020. [The present invention 1022] 1021. The isolated mammalian host cell of the present invention, which is a HSPC. [The present invention 1023] 1. A method of treating or ameliorating a lysosomal protein disease or disorder in a subject, comprising: isolating hematopoietic stem and progenitor cells (HSPCs) from bone marrow from the subject; introducing a functional human lysosomal transmembrane gene into said HSPCs, said gene encoding a protein corresponding to said lysosomal protein disease or disorder; and Transplanting the HSPCs back into the subject. thereby treating or ameliorating said lysosomal protein disease or disorder. [The present invention 1024] (a) if the lysosomal transmembrane protein disease or disorder is cystinosis, then the functional human lysosomal transmembrane gene is CTNS; (b) if the lysosomal transmembrane protein disease or disorder is Salla disease or infantile sialic acid storage disorder, the functional human lysosomal transmembrane gene is SLC17A5; (c) if the lysosomal transmembrane protein disease or disorder is cobalaminopathy type F, the functional human lysosomal transmembrane gene is LMBRD1; (d) if the lysosomal transmembrane protein disease or disorder is late-onset childhood neuronal ceroid lipofuscinosis, the functional human lysosomal transmembrane gene is MFSD8; (e) if the lysosomal transmembrane protein disease or disorder is juvenile neuronal ceroid lipofuscinosis, the functional human lysosomal transmembrane gene is CLN3; (f) if the lysosomal transmembrane protein disease or disorder is malignant infantile osteopetrosis, the functional human lysosomal transmembrane gene is CLCN7 or OSTM1; (g) if the lysosomal transmembrane protein disease or disorder is mucolipidosis IV, the functional human lysosomal transmembrane gene is MCOLN1; (h) if the lysosomal transmembrane protein disease or disorder is mucopolysaccharidosis type IIC, the functional human lysosomal transmembrane protein gene is HGSNAT; (i) if the lysosomal transmembrane protein disease or disorder is Niemann-Pick disease type C, the functional human lysosomal transmembrane protein gene is NPC1; and (j) when the lysosomal transmembrane protein disease or disorder is Danon disease, the functional human lysosomal transmembrane gene is LAMP2; The method of the present invention 1023. [The present invention 1025] The method of claim 1023, wherein the HSPCs are CD34+ cells. [The present invention 1026] The method of claim 1024, wherein the lysosomal protein disease or disorder is cystinosis and the functional human lysosomal transmembrane gene is CTNS. [The present invention 1027] The method of claim 1024, wherein the lysosomal protein disease or disorder is Danon disease and the functional human lysosomal transmembrane gene is LAMP2. [The present invention 1028] The method of claim 1026, wherein the step of introducing a functional human CTNS gene into the HSPCs comprises using a vector. [The present invention 1029] The method of claim 1028, wherein the vector is a viral vector selected from the group consisting of a lentiviral vector, an adenoviral vector, and an AAV vector. [The present invention 1030] The method of claim 1029, wherein the vector is a lentiviral vector. [The present invention 1031] The method of claim 1030, wherein the vector is a self-inactivating (SIN) lentiviral vector. [The present invention 1032] The method of claim 1026, wherein the level of cystine in the subject's eye, skin, white blood cells, parenchymal tissue, or gastrointestinal tract is reduced following treatment. [The present invention 1033] The dose is approximately 1.0 × 10 6 ~5.0×10 6 The method of claim 10, wherein the cell density is 1026 cells / kg. [The present invention 1034] The dosage is approximately 2.5 x 10 administered as a single dose. 6 The method of the present invention, wherein the cell density is 1033 cells / kg. [The present invention 1035] The method of claim 1026, wherein the subject is suffering from nephropathic cystinosis. [The present invention 1036] The method of claim 1026, wherein the subject was undergoing cysteamine therapy prior to treatment. [The present invention 1037] The method of claim 1036, wherein the subject is receiving oral cysteamine therapy. [The present invention 1038] The method of claim 1026, wherein the administration is intravenous. [The present invention 1039] The method of claim 1026, wherein cystine or cystine crystals are measured in the eye, skin, white blood cells, parenchymal tissue, and / or gastrointestinal tract before and / or after treatment. [The present invention 1040] The method of the present invention, wherein cystine levels are measured before, during, and / or after treatment. [The present invention 1041] The method of claim 1039, wherein cystine levels are measured using a biological sample. [The present invention 1042] The method of claim 1041, wherein the biological sample is blood, a rectal biopsy, or buccal mucosa. [The present invention 1043] The method of claim 1039, wherein cystine crystals are measured using in vivo confocal microscopy. [The present invention 1044] 1. A method of treating or ameliorating a lysosomal protein disease or disorder in a subject, comprising: generating a functional human lysosomal transmembrane gene in the subject using gene editing. A method comprising: [The present invention 1045] (a) if the lysosomal transmembrane protein disease or disorder is cystinosis, then the functional human lysosomal transmembrane gene is CTNS; (b) if the lysosomal transmembrane protein disease or disorder is Salla disease or infantile sialic acid storage disorder, the functional human lysosomal transmembrane gene is SLC17A5; (c) if the lysosomal transmembrane protein disease or disorder is cobalaminopathy type F, the functional human lysosomal transmembrane gene is LMBRD1; (d) if the lysosomal transmembrane protein disease or disorder is late-onset childhood neuronal ceroid lipofuscinosis, the functional human lysosomal transmembrane gene is MFSD8; (e) if the lysosomal transmembrane protein disease or disorder is juvenile neuronal ceroid lipofuscinosis, the functional human lysosomal transmembrane gene is CLN3; (f) if the lysosomal transmembrane protein disease or disorder is malignant infantile osteopetrosis, the functional human lysosomal transmembrane gene is CLCN7 or OSTM1; (g) if the lysosomal transmembrane protein disease or disorder is mucolipidosis IV, the functional human lysosomal transmembrane gene is MCOLN1; (h) if the lysosomal transmembrane protein disease or disorder is mucopolysaccharidosis type IIC, the functional human lysosomal transmembrane protein gene is HGSNAT; (i) if the lysosomal transmembrane protein disease or disorder is Niemann-Pick disease type C, the functional human lysosomal transmembrane protein gene is NPC1; and (j) when the lysosomal transmembrane protein disease or disorder is Danon disease, the functional human lysosomal transmembrane gene is LAMP2; The method of the present invention 1044. [The present invention 1046] 1. A method of treating or ameliorating a lysosomal protein disease or disorder in a subject, comprising: contacting a cell from the subject that expresses a defective lysosomal transmembrane protein with a vector encoding a gene editing system that, when transfected into the cell, removes a trinucleotide expansion mutation in an endogenous gene encoding the lysosomal transmembrane protein. thereby treating a lysosomal protein disease or disorder. [The present invention 1047] (a) If the lysosomal transmembrane protein disease or disorder is cystinosis, the lysosomal transmembrane protein is cystinosine (CTNS); (b) if the lysosomal transmembrane protein disease or disorder is Salla disease or infantile sialic acid storage disorder, the lysosomal transmembrane protein is sialin (SLC17A5); (c) if the lysosomal transmembrane protein disease or disorder is cobalaminopathy type F, the lysosomal transmembrane protein is LMBD1; (d) if the lysosomal transmembrane protein disease or disorder is late-onset childhood neuronal ceroid lipofuscinosis, the lysosomal transmembrane protein is CLN7; (e) when the lysosomal transmembrane protein disease or disorder is juvenile neuronal ceroid lipofuscinosis, the lysosomal transmembrane protein is battenin (CLN3); (f) when the lysosomal transmembrane protein disease or disorder is malignant infantile osteopetrosis, the lysosomal transmembrane protein is ClC-7 or OSTM1; (g) when the lysosomal transmembrane protein disease or disorder is mucolipidosis IV, the lysosomal transmembrane protein is TRPML-1; (h) when the lysosomal transmembrane protein disease or disorder is mucopolysaccharidosis type IIC, the lysosomal transmembrane protein is HGSNAT; (i) when the lysosomal transmembrane protein disease or disorder is Niemann-Pick disease type C, the lysosomal transmembrane protein is NPC-1; and (j) when the lysosomal transmembrane protein disease or disorder is Danon disease, the lysosomal transmembrane protein is LAMP2; The method of the present invention 1046. [The present invention 1048] The method of claim 1047, wherein the gene editing system is selected from the group consisting of CRISPR / Cas, zinc finger nucleases, and transcription activator-like effector nucleases. [The present invention 1049] The method of claim 1047, wherein the contacting step comprises administering to the subject an effective amount of the vector. [The present invention 1050] The method of claim 1047, wherein the contacting step comprises obtaining a cell sample from a subject, transfecting the cell sample with a gene editing system, and then transplanting the transfected cells into the subject. [The present invention 1051] The method of claim 1046, wherein the cell sample is selected from the group consisting of blood cells and HSPCs. [Brief description of the drawings]

[0017] [Figure 1]Figures 1A-1D are images showing histological analysis of kidney sections from 15-month-old Ctns- / - mice. Figures 1A and 1B show the results of hematoxylin & eosin staining, which revealed significant abnormalities in Ctns- / - mice (Figure 1A), whereas Ctns- / - mice transplanted with HSCs showed only focal abnormalities (Figure 1B). Figures 1C and 1D show the results of methylene blue staining, which revealed the presence of large amounts of cystine crystals in the kidneys of Ctns- / - mice (Figure 1C), and only a few in treated Ctns- / - mice (Figure 1D). [Diagram 2] Figures 2A and 2B are images and graphs showing cystine crystals in the cornea. Figure 2A shows corneal IVCM images from the side of Ctns- / - control and low and high HSC transplanted mice. Figure 2B shows surface crystal quantification within each layer for IVCM corneal full scans obtained from both eyes of Ctns- / - control and transplanted (low and high) mice. Error bars represent SEM (*p<0.05, **p<0.005). [Diagram 3] 1 is a graphical representation of the results of a thyroid study: measurements of cystine content (left panel) and TSH levels (right panel) in Ctns− / − mice compared to wild-type mice (WT) and Ctns− / − mice transplanted with Ctns-expressing HSCs (transplanted Ctns− / −). [Figure 4] Images and graphs showing the effect of HSC transplantation on the digestive tract in Ctns- / - mice. Left panel: Representative confocal photographs of the colon: abundant HSC-derived cells expressing GFP can be seen. Right panel: Cystine content in the colon and intestine of HSC- / - mice transplanted with HSC compared to controls. *p<0.05. [Figure 5A]Figures 5A-5D are graphs and images showing that cystinosine transfer through TNTs is the preferred mode of cross-correction. Figures 5A and 5B show histograms (N=4 replicates each) depicting the percent reduction in cystine content in DsRed-Ctns- / - fibroblasts (recipient cells) when co-cultured with either GFP-MSCs or GFP-macrophages (donor cells) in a contact co-culture assay (Figure 5A) or separated by a 1 μm port transwell filter in a transwell assay (Figure 5B). Values ​​are mean ± standard deviation. *p<0.05; **p<0.01; ***p<0.005. Figure 5C shows a confocal image of a TNT (arrowhead) extending from a GFP-macrophage to a DsRed-Ctns- / - fibroblast. Figure 5D shows representative frames from a confocal movie showing the TNT-mediated transfer of cystinosin-GFP-contacted vesicles from CTNS-GFP-expressing macrophages toward Ctns- / - fibroblasts (arrowheads). Bars: (Figure 5C) 30 μm; (Figure 5D) 20 μm. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6A]Figures 6A-6C are images showing migration through TNTs in vivo in kidney studies. Figure 6A shows confocal images of kidneys from 8-month-old Ctns- / - mice 6 months after transplantation of GFPWT HSPCs. GFP is green and laminin is red. PTCs (lumen, #) were labeled by Lotus Tetragonobus-lectin (LT) (blue). Figures 6A-a1, 6A-a2, and 6A-a3 show that eGFP-expressing HSC-derived cells show numerous outgrowths. Arrowheads indicate TBL passage. Apoptotic PTCs (*). Figure 6A-a3 shows that green structures expressing GFP are located within the PTCs. Figures 6B-6D show Z-stack confocal images of kidneys obtained from Ctns- / - mice transplanted with DsRed-Ctns- / - HSPCs (control, Figure 6B) or Ctns- / - mice transplanted with DsRed-Ctns- / - HSPCs transduced by lentivirus to express cystinosin-GFP and stained for phalloidin (Figure 6C). Vesicles containing cystinosin-GFP are abundantly present in the cytoplasm of PTCs (Figure 6C). Figures 6B and 6C show nuclei stained in blue (DAPI). Scale bars: 5 μm (Figure 6A), 10 μm (Figures 6B and 6C). [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 7] 1 is a diagram showing the structure of the pCCL-CTNS lentiviral vector. SIN-LTR = self-inactivating long terminal repeat; Ψ = psi sequence; RRE = reverse response element; cPPT = central polypurine tract; EFS = short elongation factor 1 alpha; CTNS cDNA = human CTNS cDNA; WPRE = woodchuck hepatitis posttranscriptional regulatory element. [Figure 8A]8A-8D are graphs and images showing quantification of cystine and cystine crystals in male kidneys. FIG. 8A shows cystine content in untreated Ctns− / − mice (KO) compared to pCCL-CTNS-HSC treatment. FIG. 8B shows quantification of cystine crystals on kidney sections stained with methylene blue. Abundant cystine crystals were observed in kidney sections from untreated Ctns− / − mice (FIG. 8C) in contrast to pCCLCTNS-treated mice (FIG. 8D). Error bars are defined as mean + SD. *P<0.05. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 9A-1] Figures 9A-9B are graphs showing the results of in vivo toxicology studies. Figures 9A-1 and 9A-2 show the body weights of pCCL-CTNS-transduced HSC-treated and mock-treated Ctns- / - males (Figure 9A-1) and Ctns- / - females (Figure 9A-2). Figure 9B shows cystine content in tissues of pCCL-CTNS-transduced HSC-treated and mock-treated Ctns- / - mice. [Figure 9A-2] See legend to Figure 9A-1. [Figure 9B] See legend to Figure 9A-1. [Figure 10A] 10A-10E are images showing LAMP2 expression in the hearts and skeletal muscles of WT-HSPC-transplant recipients. 10A-10C are images showing LAMP2 expression in the hearts of WT (FIG. 10A), KO (FIG. 10B), and WT-HSPC-transplanted (FIG. 10C) showing LAMP2-expressing vesicles in cardiomyocytes adjacent to WT-GFP+ macrophages. Arrowheads indicate RFP+ vacuoles. Western blots of heart (FIG. 10D) and skeletal muscle (FIG. 10E) lysates show that LAMP2 expression in mouse recipients of WT-HSPC transplants is restored to near WT levels. [Figure 10B]See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 10D] See legend to Figure 10A. [Figure 10E] See legend to Figure 10A. [Figure 11] Physiological assessment results: Grip strength of WT-HSPC mouse recipients is restored compared to KO (untreated) and KO-HSPC recipient mice. *p<0.05 vs. WT; #p<0.05 vs. WT-HSPC group. [Figure 12A] Figures 12A-12D are images and graphs showing that increased autophagic flux is restored after WT BMT. Figure 12A shows representative EM images of hearts from WT, KO, WT-HSPC-transplanted, and KO-HSPC-transplanted mice. Figure 12B shows quantification of EM images demonstrating that AV accumulation in WT-HSPC mice is restored to nearly WT levels. Figures 12C and 12D show western blots and results demonstrating reduced LC-II / GAPDH levels in WT-HSPC-transplanted mice compared to KO mice. *p<0.05 vs. WT; #p<0.05 vs. KO. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 12D] See legend to Figure 12A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description of the Invention The present invention is based in part on the finding that self-inactivating (SIN) lentiviral vectors containing coding human cystinosine (CTNS) or LAMP-2 cDNA and functional promoter can be used to ex vivo gene correct a patient's autologous hematopoietic stem and progenitor cells (HSPCs), which can then be re-implanted into the patient to repopulate their bone marrow, which will become a reservoir of "healthy" cells for the rest of the patient's life. These cells are mobilized and integrated into diseased tissues, brain, muscle, heart, and rescue them. Although autologous HSPCs are used in the illustrative examples herein, those skilled in the art will recognize that other HSPCs (e.g., allogeneic) would be similarly useful.

[0019] Before describing the compositions and methods of the present invention, it is to be understood that the invention is not limited to the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0020] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods and / or steps of the type described herein that would become apparent to those skilled in the art upon reading this disclosure, and so forth.

[0021] The term "comprising," which is used synonymously with "including," "containing," or "characterized by," is inclusive or open-ended language and does not exclude additional unrecited elements or method steps. The phrase "consisting of" excludes any elements, steps, or ingredients not specified in the claim. The phrase "consisting essentially of" limits the scope of the claim to those materials or steps specified, and those that do not materially affect the basic and novel characteristics of the claimed invention. The present disclosure contemplates embodiments of the compositions and methods of the invention that correspond to the scope of each of these phrases. Thus, a composition or method that includes recited elements or steps contemplates certain embodiments in which the composition or method consists essentially of or consists of those elements or steps.

[0022] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below.

[0023] The term "subject" or "host organism" as used herein refers to any individual or patient on which the method of the present invention is carried out.Generally, the subject is a human being, but those skilled in the art will recognize that the subject can also be an animal.Therefore, other animals are included in the definition of subject, including mammals such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, livestock (including cows, horses, goats, sheep, pigs, etc.), and primates (including monkeys, chimpanzees, orangutans, and gorillas).

[0024] The term "biological sample" refers to any sample taken from a participant, including but not limited to cells, blood, tissue, skin, urine, etc., or hair.

[0025] The term "buccal mucosa" means the inner lining of the cheeks and floor of the mouth.

[0026] The term "therapeutically effective amount" or "effective amount" refers to an amount of a compound or pharmaceutical composition that elicits a biological or medical response in a tissue, system, animal, or human that is desired by a researcher, veterinarian, physician, or other clinician. Thus, the term "therapeutically effective amount" is used herein to refer to any amount of a formulation that, when applied repeatedly to the affected area over a period of time, causes substantial improvement of a disease state. This amount varies depending on the condition being treated, the stage of progression of the condition, and the type and concentration of the formulation applied. The appropriate amount in any given example will be readily apparent to those skilled in the art or can be determined by routine experimentation. In the case of cystinosine, an example of a therapeutically effective amount of an agent, such as a population of hematopoietic stem cells that have been transduced, genetically corrected, or otherwise modified to express a human cystinosine transgene, is an amount sufficient to reduce the amount of cystine (e.g., crystalline cystine) in the lysosomes of a patient's cells, such as kidney, liver, lung, spleen, muscle, brain, and / or heart cells.

[0027] "Dosage" or "dose" is defined to include, and is included within, a specified magnitude, frequency, or exposure level.

[0028] As used herein, a "therapeutic effect" encompasses the therapeutic benefits and / or prophylactic benefits described herein.

[0029] The term "administration" or "administering" is defined to include the act of providing a compound or pharmaceutical composition of the present invention to a subject in need of treatment. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually orally or by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and substernal injection and infusion. As used herein, the phrases "systemic administration", "administered systemically", "peripheral administration", and "administered peripherally" refer to administration of a compound, drug, or other substance other than direct administration to the central nervous system, e.g., subcutaneous administration, so that it enters the subject's body and is therefore subject to metabolism and other similar processes.

[0030] If cell type specific viral vectors are not available, vectors can be modified to express receptors (or ligands) specific to the ligands (or receptors) expressed on target cells, or can be encapsulated in liposomes that can also be modified to contain such ligands (or receptors). Peptide agents can be introduced into cells by a variety of methods, including engineering peptides to contain protein transduction domains, such as the human immunodeficiency virus TAT ​​protein transduction domain, which can facilitate the transfer of peptides into cells. In addition, there are also a variety of biomaterial-based technologies, such as nanocages and pharmacological delivery wafers (as used in brain cancer chemotherapy drugs), which can also be modified to accommodate this technology.

[0031] The most commonly evaluated viral vectors for gene transfer are DNA-based adenovirus (Ad) and adeno-associated virus (AAV), and RNA-based retrovirus and lentivirus. Lentivirus vectors have been most commonly used to achieve chromosomal integration.

[0032] The term "parenchymal" refers to a functional portion of an organ, which may include structural portions of the same organ and / or adjacent organs.

[0033] As used herein, the terms "reduce" and "inhibit" are used together. This is because it is recognized that in some cases, the decrease can be reduced to below the detection level of a particular assay. Therefore, it may not always be clear whether expression level or activity is "reduced" to below the detection level of a certain assay or is "inhibited" completely. Nevertheless, it can be clearly determined after treatment by the method of the present invention.

[0034] As used herein, "treatment" or "treating" refers to administering a composition to a subject or body having an unwanted condition. The condition can include a disease or disorder. "Prevention" or "preventing" refers to administering a composition to a subject or body having a risk of the condition. The condition can include a predisposition to a disease or disorder. The effect of administering a composition to a subject (either treating and / or preventing) can be, but is not limited to, arresting one or more symptoms of the condition, reducing or preventing one or more symptoms of the condition, reducing the severity of the condition, eliminating the condition altogether, stabilizing or delaying the development or progression of a particular event or trait, or minimizing the likelihood of a particular event or trait occurring.

[0035] As used herein, the term "genetic modification" is used to mean any manipulation of the genetic material of an organism in a manner that does not occur under natural conditions. Methods for carrying out such manipulations are known to those skilled in the art, and include, but are not limited to, the technique of using vectors to transform cells with nucleic acid sequences of interest. Various forms of gene editing are included in this definition, using engineered nucleases or "molecular scissors" to insert, delete, or replace DNA in the genome of an organism. These nucleases create site-specific double-strand breaks (DSBs) at desired positions in the genome. The induced double-strand breaks are repaired through non-homologous end joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations (i.e., editing).

[0036] There are several families of engineered nucleases used in gene editing, including but not limited to meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), CRISPR-Cas systems, and ARCUS.However, it should be understood that any known gene editing system that utilizes engineered nucleases can be used in the methods described herein.

[0037] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an acronym for DNA loci that contain multiple short direct repeats of base sequences. Prokaryotic CRISPR / Cas system has been adapted for use as gene editing (silencing, enhancing or modifying specific genes) for use in eukaryotes (see, for example, Cong, Science, 15:339(6121):819-823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012)). By transfecting cells with Cas gene and specifically designed CRISPR-containing elements, nucleic acid sequences can be cut and modified at any desired position. Methods of preparing compositions for use in genome editing using the CRISPR / Cas system are described in detail in U.S. Patent Application Publication No. 2016 / 0340661, U.S. Patent Application Publication No. 2016 / 0340662, U.S. Patent Application Publication No. 2016 / 0354487, U.S. Patent Application Publication No. 2016 / 0355796, U.S. Patent Application Publication No. 2016 / 0355797, and WO 2014 / 018423, which are specifically incorporated by reference in their entireties.

[0038] Thus, as used herein, "CRISPR system" refers collectively to the transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (which in endogenous CRISPR systems encompass "direct repeat sequences" and partial direct repeat sequences processed by tracrRNA), guide sequences (also called "spacers", "guide RNAs", or "gRNAs" in endogenous CRISPR systems), or other sequences and transcripts derived from the CRISPR locus. One or more tracr mate sequences (e.g., direct repeat sequences-spacer-direct repeat sequences) operably linked to a guide sequence may also be referred to as "pre-crRNA" (pre-CRISPR RNA) before processing, or as crRNA after processing by nucleases.

[0039] In some embodiments, as described in Cong, Science, 15:339(6121):819-823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012), the tracrRNA and crRNA combine to form a chimeric crRNA-tracrRNA hybrid in which the mature crRNA is fused to a partial tracrRNA via a synthetic stem-loop to mimic the natural crRNA:tracrRNA duplex. A single fusion crRNA-tracrRNA construct may also be referred to as a guide RNA or gRNA (or single guide RNA (sgRNA)). Within the sgRNA, the crRNA portion may be identified as the "target sequence" and the tracrRNA is often referred to as the "scaffold."

[0040] Many sources of information are available to help practitioners determine appropriate target sites after the desired DNA target sequence is identified. For example, many public sources of information are available, including a bioinformatics-generated list of about 190,000 potential sgRNAs that target more than 40% of human exons, to help practitioners select target sites and design related sgRNAs to affect nicks or double-strand breaks at those sites. See also crispr.u-psud.fr, a tool designed to help scientists find CRISPR target sites in various species and create appropriate crRNA sequences.

[0041] In some embodiments, one or more vectors driving the expression of one or more elements of the CRISPR system are introduced into the target cell, which results in the expression of the elements of the CRISPR system and directs the formation of a CRISPR complex at one or more target sites. Although details can vary for different engineered CRISPR systems, the general methodology is similar. Practitioners interested in targeting DNA sequences using CRISPR technology can insert a short DNA fragment containing the target sequence into a guide RNA expression plasmid. The sgRNA expression plasmid contains the target sequence (about 20 nucleotides), a form of tracrRNA sequence (scaffold), as well as a suitable promoter and elements necessary for proper processing in eukaryotic cells. Such vectors are commercially available (see, for example, Addgene). Many of the systems rely on custom-made complementary oligos that are annealed to form double-stranded DNA and then cloned into the sgRNA expression plasmid. Co-expression of the sgRNA and the appropriate Cas enzyme from the same or separate plasmids in the transfected cell results in single- or double-stranded cleavage (depending on the activity of the Cas enzyme) at the desired target site.

[0042] Zinc finger nucleases (ZFNs) are artificial restriction enzymes created by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The zinc finger domain can be engineered to target specific desired DNA sequences, allowing zinc finger nucleases to target unique sequences within complex genomes. By exploiting endogenous DNA repair mechanisms, these reagents can be used to precisely modify the genomes of higher organisms. The most common cleavage domain is the type IIS enzyme Fok 1. Fok 1 catalyzes double-stranded cleavage of DNA, 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. See, for example, U.S. Patent Nos. 5,356,802; 5,436,150; and 5,487,994; and Li et al. Proc., Natl. Acad. Sci. USA 89 (1992):4275-4279; Li et al. Proc. Natl. Acad. Sci. USA, 90:2764-2768 (1993); Kim et al. Proc. Natl. Acad. Sci. USA 91:883-887 (1994a); Kim et al. J. Biol. Chem. 269:31,978-31,982 (1994b), all of which are incorporated herein by reference. One or more of these enzymes (or enzymatically functional fragments thereof) can be used as a source of cleavage domains.

[0043] Transcription activator-like effector nucleases (TALENs) have an overall organization similar to that of ZFNs, with the main difference being that the DNA-binding domain is derived from TAL effector proteins, transcription factors from plant pathogenic bacteria. The DNA-binding domain of TALENs is a tandem array of amino acid repeats, each about 34 residues long. These repeats are very similar to each other and typically differ primarily at two positions (amino acids 12 and 13, called repeat variable diresidues or RVDs). Each RVD specifies preferential binding to one of four possible nucleotides, meaning that each TALEN repeat binds to a single base pair, except that the NN RVD is known to bind adenine in addition to guanine. TAL effector DNA binding is mechanistically less well understood than that of zinc finger proteins, but their potentially simpler code can be of great advantage to the design of engineered nucleases. TALENs also cleave as dimers, have relatively long target sequences (the shortest reported to date binds 13 nucleotides per monomer), and appear to have less stringent requirements on the length of the spacer between binding sites than ZFNs. Monomeric and dimeric TALENs can contain more than 10, more than 14, more than 20, or more than 24 repeats. Methods for engineering TALs to bind to specific nucleic acids are described in Cermak, et al, Nucl. Acids Res. 1-11 (2011); US Patent Publication No. 2011 / 0145940, which discloses TAL effectors and methods for using them to modify DNA; and Miller et al. Nature Biotechnol 29: 143 (2011), which reports the creation of TALENs for site-specific nuclease construction by linking TAL truncation mutants to the catalytic domain of Fok1 nuclease. The resulting TALENs have been shown to induce genetic modifications in immortalized human cells. General design principles for TALE binding domains can be found, for example, in WO 2011 / 072246. Each of the aforementioned references is incorporated herein by reference in its entirety.

[0044] The nuclease activity of the genome editing system described herein cleaves the target DNA, resulting in single-stranded or double-stranded breaks in the target DNA. Cells can repair double-stranded breaks in one of two ways: non-homologous end joining and homologous recombination repair. In non-homologous end joining (NHEJ), double-stranded breaks are repaired by directly linking the cut ends together. Thus, no new nucleic acid material is inserted at the site, but some nucleic acid material may be lost, resulting in a deletion. In homologous recombination repair, a donor polynucleotide with homology to the cut target DNA sequence is used as a template to repair the cut target DNA sequence, resulting in the transfer of genetic information from the donor polynucleotide to the target DNA. Thus, new nucleic acid material can be inserted / copied at the site. Thus, in some embodiments, genome editing vectors or compositions optionally include donor polynucleotides. Targeted DNA modification by NHEJ and / or homology directed repair can be used to induce gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, and the like.

[0045] Thus, DNA cleavage by a genome editing vector or composition can be used to delete nucleic acid material from a target DNA sequence by cleaving the target DNA sequence and allowing the cell to repair the sequence in the absence of an exogenously provided donor polynucleotide. Alternatively, when a genome editing composition includes a donor polynucleotide sequence that includes at least one segment with homology to a target DNA sequence, these methods can be used to add, i.e., insert or replace, nucleic acid material to a target DNA sequence (e.g., "knock in" a nucleic acid encoding a protein, siRNA, miRNA, etc.), add tags (e.g., 6xHis (SEQ ID NO: 27), fluorescent proteins (e.g., green fluorescent protein, yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), add regulatory sequences to genes (e.g., promoters, polyadenylation signals, internal ribosome entry sequences (IRES), 2A peptides, start codons, stop codons, splicing signals, localization signals, etc.), and modify nucleic acid sequences (e.g., introduce mutations), etc. Thus, these compositions can be used to modify DNA in a site-specific or "targeted" manner, e.g., gene knock-out, gene knock-in, gene editing, gene tagging, etc., as used in gene therapy.

[0046] ARCUS is a genome editing platform derived from naturally occurring genome editing enzymes called "homing endonucleases". Homing endonucleases are site-specific DNA cleavage enzymes encoded in the genomes of many eukaryotic species that can precisely recognize long DNA sequences (12-40 base pairs). These non-destructive enzymes trigger gene conversion events that modify genomes in a highly precise manner, most frequently by the insertion of novel DNA sequences. Thus, the ARCUS genome editing platform relies on engineered ARC nucleases, fully synthetic enzymes that are similar to homing endonucleases but with enhanced specificity to recognize DNA sequences within any target gene.

[0047] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0048] The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to natural amino acids. Natural amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, α-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as natural amino acids, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but maintain the same basic chemical structure as natural amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of amino acids, but function in a manner similar to natural amino acids.

[0049] Amino acids may be referred to herein by either their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted one-letter symbols.

[0050] As used herein, a "regulatory gene" or "regulatory sequence" is a nucleic acid sequence that encodes a product (eg, a transcription factor) that controls the expression of other genes.

[0051] As used herein, a "protein coding sequence" or a sequence that codes for a particular protein or polypeptide is a nucleic acid sequence that is transcribed (in the case of DNA) into mRNA and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5'-terminus (N-terminus) and a translation stop nonsense codon at the 3'-terminus (C-terminus). Coding sequences can include, but are not limited to, cDNA derived from eukaryotic mRNA, genomic DNA sequences derived from eukaryotic DNA, and synthetic nucleic acids. Usually, a transcription termination sequence is located 3' to the coding sequence.

[0052] As used herein, a "promoter" is defined as a regulatory DNA sequence, usually located upstream of a gene, which directs RNA polymerase to bind to DNA and initiate RNA synthesis, thereby realizing transcription initiation. A promoter can be a constitutively active promoter (i.e., a promoter that is constitutively active / "ON"); a promoter can be an inducible promoter (i.e., a promoter whose active / "ON" or inactive / "OFF" state is controlled by an external stimulus, such as the presence of a specific compound or protein); a promoter can be a spatially restricted promoter (i.e., a transcription control element, enhancer, etc.) (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.); and a promoter can be a temporally restricted promoter (i.e., the promoter is in "ON" or "OFF" state during a specific stage of embryo development or during a specific stage of a biological process). Thus, in various embodiments, a promoter can be a stem cell-specific promoter that drives transgene expression. For example, constitutive promoters of various strengths can be used. The expression vector and plasmid according to the present invention may comprise one or more constitutive promoters, such as viral promoters or promoters derived from mammalian genes that are normally active in promoting transcription.Exemplary promoters include, but are not limited to, human elongation factor 1α promoter (EFS), SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, endogenous cellular promoters that are heterologous to the gene of interest, cytomegalovirus (CMV) promoters such as CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, synthetic promoters, and hybrid promoters.

[0053] As used herein, the term "gene" refers to a deoxyribonucleotide sequence that comprises the coding region of a structural gene. A "gene" may also include non-translated sequences located adjacent to both the 5' and 3' ends of the coding region such that the gene corresponds to the length of the full-length mRNA. The sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. The sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains a coding region interrupted by non-coding sequences called "introns" or "intervening regions" or "intervening sequences". Introns are segments of a gene that are transcribed into heterogeneous nuclear RNA (hnRNA). Introns may include regulatory elements such as enhancers. Introns are removed or "spliced ​​out" from the nuclear or primary transcript. Introns are therefore absent in messenger RNA (mRNA) transcripts. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0054] As used herein, the terms "operably linked" and "functionally linked" are used interchangeably and refer to the functional relationship between two or more DNA segments, particularly between the gene sequence to be expressed and the sequence that controls those sequences.For example, a promoter / enhancer sequence, including any combination of cis-acting transcriptional control elements, is functionally linked to a coding sequence if it promotes or regulates the transcription of that coding sequence in a suitable host cell or other expression system.The promoter regulatory sequence that is functionally linked to the gene sequence to be transcribed is physically adjacent to the transcription sequence.

[0055] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that code for the same or essentially identical amino acid sequences, or, if the nucleic acid does not code for an amino acid sequence, essentially the same sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variants are "silent variants," which are a type of conservatively modified variant. Any nucleic acid sequence herein that codes for a polypeptide also provides a description of all possible silent variants of that nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be altered to obtain a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0056] With respect to amino acid sequences, one of skill in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants" if the alteration results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the invention.

[0057] The term "antibody" as used herein refers to polyclonal and monoclonal antibodies and their fragments and their immunological binding equivalents. The term "antibody" refers to a homogenous molecular entity or a mixture, such as a polyclonal serum product, composed of multiple different molecular entities, and broadly includes naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE) as well as recombinant antibodies, such as single-chain antibodies, chimeric antibodies and humanized antibodies, and multispecific antibodies. The term "antibody" also refers to fragments and derivatives of all the above, and may further include any modified or derivatized variants thereof that maintain the ability to specifically bind to an epitope. Antibody derivatives may include protein or chemical moieties attached to the antibody. Monoclonal antibodies can selectively bind to a target antigen or epitope. Antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, camelized antibodies, single chain antibodies (scFv), Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv) fragments, such as those produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, intrabodies, nanobodies, synthetic antibodies, and epitope-binding fragments of any of the above.

[0058] As used herein, the term "humanized mouse" (Hu mouse) refers to a mouse that has been developed to have functional human genes, cells, tissues, and / or organs.Humanized mice are often used as small animal models in biological and medical research of human therapeutic substances.Immune-deficient mice are often used as recipients of human cells or tissues, because they can relatively easily accept xenogeneic cells due to the lack of host immunity.

[0059] HSCs have the capacity for multipotency (i.e., one HSC can differentiate into all types of functional blood cells) and self-renewal (i.e., HSCs can divide and give rise to identical daughter cells without differentiating). Through a series of lineage commitment steps, HSCs gradually lose the ability to self-renew and subsequently give rise to progeny with increasingly restricted differentiation capacity, which give rise to multipotent but lineage-committed progenitor cells and, ultimately, mature, functional circulating blood cells.

[0060] The ability of hematopoietic stem and progenitor cells (HSPCs) to self-renew and differentiate is fundamental to the establishment and maintenance of hematopoiesis throughout life, and deregulation of these processes can lead to severe clinical outcomes. HSPCs are also highly beneficial due to their ability to reconstitute the hematopoietic system when transplanted, which has enabled their use in the clinical setting to treat a variety of disorders, including bone marrow failure, myeloproliferative disorders, and other acquired or genetic disorders that affect blood cells.

[0061] As used herein, "pluripotent cells" refer to cells derived from an embryo resulting from activation of cells containing DNA from all females or males, and which can be maintained in vitro for long, theoretically indefinite periods, in an undifferentiated state capable of giving rise to various differentiated tissue types, i.e., ectoderm, mesoderm, and endoderm. "Embryonic stem cells" (ES cells) are pluripotent stem cells derived from the inner cell mass of the blastocyst, i.e., the early preimplantation embryo.

[0062] As used herein, "autologous transplantation" refers to transplantation using the subject's own stem cells. These cells are collected in advance and returned at a later stage. Thus, "allogeneic transplantation" refers to transplantation in which the donor and recipient of stem cells are different people. Exemplary allogeneic cells include, but are not limited to, syngeneic cells, MHC-matched cells, etc.

[0063] As used herein, "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, and emulsions such as oil / water or water / oil emulsions, as well as various types of wetting agents.

[0064] As used herein, "lysosomal protein disorder" or "lysosomal protein disease" refers to any metabolic disorder resulting from defective lysosomal function. Such diseases / disorders, also called "lysosomal storage disorders", are typically caused by lysosomal dysfunction that usually results from the deficiency of a single enzyme required for the metabolism of lipids, glycoproteins (proteins containing sugars), or so-called mucopolysaccharides. Exemplary lysosomal storage disorders include, but are not limited to, cystinosis, Salla disease, infantile sialic acid storage disorder, cobalamin disease type F, neuronal ceroid lipofuscinosis (both late-onset childhood and juvenile forms), malignant infantile osteopetrosis, mucolipidosis IV, mucopolysaccharidosis type IIIC (Sanfilippo syndrome type C), Niemann-Pick disease type C, and Danon disease (Ruivo, et al. Biochimica et Biophysica Acta 1793 (2009) 636-649, incorporated herein by reference).

[0065] For example, cystinosis is an autosomal metabolic disease that belongs to the family of lysosomal storage disorders. Cystinosis has devastating effects on affected individuals, primarily children and young adults, even with cysteamine treatment. The prevalence of cystinosis is 1:100,000 to 1:200,000. The gene involved in cystinosis is the gene CTNS, which codes for cystinosin, a seven-transmembrane lysosomal cystine transporter. The most severe and most frequent form of cystinosis is the infantile form, also called nephropathic cystinosis. Children develop renal Fanconi syndrome, characterized by severe fluid and electrolyte disturbances, growth retardation, and rickets, at 6 to 8 months of age. The gradual loss of renal glomerular function leads to kidney failure; according to the NAPRTCS' (North American Pediatric Renal Trials and Collaborative Studies), cystinosis affects 1.4% of children undergoing dialysis (2011 Annual Dialysis Report) and 2.1% of children undergoing kidney transplants (2010 Annual Transplant Report). Cystinosis as a clinical entity is also a progressive multi-organ dysfunction caused by the accumulation of cystine in the lysosomes of all cells in the body; affected patients store 50-100 times the normal amount of cystine in their cells.

[0066] Cystine accumulation leads to the formation of cystine crystals in all tissues. Major clinical complications of cystinosis include diabetes, hypothyroidism, myopathy, and central nervous system decline. Corneal cystine crystals appear from 0-10 years of age, resulting in photophobia and visual impairment. Dysphagia is directly correlated to muscle atrophy and is the major cause of death in cystinosis. In addition to cystine accumulation, cellular dysfunctions such as abnormal vesicular trafficking, autophagy, and TFEB (Transcription Factor EB) signaling have also been described to contribute to the pathogenesis of cystinosis.

[0067] The currently used treatment for cystinosis is the drug cysteamine (mercaptoethylamine), which reduces intracellular cystine content. However, this therapy only slows the progression of the disease and has no effect on renal Fanconi syndrome or prevents end-stage renal failure in affected patients. Cysteamine has also been shown to be inefficient in improving cellular dysfunction in CTNS-deficient cells, proving that the cellular defects in cystinosis are not due solely to cystine accumulation, but also to the lack of cystinosin itself, which directly interacts with key cellular components.

[0068] Furthermore, cysteamine must be taken every 6 hours, including at night, and causes body odor and severe gastrointestinal side effects such as vomiting and diarrhea, making treatment compliance difficult. In 2013, a delayed-release formulation of cysteamine (PROCYSBI®) was approved by the FDA. PROCYSBI requires dosing every 12 hours. Although PROCYSBI® improves the quality of life of patients by reducing the frequency of dosing, the impact on the disease is similar to immediate-release cysteamine, and patients still experience gastric side effects. Furthermore, the cost of this pharmaceutical is very high, costing $300,000 to $600,000 per patient per year.

[0069] The eye lesions of cystinosis require hourly topical cysteamine eye drops, which cause irritation and burning, making compliance very difficult. The cost of the eye drops is approximately $50,000 per patient per year. Cysteamine and supplemental treatments for all complications related to cystinosis require patients to take up to 60 pills per day; often, in children, a gastric tube must be placed so that they can tolerate these medical drugs and obtain essential caloric intake. The severity and number of medical complications increases with age, resulting in new and ever-increasing symptoms and treatments. There is an endless stream of doctor appointments, G-tube feedings, frequent blood draws, growth hormone injections, bone pain, routine vomiting, eye pain, and severe gastrointestinal side effects. As the disease progresses, the patient becomes physically weaker. The most severe complications in adults are myopathy, lung problems, and the development of corneal cystinosis. Patients with kidney failure require dialysis or transplantation, both of which have significant adverse health effects and, due to the extreme shortage of donor organs, patients may wait 3-6 years for a transplant. Thus, the current standard of care does not prevent disease progression and still significantly impacts the quality of life of patients with cystinosis, who die in early adulthood.

[0070] Danon disease has many similarities to other lysosomal membrane protein diseases and has been characterized as a disorder of autophagy affecting the degradation of many cellular components, and therefore not resulting in the accumulation of a single substrate. More recently, Danon disease has been described as an autophagic vacuolar myopathy. Danon disease is caused by mutations in the gene encoding the lysosomal associated membrane protein 2 (LAMP-2) protein, which results in reduced expression of LAMP-2. Reduced LAMP-2 expression disrupts autophagic flux, impairing the ability of cells to respond to stress and remove damaged cellular components.

[0071] Thus, the present disclosure demonstrates that one-time hematopoietic stem and progenitor cell (HSPC) transplantation has the potential to be a lifelong curative therapy for diseases or disorders associated with defective lysosomal transmembrane proteins. This therapy may also prevent kidney transplants and long-term complications associated with cystinosis, and unexpectedly includes the elimination of corneal cystine crystals. This should also allow patients to discontinue the use of oral cysteamine, cysteamine eye drops, and any other medical drugs used to treat symptoms associated with the disease. Thus, the quality of life of patients will be greatly improved, and the cost of therapeutic drugs will be significantly reduced.

[0072] Due to the multisystemic nature of cystinosis and all the drugs required to compensate for the lack of the protein cystinosine in all tissues, a gene therapy approach was investigated. Gene therapy has the potential to be an important new approach in the third millennium to treat both rare and common critical diseases, as its reach far exceeds that of conventional drugs and offers the promise of stem cell-based curative therapy with limited risks compared to allogeneic HSC transplantation. Hematopoietic stem and progenitor cells (HSPCs) are therefore ideal candidates for use in regenerative medicine and cell replacement therapy due to their ease of isolation, self-renewal capacity, and safety. Furthermore, gene therapy can address unmet medical needs, such as in the case of cystinosis, particularly as this strategy overcomes the lack of matched HSC donors, making treatment potentially available to all patients.

[0073] Ctns, a rodent model of cystinosis - / - Transplantation of mice with HSCs expressing a functional Ctns gene has been shown to result in abundant incorporation of bone marrow-derived cells into tissues, a marked reduction in cystine accumulation (up to 97% elimination), and long-term kidney maintenance. - / - Mice progressed to end-stage renal failure, whereas age-matched Ctns transplanted with wild-type HSCs did not. - / -The mice maintained normal kidney function more than one year after transplantation. Large amounts of cystine crystals were consistently observed in the kidneys of untreated Ctns. - / - In contrast to mice with HSC transplantation, little to no cystine crystals were observed in the kidneys of treated mice. - / - It has also been recently demonstrated that treatment with Ctns rescues eye defects in mice. - / - Mice showed near total degradation of cystine crystals from the epithelial layer to the mid-stroma (100% to 72% reduction, respectively) as well as normal corneal thickness and intraocular pressure. The effects of transplanted HSCs on the thyroid gland were also studied. - / - Mice show sustained TSH activation combined with thyroid cell hypertrophy, hyperplasia, and vascular proliferation. In contrast, Ctns treated with transplanted HSCs - / - Mice showed normalized cystine and TSH levels and normal histology. These studies are the first proof of concept that the multiorgan degeneration associated with cystinosis can be prevented by a single HSC transplant.

[0074] Thus, the present disclosure provides a mouse model of cystinosis (Ctns - / - The present disclosure evaluates the effects of HSPC transplantation in a mouse model of cystinosis (Ctns mice). - / - Using mouse models (M2 mice), we demonstrate that transplantation of wild-type (WT) mouse mHSCs results in abundant tissue incorporation of bone marrow-derived cells, a marked reduction in tissue cystine accumulation (up to 97%), and long-term maintenance of kidney, eye, and thyroid. Given the risk of mortality and morbidity associated with allogeneic HSC transplantation, such as graft-versus-host disease (GVHD), an autologous transplantation protocol of HSCs was developed for ex vivo modification. Using pCCL-CTNS (backbone pCCL-EFS-X-WPRE), a self-inactivating lentiviral vector (SIN-LV) to introduce a functional form of CTNS cDNA, we expressed the Ctns gene. - / - Efficacy was demonstrated in mice.

[0075] Human CD34 isolated from peripheral blood of healthy donors and patients with cystinosis + In vitro studies have been completed using HSPCs and Ctns - / - Significant progress has been made in serial transplantation in mice. Thus, the data provided herein demonstrate that CD34+ cystinosis cells derived from peripheral blood stem cells (PBSCs) mobilized by G-CSF from cystinosis patients and modified by ex vivo transduction with pCCL-CTNS LVs are + Demonstrate the efficacy of HSC transplantation.

[0076] Both cystinosis and Danon disease occur as a result of loss-of-function mutations in transmembrane lysosomal proteins, cystinosin and LAMP-2, respectively. In fact, cystinosin is localized in LAMP-2-positive vesicles that are transported during cross-correction. Thus, the present disclosure also shows that bone marrow was harvested from Danon disease patients and sorted for CD34+ hematopoietic stem cells (HPSCs). After harvesting, patient HPSCs are genetically modified using viral transduction vectors, including but not limited to lentiviruses and other retroviruses, carrying any normal range variant of the LAMP-2 gene, and / or any of the LAMP-2 splice isoforms (e.g., LAMP-2A, LAMP-2B, LAMP-2C) (hereinafter collectively referred to as "wild-type LAMP-2" or "WT LAMP-2") are inserted into the genome of the harvested HPSCs. After infection, the viral vector inserts the wild-type LAMP-2 transgene into a specific site in the host cell genome that limits genome disruption. This insertion allows the wild-type LAMP-2 transgene to then be stably expressed by the host cell. After translation, the wild-type LAMP-2 protein is transported to the lysosomal membrane, where it tucks away and assumes its normal intracellular location. Introducing the wild-type LAMP-2 protein into the lysosomal membrane restores autophagic flux, allowing the cell to function normally.

[0077] After the wild-type LAMP-2 gene is introduced, the HPSCs are transplanted back into the patient from whom they were taken. These cells then re-engraft in the patient's bone marrow and begin to produce progenitor cells. Some of these progenitor cells differentiate into monocytes with the wild-type LAMP-2 gene. Monocytes with the wild-type LAMP-2 gene enter the blood circulation and then into peripheral tissues where they transform into tissue-resident macrophages. These macrophages transfer their lysosomes with membrane-bound wild-type LAMP-2 protein to diseased peripheral cells through a variety of mechanisms, including but not limited to the formation of tunneling nanotubes, vesicle release, and direct cell-cell adhesion. Wild-type LAMP-2 protein may also be transferred between macrophages and diseased peripheral cells in other forms, including but not limited to as a free protein or bound to other proteins, membranes, or organelles. Delivery of wild-type LAMP-2-containing lysosomes or other forms of wild-type LAMP-2 restores normal autophagic flux in diseased cells, partially or completely ameliorating the Danon disease phenotype.

[0078] Thus, in one aspect, the present invention provides a method for treating a lysosomal transmembrane protein disease or disorder in a subject. The method includes introducing a functional human transmembrane protein corresponding to the disorder to be treated ex vivo into the HSPCs of the subject, and then transplanting the HSPCs into the subject, thereby treating the lysosomal transmembrane protein disease or disorder. Thus, for example, when the disease or disorder to be treated is cystinosis, the functional human transmembrane protein to be introduced is CTNS. In various embodiments, the vector is a self-inactivating (SIN) lentiviral vector, such as pCCL-CTNS (for CTNS). Similarly, when the disease or disorder to be treated is Danon disease, the functional human transmembrane protein to be introduced is LAMP-2. In various embodiments, the introducing step may include contacting the HSPCs with a vector that includes a polynucleotide encoding a functional protein (e.g., CTNS or LAMP-2) and a functional promoter (e.g., a ubiquitous promoter or an endogenous promoter of the functional protein), and expressing the functional protein. Thus, the present disclosure provides methods for autologous transplantation of ex vivo genetically modified HSPCs to introduce functional proteins associated with specific lysosomal transmembrane protein diseases or disorders.

[0079] In various embodiments, lysosomal transmembrane protein diseases or disorders include, but are not limited to, cystinosis, Salla disease, infantile sialic acid storage disorder, cobalaminopathy type F, neuronal ceroid lipofuscinosis (both late-onset childhood and juvenile forms), malignant infantile osteopetrosis, mucolipidosis IV, mucopolysaccharidosis type IIIC (Sanfilippo syndrome type C), Niemann-Pick disease type C, and Danon disease. Without being bound by theory, in cystinosis and free sialic acid storage disease, the transporters of cystine and acidic monosaccharides are disturbed or inhibited, respectively. In cobalaminopathy type F and mucopolysaccharidosis type IIIC, the postulated cobalamin transporter and the acetyl group hybrid transporter / transferase, respectively, are defective. In neurodegenerative osteopetrosis, mutations in the proton / chloride ion exchanger impair the charge balance required for sustained proton pumping by V-type ATPase, thus neutralizing bone resorption pits. However, the mechanisms that lead to lysosomal accumulation and neurodegeneration remain unclear. Mucolipidosis type IV is caused by mutations in a lysosomal cation channel named TRPML1; its gating properties are still poorly understood, and the ionic species that link this channel to lipid accumulation and membrane trafficking defects are debated. Finally, the autophagy defects in Danon disease appear to occur as a result of the role of LAMP2 in lysosome / autophagosome fusion, possibly concomitant with its role in dynein-based centripetal motility (Ruivo, et al. Biochimica et Biophysica Acta 1793 (2009) 636-649, incorporated herein by reference).

[0080] Table 1 sets forth exemplary lysosomal transmembrane protein diseases or disorders that are to be treated by ex vivo introduction of the corresponding functional human transmembrane protein.

[0081] [Table 1]

[0082] Lentivirus-derived vectors have replaced gamma-retroviral vectors for gene therapy due to their superior gene transfer efficiency and more favorable biosafety profile. In fact, all cases of leukemogenic complications observed so far in clinical trials or animal models involved the use of retroviral vectors with LTRs containing strong enhancers / promoters capable of inducing distal enhancer activation. In contrast, third-generation lentiviral vectors with deletions in the LTR, SIN-LV, contain only one internal enhancer / promoter, thus reducing the incidence of interactions with nearby cellular genes and therefore the risk of oncogenic integration. SIN-LV is also designed to prevent the possibility of developing replication-competent lentivirus (RCL) during the generation of viral supernatants with the three packaging plasmids required for their generation. Lentiviral vectors efficiently transduce HSPCs and do not alter their repopulation properties, making this type of vector an attractive vehicle for stem cell gene therapy.

[0083] Clinical trials using SIN-LV to gene correct human HSPCs are underway in the United States and Europe for several conditions, including HIV-1, β-thalassemia, immunodeficiency, metabolic diseases, and cancer. In the case of immunodeficiency disorders, HSPCs modified by SIN-LV have been transplanted in 35 patients so far. In a clinical trial in patients with adrenoleukodystrophy (ALD), stable gene correction was achieved in approximately 20% of hematopoietic cells in two patients. Cerebral demyelination was suppressed without further progression during a 3-year follow-up examination. This corresponds to a clinical outcome comparable to that observed after allogeneic transplantation; there was no evidence of clonal dominance. Recently, a clinical trial was reported for Wiskott-Aldrich syndrome in three patients 32 months after transplantation. Stable and long-term engraftment (25–50%) of gene-modified HSPCs resulted in improved platelet counts, protection from bleeding and infection, and disappearance of eczema. Another clinical success was recently reported in three presymptomatic patients with metachromatic leukodystrophy. Engraftment of blood cells derived from the transduced cells achieved 45-80% and protein activity in cerebrospinal fluid returned to above normal levels for up to 24 months, with clear therapeutic benefit.

[0084] Recent successful gene therapy using AAV vectors in MCK mice not only prevented heart failure when given to presymptomatic animals but also ameliorated cardiomyopathy when given after disease onset. Although promising, this approach has potential concerns regarding safety as well as procurement and delivery: i) local delivery by direct virus injection at the diseased site poses several challenges in accessing sites such as the heart and brain and provides only tissue-specific rescue, ii) systemic AAV delivery remains challenging in humans due to the need for high levels of vector, which raises vector synthesis and safety concerns. In contrast, HSPC gene therapy approaches have important advantages: i) a single injection of stem cells treats all complications, ii) gene correction occurs in a controlled ex vivo environment, allowing the cells to be characterized prior to transplantation, iii) gene-corrected HSPCs reside in the bone marrow niche after transplantation, where they self-renew and become a reservoir of healthy cells for the patient's lifespan, and iv) avoids immune responses compared to allogeneic transplantation. Therefore, autologous HSPC gene therapy may provide a cure for lysosomal transmembrane protein diseases or disorders.

[0085] The amino acid and nucleic acid sequences of the human proteins set forth in Table 1 are known in the art. GenBank Accession No. Y15924.1, human CTNS gene, exon 3, adjacent intron regions and concatenated CDS, which provides the following amino acid sequence (SEQ ID NO: 1): TIFF2025039642000003.tif18152 provides the following nucleic acid sequence (SEQ ID NO: 2), GenBank Accession No. AJ222967.1, human CTNS mRNA: TIFF2025039642000004.tif142152 provides the following amino acid sequence (SEQ ID NO: 3), GenBank Accession No. CAB62540.1, human sialin: TIFF2025039642000005.tif29152 provides the following nucleic acid sequence (SEQ ID NO: 4), GenBank Accession No. AJ387747.1, human sialin mRNA: TIFF2025039642000006.tif135152 provides the following amino acid sequence (SEQ ID NO: 5), GenBank Accession No. CCP79466.1, human LMBD1: TIFF2025039642000007.tif29152 provides the following nucleic acid sequence (SEQ ID NO: 6), GenBank Accession No. HAAF01007642.1, transcribed RNA of human LMBD1: TIFF2025039642000008.tif109152 provides the following amino acid sequence (SEQ ID NO: 7), GenBank Accession No. AAH295036.1, human CLN7: TIFF2025039642000009.tif29152 provides the following nucleic acid sequence (SEQ ID NO: 8), GenBank Accession No. BC029503.1, human CLN7 mRNA: TIFF2025039642000010.tif104152 provides the following amino acid sequence (SEQ ID NO: 9), GenBank Accession No. AAB51075.1, human CLN3: TIFF2025039642000011.tif22152 provides the following nucleic acid sequence (SEQ ID NO: 10), GenBank Accession No. U32680.1, human CLN3 mRNA full coding region: TIFF2025039642000012.tif93152 provides the following amino acid sequence (SEQ ID NO: 11), GenBank Accession No. AAF34711.1, human CLCN7: TIFF2025039642000013.tif41152 provides the following nucleic acid sequence (SEQ ID NO: 12), GenBank Accession No. AF224741.1, human CLCN7 mRNA full coding region: TIFF2025039642000014.tif176152 provides the following amino acid sequence (SEQ ID NO: 13), GenBank Accession No. AAH68581.1, human OSTM1: TIFF2025039642000015.tif18152 provides the following nucleic acid sequence (SEQ ID NO: 14), GenBank Accession No. BC068581.1, human OSTM1 mRNA: TIFF2025039642000016.tif154152 provides the following amino acid sequence (SEQ ID NO: 15), GenBank Accession No. AAG00797.1, human MCOLN1: TIFF2025039642000017.tif29152 provides the following nucleic acid sequence (SEQ ID NO: 16), GenBank Accession No. AF287269.1, human MCOLN1 mRNA, complete coding region: TIFF2025039642000018.tif113152 provides the following amino acid sequence (SEQ ID NO: 17), GenBank Accession No. Q68CP4.2, human HGSNAT: TIFF2025039642000019.tif37152 provides the following nucleic acid sequence (SEQ ID NO: 18), GenBank Accession No. NM_152419, human HGSNAT mRNA: TIFF2025039642000020.tif60152TIFF2025039642000021.tif221152Provides the following amino acid sequence (SEQ ID NO: 19), GenBank Accession No. AAB63982.1, Human NPC1: TIFF2025039642000022.tif63152 provides the following nucleic acid sequence (SEQ ID NO: 20), GenBank Accession No. AF002020.1, human NPC1 mRNA, entire coding region: TIFF2025039642000023.tif135152TIFF2025039642000024.tif116152Provides the following amino acid sequence (SEQ ID NO: 21), GenBank Accession No. CAA54416.1, Human LAMP-2A: TIFF2025039642000025.tif22152 provides the following nucleic acid sequence (SEQ ID NO: 22), GenBank Accession No. X77196.1, human LAMP2 mRNA: TIFF2025039642000026.tif101152 provides the following amino acid sequence (SEQ ID NO: 23), GenBank Accession No. AAA91149.1, human LAMP-2B: TIFF2025039642000027.tif22152 provides the following nucleic acid sequence (SEQ ID NO: 24), GenBank Accession No. U36336.1, human LAMP-2B mRNA, entire coding region: TIFF2025039642000028.tif218152 provides the following amino acid sequence (SEQ ID NO: 25), GenBank Accession No. AAS67876.1, human LAMP-2C: TIFF2025039642000029.tif22152 provides the following nucleic acid sequence (SEQ ID NO: 26), GenBank Accession No. AY561849.1, human LAMP-2C mRNA, full coding region: TIFF2025039642000030.tif67152

[0086] In another aspect, a method for treating a lysosomal transmembrane protein disease or disorder in a subject includes contacting a cell from the subject that expresses a protein (see Table 1) associated with a particular disease or disorder with a vector that encodes a gene editing system that, when transfected into the cell, removes a mutation (e.g., trinucleotide repeat expansion mutation) of the endogenous protein, thereby treating the lysosomal transmembrane protein disease or disorder. In various embodiments, the gene editing system is selected from the group consisting of CRISPR / Cas, zinc finger nuclease, and transcription activator-like effector nuclease. The contacting step can be performed ex vivo by first obtaining a cell sample from the subject, transfecting the gene editing system into the cell sample, and then transplanting the transfected cell into the subject, thereby treating the lysosomal transmembrane protein disease or disorder. The cell sample can be any cell that expresses a protein associated with a lysosomal transmembrane protein disease or disorder, such as the subject's blood cells or HSPCs.

[0087] In another aspect, the present invention provides a method for treating or ameliorating a lysosomal protein disease or disorder in a subject, comprising transplanting into the subject a population of HSPCs that have been genetically modified by introducing a transgene encoding a corresponding functional human lysosomal transmembrane protein, thereby treating the lysosomal transmembrane protein disease or disorder. Thus, if the lysosomal transmembrane protein disease or disorder is cystinosis, the functional human lysosomal transmembrane gene is CTNS; if the lysosomal transmembrane protein disease or disorder is Salla disease or infantile sialic acid storage disorder, the functional human lysosomal transmembrane gene is SLC17A5; if the lysosomal transmembrane protein disease or disorder is cobalaminopathy type F, the functional human lysosomal transmembrane gene is LMBRD1; if the lysosomal transmembrane protein disease or disorder is late-onset childhood neuronal ceroid lipofuscinosis, the functional human lysosomal transmembrane gene is MFSD8; if the lysosomal transmembrane protein disease or disorder is juvenile neuronal ceroid lipofuscinosis, the functional human lysosomal transmembrane gene is MFSD8. the gene is CLN3; if the lysosomal transmembrane protein disease or disorder is malignant infantile osteopetrosis, the functional human lysosomal transmembrane gene is CLCN7 or OSTM1; if the lysosomal transmembrane protein disease or disorder is mucolipidosis IV, the functional human lysosomal transmembrane gene is MCOLN1; if the lysosomal transmembrane protein disease or disorder is mucopolysaccharidosis type IIC, the functional human lysosomal transmembrane gene is HGSNAT; if the lysosomal transmembrane protein disease or disorder is Niemann-Pick disease type C, the functional human lysosomal transmembrane gene is NPC1; if the lysosomal transmembrane protein disease or disorder is Danon disease, the functional human lysosomal transmembrane gene is LAMP2. In various embodiments, HSPCs are isolated from a subject, for example, from the bone marrow of a subject.

[0088] Although the present invention has been demonstrated with respect to cystinosis and Danon disease, it should be understood that these methods are applicable to any of the diseases or disorders described in Table 1. Thus, this strategy turns HSPCs into intelligent and widespread carriers that provide stable and sustained cross-correction after differentiation into monocytes. Monocytes enter the blood circulation and then into peripheral tissues where they transform into tissue-resident macrophages. These macrophages transfer their lysosomes bearing the respective proteins to diseased peripheral cells through various mechanisms including but not limited to tunneling nanotube formation, vesicle release, and direct cell-cell adhesion. Thus, this study demonstrates the success of HSPC gene therapy strategy to treat lysosomal transmembrane protein diseases or disorders.

[0089] The following examples are intended to illustrate, but not limit, the invention. EXAMPLES

[0090] Example 1 Preclinical models of cystinosis for testing therapeutic approaches We tested a stem cell therapeutic approach in a mouse model of cystinosis, Ctns - / - The mouse model was engineered to produce defective cystinosin, and therefore unable to properly transport cystine out of lysosomes. This defect leads to the accumulation of cystine and the formation of cystine crystals characteristic of cystinosis. Cystine accumulation is present from birth and increases with age. The original Ctns - / - Mice were backcrossed to inbred C57BL / 6 Ctns - / - The inbred C57BL / 6 Ctns mice were generated. - / -Mice begin to develop renal insufficiency from 6 months of age, as observed biochemically (increased serum urea and creatinine) and histologically, with these mice reaching end-stage renal failure by 18 months of age. Renal Fanconi syndrome also begins around 6 months of age (polyuria, phosphaturia, and proteinuria), and proximal tubule cells appear to dedifferentiate and display the typical "swan-neck deformation" seen in mice and humans with cystinosis, resulting in glomeruli that are not connected to the tubules. Finally, Ctns - / - Massive infiltration of inflammatory cells can be observed in the kidneys of mice. - / - The mice also develop eye defects with cystine crystal deposits in the cornea and thyroid dysfunction, similar to those observed in affected patients.

[0091] Example 2 Effects of BMC, HSC, and MSC transplantation on cystinosis To identify the appropriate cell population for transplantation in the setting of cystinosis, we performed syngeneic bone marrow cell (BMC) transplantation, Sca1 + Hematopoietic stem cell (HSC) and mesenchymal stem cell (MSC) transplants in 2-month-old irradiated Ctns - / - These cells were cultured in green fluorescent protein (GFP) transgenic wild-type (WT) mice or Ctns mice as a control. - / - MSCs were isolated from either WT BMC or WT HSC-treated mice. Disease parameters were analyzed 4 months after transplantation. The beneficial effect of MSCs on disease was only short-term and limited. In contrast, tissue cystine content was significantly reduced (57%-94% reduction depending on the tissue) in all examined organs of WT BMC- and WT HSC-treated mice. Abundant GFP + Bone marrow-derived cells were present in all organs and kidney function was improved, providing the first proof of concept that HSCs could rescue cystinosis even though cystinosin is a transmembrane lysosomal protein rather than a secreted enzyme.

[0092] Example 3 Ctns - / - Long-term effects of HSC transplantation in mice It was then determined whether this treatment could be sustained for the life of the mouse and result in multi-organ maintenance.

[0093] Renal analysis: Transplantation of WT HSCs was able to achieve long-term protection of kidney function and structure, preventing the progression of kidney disease up to 15 months after transplantation (the final time point tested; Figure 1). However, effective therapy relies on achieving a relatively high level of donor-derived blood cell engraftment (>50%) in terms of Ctns-expressing cells, which is directly related to the amount of Ctns-expressing cells present in the kidney. In contrast, kidney maintenance was not dependent on the age of the mice at the time of transplantation. In fact, mice up to 10 months old were able to exhibit normal kidney function after stem cell therapy, suggesting that the kidney can be salvaged if tissue damage is not consolidated. We also demonstrated a significant reduction in cystine content in all tissues (from 54% in kidney to 96.5% in liver), demonstrating that this treatment, which is essentially a single HSC transplant, results in long-term and stable low levels of tissue cystine over the life of the mouse. Furthermore, untreated Ctns - / - Large amounts of cystine crystals were consistently observed in kidneys from mice, whereas few to no cystine crystals were observed in kidneys from treated mice.

[0094] Eye analysis: GFP + WT HSC transplantation inhibits Ctns - / - Mouse eyes were maintained long-term. Abundant GFP+ bone marrow-derived cells were detected not only in the corneas of treated mice, but also in the sclera, ciliary body, retina, choroid, and lens. In vivo confocal microscopy (IVCM) was performed in live mice to quantify cystine crystals in the cornea. Effective therapy was dependent on the level of donor-derived blood cell engraftment, as previously demonstrated for the kidney. Ctns with low levels of engraftment - / -Mice with high levels of engraftment (>50%; HIGH; n=5) showed almost total resolution of crystals from the epithelial layer to the middle stroma (100% to 72% clearance, respectively; Figure 2). At 1 year post-transplant, HSC-treated Ctns - / - The mice showed normal corneal thickness and structure as well as normal intraocular pressure. This study was the first demonstration that transplanted HSCs could rescue corneal defects. This study brings new perspectives for ocular regenerative medicine.

[0095] Thyroid analysis: The thyroid gland is also affected in cystinosis, so Ctns - / - We analyzed the function and structure of the thyroid gland in mice and in mice transplanted with HSCs. Sustained activation of thyroid-stimulating hormone (TSH) with morphological evidence of increased thyroglobulin synthesis was shown to mediate the activation of Ctns. - / - In mice, follicular changes included thyrocyte hypertrophy, hyperplasia, colloid depletion, and vascular proliferation. In contrast, Ctns treated with HSC transplantation - / - Mice exhibited virtually normal histology and normalization of cystine and TSH levels (Figure 3).

[0096] Gastrointestinal Analysis: Biopsies of the gastrointestinal mucosa can be used to measure the engraftment of genetically modified stem cell tissues and their impact on cystine and cystine crystal levels in subjects enrolled in clinical trials of HSC gene therapy for cystinosis. Histological techniques for evaluating tissue cystine crystal levels in intestinal mucosa biopsies have been described previously. It has been shown that cystine crystal counts can be correlated with renal function and help evaluate the response to cysteamine treatment. Therefore, rectal biopsies are planned before and every 6 months after transplantation of gene-corrected HSCs into subjects suffering from cystinosis. Up to nine biopsies can be obtained at one time, and therefore it is possible to measure vector copy number (VCN), CTNS expression, cystine content, and cystine crystals in this tissue at each time point. To ascertain whether this tissue represents the efficacy of the treatment, Ctns- / - We investigated the effect of GFP+ WT HSC transplantation on the gastrointestinal tract in mice. Six months after transplantation, abundant GFP+ HSC-derived cells were observed in both intestinal and colonic tissues, with a significantly reduced cystine content in treated mice compared to these compartments in controls (Figure 4).

[0097] Skin Analysis: In vivo confocal microscopy is used on the skin as a non-invasive imaging technique to visualize and quantify tissue cystine crystals before and after HSC transplantation in subjects with cystinosis enrolled in clinical trials. Chiaverini et al. (Journal of the American Academy of Dermatology 68, e111 (2013)) showed that this technique can detect cutaneous cystine deposits in cystinosis patients. To this end, cystinosis patients were studied using a reflectance confocal imager (Caliber VIVASCOPE® 3000) adapted for skin imaging. Engraftment of abundant GFP+ bone marrow-derived cells in the skin leading to significant cystine reduction in this tissue was demonstrated, indicating that HSC transplantation Ctns - / - This was also shown in mice (HSC-treated Ctns - / - 79±0.87 in patients with RA vs. 193±78 in controls, p<0.05).

[0098] Example 4 Myeloablative conditioning regimens: Ctns - / - Efficacy and toxicity in mice Ctns was induced by busulfan (Bu) and cyclophosphamide (Cy), myeloablative drugs currently used in clinical practice for HSC transplantation. - / - Mice were exposed to test whether drug-mediated myeloablation allowed efficient engraftment of Ctns-expressing HSCs in a preclinical model, reduced tissue cystine, and determined whether any unexpected toxicities occurred due to cystinosis. - / - Mice and WT mice as controls were injected intraperitoneally (IP). Mice were analyzed 4 months after transplantation to demonstrate: i) Ctns- / - Mice showed no toxicity to Bu or Cy compared to WT controls; ii) renal function was similar to age-matched untreated WT controls; iii) myeloablation was successful in both cases, with donor cell engraftment measured in peripheral blood reaching 94.2 ± 1.6% with Bu / Cy and 94.0 ± 0.8% with Bu alone; iv) treated Ctns - / - In mice, cystine was significantly reduced in all tissues examined compared with untreated controls. Thus, Bu and Cy were not toxic in a mouse model of cystinosis and Ctns - / - Drug-mediated myeloablation and HSC transplantation in mice significantly reduced cystine in all tissues.

[0099] Myeloablative dosing can be done using busulfan alone without cyclophosphamide. Cyclophosphamide does not eliminate hematopoietic stem cells (i.e., does not create engraftment space) and is immunosuppressive and antileukemic. Because HSC transplants are autologous and not leukemia-directed, cyclophosphamide is not required, which adds unnecessary toxicity to the conditioning regimen. Furthermore, severe nephrotoxicity is uncommon as a direct result of commonly used conditioning regimen agents such as busulfan. It is of note that Dr. Donald Kohn's sickle cell trial (ClinicalTrials.gov Identifier: NCT02247843) uses busulfan alone, and bluebird bio, Inc.'s sickle cell and thalassemia trial (ClinicalTrials.gov Identifier: NCT02151526) also uses busulfan without cyclophosphamide.

[0100] Example 5 Mechanism of therapeutic action The degree of efficacy of HSCs to rescue cystinosis was surprising, especially considering that the ability of HSC transplantation to rescue non-hematopoietic tissues remains controversial, and that cystinosin is a transmembrane lysosomal protein. To elucidate the mechanism of HSC-mediated tissue repair, we transformed Ctns to ubiquitously express the DsRed reporter gene. - / - A novel mouse model was developed in which mice were backcrossed onto a DsRed background (Harrison et al., Mol Ther 21, 433 (2013)). When GFP-expressing HSCs derived from GFP transgenic mice were transplanted, this resulted in a bifluorescent mouse model that not only allowed the fate of transplanted HSCs to be tracked in an in vivo environment, but also allowed events such as fusion, differentiation, and transdifferentiation to be identified with high sensitivity and clearly discriminated.

[0101] Using this model, it was first shown that HSCs differentiate into macrophages in tissues (Naphade et al., Stem Cells 33, 301 (2015)). We then used WT GFP macrophages and DsRed-Ctns - / - In vitro co-culture experiments were performed with fibroblasts. WT macrophages were co-cultured with Ctns - / - When co-cultured with fibroblasts, cystine levels were reduced by about 75% in FACS-sorted fibroblasts (Figure 5A). In contrast, when the two populations were physically separated using a transwell permeable to microvesicles, cystine levels were reduced by only about 20% (Figure 5B). These findings indicate that cross-modification occurs even though cystinosin is a lysosomal transmembrane protein, and that direct cell-cell contact is the major pathway for cross-modification. Using confocal microscopy (Figure 5C), we observed that macrophages extend long membrane protrusions (~40 μm) called tunneling nanotubes (TNTs) that establish contact with fibroblasts. To determine whether TNTs could mediate the physical transfer of vesicles carrying cystinosin, we used DsRed-Ctns - / -Fibroblasts were co-cultured with macrophages stably transduced with a lentiviral vector expressing a cystinosin-GFP fusion protein (CTNS-GFP-macrophages). Confocal microscopy of live cells revealed that vesicles containing cystinosin-GFP were transported along TNTs by the DsRed-Ctns protein. - / - It was revealed that the vesicles could move toward the fibroblasts (Figure 5D). LysoTracker staining identified these vesicles as lysosomes (Naphade et al., Stem Cells 33, 301 (2015)).

[0102] Little is known about TNT in vivo. - / - To explain long-term kidney maintenance in mice, we investigated whether intercellular vesicular exchange using nanotubules could be detected in vivo. Our initial focus was on the kidney, not only because cystinosis occurs early in proximal tubule cells (PTCs), but also because of their physical isolation by the dense tubular basal layer (TBL). In two-color transplanted mice, GFP+ bone marrow-derived cells were observed around but never inside proximal tubules, and numerous tubular extensions emanated from HSC-derived macrophages and passed through the TBL (Figure 6A-a1-6A-a3). GFP-containing structures were observed inside PTCs, suggesting a physical transfer of cytoplasm from macrophages into the epithelium (Figure 6A-a3). To test this hypothesis, we used DsRed-Ctns stably expressing a cystinosine-GFP fusion protein. - / - HSPCs (Figure 4C) or DsRed-Ctns - / - Ctns in HSPCs (Figure 4B) - / - We then transplanted the cystinosin-GFP vesicles into mice. Many cystinosin-GFP vesicles were observed in the PTCs (Figure 4C) (Naphade et al., Stem Cells 33, 301 (2015)). This is the first evidence for the direct transfer of a protein from interstitial macrophages to epithelial cells via TNTs through the TBL to correct the genetic defect that leads to PTC degeneration. Similar data have been reported for the direct transfer of a protein from Ctns to epithelial cells via TNTs through the TBL to correct the genetic defect that leads to PTC degeneration. - / -Insights have been gained into the mechanisms of HSC-mediated therapeutic action for eye defects in mice (Rocca et al., Investigative ophthalmology & visual science 56, 7214 (2015)) and thyroid rescue (Chevronnay et al., Endocrinology In press, (2016)). These findings on HSC-mediated tissue repair should be applicable to other multicompartmental disorders with defective organelles, thus providing new perspectives for regenerative medicine.

[0103] Example 6 Clinical research on hematopoietic stem cell transplantation The aforementioned study represents the first proof of concept for the use of HSC transplantation as a therapy for cystinosis. To minimize the risk of graft-versus-host disease (GVHD), subjects are required to have a sibling bone marrow donor who is HLA-matched for all alleles. The study was designed to include six subjects who were either adults aged 18 years or older with significant signs of disease progression or adolescents aged 13-17 years who could not tolerate cysteamine. However, given the rarity of the disease and the strict donor conditions, no candidate so far was a perfect match for their sibling. Moreover, the risk-benefit ratio of allogeneic HSC transplantation may not be ideal for younger patients, where the introduction of the regular use of the drug cysteamine has allowed patients to live into adulthood, albeit with considerable medical problems (Cherqui, Kidney Int 81, 127 (2012)). Indeed, the risk of morbidity and mortality associated with allogeneic transplantation is significant. The major complication is GVHD; recent studies have shown that acute GVHD grade II-IV occurs in 20%-32% of patients and chronic GVHD occurs in 16%-59%, both of which have a significant impact on recipient survival (Cutler et al., Blood 109, 3108 (2007); Geyer et al., Br J Haematol 155, 218 (2011); and Schleuning et al., Bone Marrow Transplant 43, 717 (2009)). Therefore, a preferred candidate therapy would utilize the patient's own stem cells for autologous HSC transplantation, thereby mitigating the risk of graft rejection and GVHD.

[0104] Example 7 Viral Vector Selection Considering the risks associated with allogeneic HSC transplantation and reviewing the preclinical data on HSC gene therapy, transplantation of autologous HSCs engineered to express functional cystinosin represents a safer approach.

[0105] For gene therapy, lentivirus-derived vectors have replaced gamma-retroviral vectors due to their superior gene transfer efficiency and improved biosafety profile (Case et al., Proc Natl Acad Sci USA 96, 2988 (1999); Miyoshi, et al. Science 283, 682 (1999); Naldini et al., Science 272, 263 (1996); and Varma et al., Nature 389, 239 (1997)). Specifically: 1. All cases of leukemogenic complications observed to date in gene therapy clinical trials or animal models have involved the use of gamma-retroviral vectors, such as Moloney Leukemia Virus (MLV) retroviruses, which have long terminal repeats (LTRs) containing strong enhancer / promoters capable of inducing distal enhancer activation (Hacein-Bey-Abina et al., J Clin Invest 118, 3132 (2008); Li et al., Science 296, 497 (2002)). 2. In contrast, self-inactivating (SIN) lentiviral vectors (LV), third generation lentiviral vectors with deletions in the LTR, contain only one internal enhancer / promoter, reducing the incidence of interactions with nearby cellular genes and therefore the risk of oncogenic integration (Modlich et al., Blood 108, 2545 (2006); Montini et al., J Clin Invest 119, 964 (2009)). Moreover, in contrast to MLV, lentiviruses have not been associated with oncogenesis. Importantly, leukemia has not been recognized as a side effect in HIV patients, even though it is known that memory T cells can harbor integrated viruses for many years. 3. SIN-LTR is also designed to prevent the possibility of developing replication-competent lentivirus (RCL) during the production of viral supernatant. In fact, a transient transfection system containing three packaging plasmids is usually used for vector production - gag, pol, and rev (Dull et al., J Virol 72, 8463 (1998)). A fourth plasmid containing genes encoding the envelope and vesicular stomatitis virus glycoprotein (VSV-G) is frequently used as the envelope selection. So far, no RCL has been reported for this commonly used virus production system in patients after infusion of vector-transduced cell products (Sastry et al., Mol Ther 8, 830 (2003)). 4. LV efficiently transduces HSCs and does not alter their repopulation properties (Montini et al., J Clin Invest 119, 964 (2009); Gonzalez-Murillo et al., Blood 112, 3138 (2008)). 5. Clinical trials using SIN-LV to transduce human HSCs are underway in the United States and Europe for several conditions, including HIV-1, β-thalassemia, immunodeficiency, and cancer (DiGiusto et al., Viruses 5, 2898 (2013); Drakopoulou et al., Current molecular medicine 13, 1314 (2013); Porter et al., N Engl J Med 365, 725 (2011); and Zhang et al., Gene Ther 20, 963 (2013)). In the case of immunodeficiency disorders, SIN-LV modified HSCs have been transplanted into 35 patients to date (Bigger et al., Discovery medicine 17, 207 (2014)). A clinical trial of ex vivo HSC correction with SIN-LV in patients with X-adrenoleukodystrophy showed that cerebral demyelination was suppressed without further progression during a 3-year follow-up in two enrolled patients; and there was no evidence of clonal dominance (Cartier et al., Methods Enzymol 507, 187 (2012); Cartier et al., Science 326, 818 (2009)). Recently, a clinical trial was reported with Wiskott-Aldrich in three patients 32 months after transplantation. Stable and long-term engraftment (25-50%) of genetically modified HSC resulted in improved platelet counts, protection from bleeding and infection, and disappearance of eczema (Aiuti et al., Science 341, 1233151 (2013)). Another clinical success was recently reported in three presymptomatic patients with metachromatic leukodystrophy. Transduced cells achieved 45-80% engraftment of donor blood cells and protein activity in cerebrospinal fluid was returned to above normal levels for up to 24 months, with clear therapeutic benefit (Biffi et al., Science 341, 1233158 (2013)).

[0106] The pCCL-CTNS lentiviral vector, a third generation SIN-lentiviral vector into which the human CTNS cDNA was subcloned (FIG. 7), was prepared for use. The vector backbone pCCL-EFS-X-WPRE described by Zufferey et al. (J Virol 72, 9873 (1998)) was provided by Dr. Donald Kohn (UCLA). A central polypurine tract (cPPT) fragment was added to the CCL vector backbone to increase the nuclear import of viral DNA (Demaison et al., Hum Gene Ther 13, 803 (2002)). The Woodchuck Hepatitis Virus post-translational regulatory element (WPRE) is present to enhance titer and gene expression. However, its open reading frame was removed (Zanta-Boussif et al., Gene Ther 16, 605 (2009)) due to its overlap with the woodchuck hepatitis virus X protein, a transcriptional activator involved in the development of liver tumors (Kingsman et al., Gene Ther 12, 3 (2005)). Transgene expression is driven by the ubiquitously expressed intronless short human elongation factor 1α promoter (EFS, 242 bp) (Wakabayashi-Ito, S. Nagata, J Biol Chem 269, 29831 (1994)). The EFS promoter, lacking the introns and enhancers of the large elements used in many expression plasmids, was shown to direct high levels of transcription of reporter genes in mouse HSCs and to have significantly reduced transactivation potential compared to gamma-retroviral LTRs (Zychlinski et al., Mol Ther, (2008)).

[0107] Vectors with this backbone will be used in clinical trials led by Dr. Kohn: i) autologous transplantation of bone marrow CD34+ stem / progenitor cells after addition of normal human ADA cDNA with EFS-ADA lentiviral vector for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) (BB IND 15440; NCT01852071); ii) autologous bone marrow stem cells (CD34+) transduced with a self-inactivating (SIN) lentiviral vector expressing human β-globin (LENTI / βAS3-FB) and cultured with cytokines after busulfan treatment (BB IND 16028; NCT02247843).

[0108] Example 8 Preclinical studies on transplantation of HSCs transduced with pCCL-CTN Ctns - / - Sca1 isolated from mouse + HSCs were transduced ex vivo with pCCL-CTNS using our optimized protocol for mHSCs, and 1- to 4-month-old Ctns were cultured. - / - The cystine content in the brain, eye, heart, kidney, liver, muscle, and spleen was analyzed 4 months (group 1; n = 8) and 8 months (group 2; n = 12) after transplantation. - / - Mice (n = 7 and n = 12) were used, or Ctns transplanted with WT HSCs. - / - Mice (n=4 and n=4) were used. The reduction in cystine content was consistent with the expression of Ctns in all tissues examined in mice treated with pCCL-CTNS-transduced HSCs. - / - The difference was statistically significant compared to the control (Figure 8A). - / - The effect of HSCs on tissue cystine levels was also tested to exclude the possibility that the presence of any transgene could result in cystine depletion. - / - In mice transplanted with HSCs, untreated Ctns - / -No reduction was observed in any tissue compared to mice (Harrison et al., Mol Ther 21, 433 (2013)).

[0109] Renal glomerular and tubular function was assessed by measuring serum creatinine, urea, and phosphate levels, as well as creatinine clearance in 24-hour urine, in males 8 months after transplantation and compared with age-matched WT males (n=6). - / - All parameters were elevated and creatinine clearance was decreased in pCCL-CTNS-treated Ctns mice compared to WT mice. - / - In mice, serum creatinine, urinary phosphate, and urine volume were significantly decreased compared to controls, indicating that Ctns - / - The beneficial effects of genetically modified HSCs on kidney function in mice were demonstrated. - / - Mice demonstrated significantly fewer cystine crystals present in kidney sections compared to controls (Figures 8B and 8C). - / - It should be noted that in mice, we have shown that cystine content in female kidneys is increased five-fold compared to male kidneys, therefore kidney studies must be performed separately for males and females (Harrison et al., Mol Ther 21, 433 (2013)).

[0110] pCCL-CTNS transplanted Ctns - / -Quantitative PCR (qPCR) was performed using lentivirus-specific primers on genomic DNA isolated from blood collected from mice to measure vector copy number (VCN) per cell. The average VCN was 1.573 ± 1.868, falling within the target range of VCN 1-3. To determine whether lentivirus levels in tissues could be predicted, linear regression analysis was performed between pCCL levels in various tissues as a function of blood VCN levels. A direct correlation was demonstrated between the levels of lentivirus present in blood and those present in tissues (Harrison et al., Mol Ther 21, 433 (2013)). This is useful for tracking future subjects enrolled in clinical trials.

[0111] Example 9 Preclinical Pharmacology and Toxicology Pharmacology / toxicology studies on HSCs genetically modified ex vivo with pCCL-CTNS will be performed using one batch of pCCL-CTNS lentiviral vector preparation obtained from the Indiana University Vector Production Facility (IUVPF) manufactured under Good Manufacturing Practice (GMPc) under the direction of Dr. Kenneth Cornetta. The VCN target range proposed to the FDA for safety is between 1 and 3.

[0112] In vitro immortalization (IVIM) assays, genotoxicity tests, were performed by the Translational Trials Development and Support Laboratory at Cincinnati Children's Hospital Medical Center. The assays consist of a 2-week bulk culture expansion of transduced mouse Lin- BMCs, followed by culture in 96-well plates at a density of 100 cells / well or 10 cells / well for up to 7 weeks (Arumugam et al., Mol Ther 17, 1929 (2009); Modlich et al., Mol Ther 17, 1919 (2009)). Positive wells are counted and the frequency of cell reseeding is calculated and compared to negative (mock transduced) and positive (MLV vector) controls. IVIM assays were performed in triplicate with GMPc pCCL-CTNS preparations with a VCN range between 1 and 3. No immortalized clones were generated using this construct, thus demonstrating a good safety profile.

[0113] Currently, in vivo pharmacology / toxicology studies are being conducted on Ctns with serial transplants. - / - Sca1 in mice + Similar cell therapy products based on mHSCs have been used. 15-20 Ctns were used as primary recipients. - / - Mice (10 males and 10 females) were transduced with pCCL-CTNS. - / - 20 mice were transplanted with mHSCs (VCNs 1–3) and mock-transduced Ctns. - / - The bone marrow cells from each of these mice were then transplanted into secondary Ctns. - / - The primary and secondary mice will be fully analyzed at 6 months post-transplantation with comprehensive molecular, clinical, and histological analysis. To date, we have 32 primary recipients that have reached the 6 month time point: Ctns transduced with pCCL-CTNS- / - 11 Ctns transplanted with mHSCs - / - Mice (VCN included in 1-3) and 21 mock-treated mice, as well as 18 secondary mice. No adverse events have been observed so far, and the data indicate the efficacy of the product, with mice treated with pCCL-CTNS-HSCs weighing more and with cystine content in the tissues examined being significantly less than mock-treated controls (Figures 9A-9B). Therefore, we performed a randomized controlled study of up to 9 additional Ctns treated with pCCL-CTNS-transduced HSCs. - / - The 6 month time point must be reached for the primary recipients and for 15 secondary recipients.

[0114] Example 10 Manufacturing: Process Development GMPc pCCL-CTNS preparation was used to identify human CD34 +A protocol was optimized to transduce HSCs to obtain a VCN comprised between 1 and 3. The protocol involved one-hit vector transduction for 20 h at an MOI of 20. Colony forming unit (CFU) assays were then performed with human CD34+ peripheral blood stem cells (PBSCs) isolated from five healthy donors and four cystinosis patients, neither of which showed any abnormal proliferation or differentiation capacity in the case of pCCL-CTNS LVs compared to mock-transduced controls. Furthermore, vector integration site (VIS) analysis in the patient cells did not show enrichment of integration sites near the 5' end of the proto-oncogene. However, this protocol resulted in a mean VCN of 2 in healthy CD34+ cells, whereas the mean VCN in the cystinosis patients was 0.96. Therefore, the protocol was further optimized using cystinosis patient cells to achieve higher levels of transduction, and we demonstrated that the higher the level of CTNS expressing cells, the better the therapeutic response (Yeagy et al., Kidney Int 79, 1198 (2011); Rocca et al., Investigative ophthalmology & visual science 56, 7214 (2015); Harrison et al., Mol Ther 21, 433 (2013)). This protocol involved two-hit vector transduction for a total of 24 hours at MOI 20, respectively, resulting in an average VCN of 1.9 for patient cells. The CFU assay and VIS should then be repeated using this new protocol.

[0115] For clinical trials, the transduction protocol will be performed according to standard operating procedures in a GMP facility, using the optimal protocol for CD34+ cells from cystinosis patients. It should be noted that before enrolling the first patient, optimal conditions for large-scale transduction using GMP-grade pCCL-CTNS vector preparations and the optimal protocol will be verified in proficiency testing with human CD34+ cells from healthy donors in a small-scale and GMP facility. The clinical trial will include six patients affected by cystinosis, four adults and two adolescents. This is believed to be the first human clinical trial of an autologous stem cell and gene therapy treatment strategy for cystinosis. If successful, this treatment could be a lifelong therapy that could eliminate or reduce kidney deterioration and the need for kidney transplants, as well as the long-term complications associated with cystinosis. Furthermore, the CD34 cells modified by pCCL-CTNS have been shown to be highly effective in treating cystinosis. + The mechanisms by which HSC transplantation provides beneficial and protective effects may be applicable to other inherited multi-organ degenerative disorders.

[0116] Example 11 HSPC transplantation for Danon disease The goal of this experiment is to determine whether Danon disease can be rescued by HSPC transplantation and whether lysosomal cross-correction occurs. Using the mouse model described herein, it is demonstrated that the hearts of LAMP2 KO mice exhibit an increased number of abnormal mitochondria and impaired mitophagy and mitochondrial respiration, consistent with previous studies in induced pluripotent stem cell (hiPSC)-derived cardiomyocytes obtained from Danon disease patients (Cherqui, Kidney Int 81, 127 (2012)), confirming the similarity of the mouse model to the human disease.

[0117] To assess the ability of WT HSPCs to rescue Danon disease, lethally irradiated 2-month-old LAMP2 KO mice were transplanted with Sca1 HSPCs isolated from congenic C57BL / 6 WT (Tg(ACTB-EGFP)1Osb / J obtained from the Jackson Laboratory), which ubiquitously express cytoplasmic eGFP, using transplantation protocols previously described (Yeagy et al., Kidney Int 79, 1198 (2011); Naphade et al., Stem Cells 33, 301 (2015); Case et al., Proc Natl Acad Sci USA 96, 2988 (1999)). + As a negative control, LAMP2 KO mice were transplanted with HSPCs (WT-HSPCs). + We also transplanted HSPCs (KO-HSPCs). Skeletal muscle strength was examined using previously described techniques and demonstrated that grip strength was significantly reduced in LAMP2 KO mice compared to both WT mice and LAMP2 mice fed WT HSPCs (Figure 11). As assessed by Western blot analysis, LAMP2 protein expression was restored to near WT levels in the heart and skeletal muscle of LAMP2 KO mice transplanted with WT HSPCs (Figures 10D-10E).

[0118] To demonstrate that LAMP2 was expressed not only in donor macrophages present in the hearts of recipient LAMP2 KO mice, but also in their cardiomyocytes, we performed immunofluorescence studies, which revealed that LAMP2 was expressed in cardiomyocytes (α-actinin: white) located in the vicinity of donor macrophages. +Small vacuoles were demonstrated (Figures 10A-10C). EM analysis showed that vacuoles in LAMP2 KO mice fed with WT HSPCs were reduced compared to LAMP2 KO mice (Figures 11A and 11B), appearing similar to that in WT mice. Improved autophagic flux after WT HSPC transplantation into LAMP2 KO mice was confirmed by assessing LC3-II / GAPDH levels (Figures 11C-11D). In summary, these studies demonstrate that physiological and metabolic functions are restored in LAMP2 KO mice treated with WT HSPC transplantation.

[0119] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the appended claims.

[0120] Sequence information SEQUENCE LISTING <110> The Regents of the University of California <120> METHODS OF TREATING LYSOSOMAL DISORDERS <150> US 62 / 471,741 <151> 2017-03-15 <150> US 62 / 507,713 <151> 2017-05-17 <160> 27 <170> PatentIn version 3.5 <210> 1 <211> 367 <212> PRT <213> homo sapiens <400> 1 Met Ile Arg Asn Trp Leu Thr Ile Phe Ile Leu Phe Pro Leu Lys Leu 1 5 10 15 Val Glu Lys Cys Glu Ser Ser Val Ser Leu Thr Val Pro Pro Val Val 20 25 30 Lys Leu Glu Asn Gly Ser Ser Thr Asn Val Ser Leu Thr Leu Arg Pro 35 40 45 Pro Leu Asn Ala Thr Leu Val Ile Thr Phe Glu Ile Thr Phe Arg Ser 50 55 60 Lys Asn Ile Thr Ile Leu Glu Leu Pro Asp Glu Val Val Val Pro Pro 65 70 75 80 Gly Val Thr Asn Ser Ser Phe Gln Val Thr Ser Gln Asn Val Gly Gln 85 90 95 Leu Thr Val Tyr Leu His Gly Asn His Ser Asn Gln Thr Gly Pro Arg 100 105 110 Ile Arg Phe Leu Val Ile Arg Ser Ser Ala Ile Ser Ile Ile Asn Gln 115 120 125 Val Ile Gly Trp Ile Tyr Phe Val Ala Trp Ser Ile Ser Phe Tyr Pro 130 135 140 Gln Val Ile Met Asn 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Arg Lys Arg Pro Gly Tyr Asp Gln Leu Asn 355 360 365 <210> 2 <211> 2611 <212> DNA <213> homo sapiens <400> 2 cgcctctccc aaagtctagc cgggcagggg aacgcggtgc attcctgacc ggcacctggc 60 gaggctcatg cgtcccgtga gggcggttcc tcgagcctgg gggcgctcag attgctttgg 120 agacgctgag agaacctttg cgagagcgcc ggttgacgtg cggagtgcgg ggctccgggg 180 gactgagcag cacgagaccc catcctcccc tccgggtttt cacactgggc gaagggagga 240 ctcctgagct ctgcctcttc cagtaacatt gaggattact gtgttttgtg agagctcgct 300 aggcgcccta agcaacagag ttctgagaaa tcgagaaaca tgataaggaa ttggctgact 360 attttatcc ttttcccct gaagctcgta gagaaatgtg agtcaagcgt cagcctcact 420 gttcctcctg tcgtaaagct ggagaacggc agctcgacca acgtcagcct caccctgcgg 480 ccaccattaa atgcaaccct ggtgatcact tttgaaatca catttcgttc caaaaatatt 540 actatccttg agctccccga tgaagttgtg gtgcctcctg gagtgacaaa ctcctctttt 600 caagtgacat ctcaaaatgt tggacaactt actgtttatc tacatggaaa tcactccaat 660 cagaccggcc cgaggatacg ctttcttgtg atccgcagca gcgccattag catcataaac 720 caggtgattg gctggatcta ctttgtggcc tggtccatct ccttctaccc tcaggtgatc 780 atgaattgga ggcggaaaag tgtcattggt ctgagcttcg acttcgtggc tctgaacctg 840 acaggcttcg tggcctacag tgtattcaac atcggcctcc tctgggtgcc ctacatcaag 900 gagcagtttc tcctcaaata ccccaacgga gtgaaccccg tgaacagcaa cgacgtcttc 960 ttcagcctgc acgcggttgt cctcacgctg atcatcatcg tgcagtgctg cctgtatgag 1020 cgcggtggcc agcgcgtgtc ctggcctgcc atcggcttcc tggtgctcgc gtggctcttc 1080 gcatttgtca ccatgatcgt ggctgcagtg ggagtgatca cgtggctgca gtttctcttc 1140 tgcttctcct acatcaagct cgcagtcacg ctggtcaagt attttccaca ggcctacatg 1200 aacttttact acaaaagcac tgagggctgg agcattggca acgtgctcct ggacttcacc 1260 gggggcagct tcagcctcct gcagatgttc ctccagtcct acaacaacga ccagtggacg 1320 ctgatcttcg gagacccaac caagtttgga ctcggggtct tctccatcgt cttcgacgtc 1380 gtcttcttca tccagcactt ctgtttgtac agaaagagac cggggtatga ccagctgaac 1440 tagcacccag ggacccagtg tacccagcct ctggcctcgt gccctgctgg ggaaggcctc 1500 acccagcgaa ggccggagaa gcggttgggc cctggcacac agggctggct cagtgtgcgg 1560 acagaggaga ccactctgct cctggggcca gaggccattc aatagcctgc cttcgtccgg 1620 gcccctcctg ggcctccccg gccaggcacg tggcaccgtc gccttgacac cgccatctct 1680 tttctttaag gcttcaggca gcgcgcacag gctctggcag ccgtctcagg caggactggg 1740 caccaagctt gcagccgaag gccttgcccc aaactaccag cgtttctgca agcagcttga 1800 agggctgacc ttgcagccgg gtgagccaag ggcactttgc tgccaccgct gcattcccag 1860 agatcaagca gcccggtgcc gtggccagtg aactcagagg tgctggtgga cgggctagga 1920 ctttggggtt aggccatggg gctctttctc tgaaggccac tttcctgacg tactctctgt 1980 acataactca gcgtccgtga ctgcagtaac agccagccct acccagagta tttctgagcc 2040 atgaggggcc caccagattg gttctgaatt ggattcatgc ccagcgcatt agcatagtaa 2100 ctcctttcag attttttgga gggacgtttg gaagtggctt actctcttct gccctctctc 2160 ctacctccac cttctcagat gagccccatc tgagcacatc cagctgctcc ttacccagca 2220 tctggagtac aggacatagc tctctcctgc taccagtctg tgccttagag gtcgttaggc 2280 ctgccaaacg gcgaccagct cccctggagc gagggcaggc cccttccctc tctttcccca 2340 gacacctact tgagactcac caatttctgg cctgttcagg agcctcagat aagtatttgt 2400 acttgagacc acctcacaca atctgtatgg gcccaaccct gatctcaaac ctccttccct 2460 ctgcccaaag ctgtccttcc tatggcagga ggggtggggg tcccaggacg tgcctcatac 2520 atgacttgag cttgtcagtc cactgagttt ccttctacga gatcaacgcg aggggcctgt 2580 atcttgaatt aaagcctact cgcttccttt c 2611 <210> 3 <211> 495 <212> PRT <213> homo sapiens <400> 3 Met Arg Ser Pro Val Arg Asp Leu Ala Arg Asn Asp Gly Glu Glu Ser 1 5 10 15 Thr Asp Arg Thr Pro Leu Leu Pro Gly Ala Pro Arg Ala Glu Ala Ala 20 25 30 Pro Val Cys Cys Ser Ala Arg Tyr Asn Leu Ala Ile Leu Ala Phe Phe 35 40 45 Gly Phe Phe Ile Val Tyr Ala Leu Arg Val Asn Leu Ser Val Ala Leu 50 55 60 Val Asp Met Val Asp Ser Asn Thr Thr Leu Glu Asp Asn Arg Thr Ser 65 70 75 80 Lys Ala Cys Pro Glu His Ser Ala Pro Ile Lys Val His His Asn Gln 85 90 95 Thr Gly Lys Lys Tyr Gln Trp Asp Ala Glu Thr Gln Gly Trp Ile Leu 100 105 110 Gly Ser Phe Phe Tyr Gly Tyr Ile Ile Thr Gln Ile Pro Gly Gly Tyr 115 120 125 Val Ala Ser Lys Ile Gly Gly Lys Met Leu Leu Gly Phe Gly Ile Leu 130 135 140 Gly Thr Ala Val Leu Thr Leu Phe Thr Pro Ile Ala Ala Asp Leu Gly 145 150 155 160 Val Gly Pro Leu Ile Val Leu Arg Ala Leu Glu Gly Leu Gly Glu Gly 165 170 175 Val Thr Phe Pro Ala Met His Ala Met Trp Ser Ser Trp Ala Pro Pro 180 185 190 Leu Glu Arg Ser Lys Leu Leu Ser Ile Ser Tyr Ala Gly Ala Gln Leu 195 200 205 Gly Thr Val Ile Ser Leu Pro Leu Ser Gly Ile Ile Cys Tyr Tyr Met 210 215 220 Asn Trp Thr Tyr Val Phe Tyr Phe Phe Gly Thr Ile Gly Ile Phe Trp 225 230 235 240 Phe Leu Leu Trp Ile Trp Leu Val Ser Asp Thr Pro Gln Lys His Lys 245 250 255 Arg Ile Ser His Tyr Glu Lys Glu Tyr Ile Leu Ser Ser Leu Arg Asn 260 265 270 Gln Leu Ser Ser Gln Lys Ser Val Pro Trp Val Pro Ile Leu Lys Ser 275 280 285 Leu Pro Leu Trp Ala Ile Val Val Ala His Phe Ser Tyr Asn Trp Thr 290 295 300 Phe Tyr Thr Leu Leu Thr Leu Leu Pro Thr Tyr Met Lys Glu Ile Leu 305 310 315 320 Arg Phe Asn Val Gln Glu Asn Gly Phe Leu Ser Ser Leu Pro Tyr Leu 325 330 335 Gly Ser Trp Leu Cys Met Ile Leu Ser Gly Gln Ala Ala Asp Asn Leu 340 345 350 Arg Ala Lys Trp Asn Phe Ser Thr Leu Cys Val Arg Arg Ile Phe Ser 355 360 365 Leu Ile Gly Met Ile Gly Pro Ala Val Phe Leu Val Ala Ala Gly Phe 370 375 380 Ile Gly Cys Asp Tyr Ser Leu Ala Val Ala Phe Leu Thr Ile Ser Thr 385 390 395 400 Thr Leu Gly Gly Phe Cys Ser Ser Gly Phe Ser Ile Asn His Leu Asp 405 410 415 Ile Ala Pro Ser Tyr Ala Gly Ile Leu Leu Gly Ile Thr Asn Thr Phe 420 425 430 Ala Thr Ile Pro Gly Met Val Gly Pro Val Ile Ala Lys Ser Leu Thr 435 440 445 Pro Asp Asn Thr Val Gly Glu Trp Gln Thr Val Phe Tyr Ile Ala Ala 450 455 460 Ala Ile Asn Val Phe Gly Ala Ile Phe Phe Thr Leu Phe Ala Lys Gly 465,470,475,480 Glu Val Gln Asn Trp Ala Leu Asn Asp His His Gly His Arg His 485 490 495 <210> 4 <211> 2512 <212> DNA <213> homo sapiens <400> 4 cggctacttt gcgccaatcc tacgagaact cccagaactc cgcttcccta gtccaaccca 60 agccagagtt gcccacacct aagatggcgg cggggggcgg agtcggcgcg gccgcctctg 120 ggcgggaccg cggggactag acgtggccgc ggggcggtgt catcgccccc gcccgcccg 180 gtccagccag ctcggcccgg gggcttcggg ctgtcgggcc ggcgctccct tctctgccag 240 gtggcgagta cacctgctca cgtaggcgtc atgaggtctc cggttcgaga cctggcccgg 300 aacgatggcg aggagagcac ggaccgcacg cctctctac cgggcgcccc acgggccgaa 360 gccgctccag tgtgctgctc tgctcgttac aacttagcaa ttttggcctt ttttggttc 420 ttcattgtgt atgcattacg tgtgaatctg agtgttgcgt tagtggatat ggtagattca 480 aatacaactt tagaagataa tagaacttcc aaggcgtgtc cagagcattc tgctcccata 540 aaagttcatc ataatcaaac gggtaagaag taccaatggg atgcagaaac tcaaggatgg 600 attcggtt ccttttttta tggctacatc atcacacaga ttcctggagg atatgttgcc 660 agcaaatag gggggaaaat gctgctagga tttgggatcc ttggcactgc tgtcctcacc 720 ctgttcactc ccattgctgc agatttagga gttggaccac tcattgtact cagagcacta 780 gaaggactag gagagggtgt tacatttcca gccatgcatg ccatgtggtc ttcttgggct 840 ccccctctg aaagaagcaa acttcttagc atttcatatg caggagcaca gcttgggaca 900 gtaatttctc ttcctctttc tggaataatt tgctactata tgaattggac tttgtcttc 960 tactttttg gtactattgg aatattttgg tttctttgt ggatctggtt agttagtgac 1020 acaccacaaa aacacaagag aatttcccat tatgaaaagg aatacattct ttcatcatta 1080 agaaatcagc tttcttcaca gaagtcagtg ccgtgggtac ccattttaaa atccctgcca 1140 ctttgggcta tcgtagttgc acacttttct tacaactgga ctttttatac tttattgaca 1200 ttattgccta cttatatgaa ggagatccta aggttcaatg ttcaagagaa tgggttttta 1260 tcttcattgc cttatttagg ctcttggtta tgtatgatcc tgtctggtca agctgctgac 1320 aatttaaggg caaaatggaa tttttcaact ttatgtgttc gcagaatttt tagccttata 1380 ggaatgattg gacctgcagt attcctggta gctgctggct tcattggctg tgattattct 1440 ttggccgttg ctttcctaac tatatcaaca acactgggag gcttttgctc ttctggattt 1500 agcatcaacc atctggatat tgctccttcg tatgctggta tcctcctggg catcacaaat 1560 acatttgcca ctattccagg aatggttggg cccgtcattg ctaaaagtct gacccctgat 1620 aacactgttg gagaatggca aaccgtgttc tatattgctg ctgctattaa tgtttttggt 1680 gccattttct ttacactatt cgccaaaggt gaagtacaaa actgggctct caatgatcac 1740 catggacaca gacactgaag gaaccaataa ataatcctgc ctctattaat gtatttttat 1800 tttcatgta acctcaagt gccttctgta ttgtgtaagc attctatgtc ttttttaat 1860 tgtacttgta ttagattttt aaggcctata atcatgaat atcactagtt gccagaataa 1920 taaaatgaac tgtgtttaat tatgaataat atgtaagcta ggactctac tttaggttca 1980 catacctgcc tgctagtcgg gcaacatgaa gtaggacagt tctgttgat tttaggcc 2040 attackaagg gatgagctg aacagacct cctgatacct ttgcttatt aaactagatg 2100 atattctca ggtactgata aacacctgtt gttgttcact ttcctcataa aaattgtcag 2160 ctctctctga cacttagacc tcaacttta gcatctctgt ggagctgcca tccactgtat 2220 aatttcgcct ggcactgga ctgagggag tgtgccagg cagctgccaa gcactccctc 2280 cctggcttca gggtcagagt gcccagcgtt tatcagaggc agcatccaag cccagagcca 2340 gtgtcgactc tcggctgt gcttttcctc tgagggct tcaatgtgta gataaagccc 2400 tgagtaggca agagcagtga gatccactgc tatggtctg atacatcctc aaactttccc 2460 ttcccagcac agaggaatat tggctggcat gcaacctgca aaagaaaaat gc 2512 <210> 5 <211> 537 <212> PRT <213> homo sapiens <400> 5 Gly Ala Ala Ser Ala Glu Leu Val Ile Gly Trp Cys Ile Phe Gly Pro 1 5 10 15 Leu Leu Leu Wings Where Wings Cys Trp And Tyr Val Arg Lys Tyr 20 25 30 Gln Ser Gln Arg Glu Ser Glu Val Val Val Ser 35 40 45 Ser Leu Wing Ile Leo Ile Thr Ser Leo Wing Leo Pro Val Asp Ile 50 55 60 Phe Leu Val Ser Tyr Met Lys Asn Gln Asn Gly Thr Phe Lys Asp Trp 65 70 75 80 Only Asn Only Asn Will Be Arg Gln And Glu Asp Thr Will Leu Tyr Gly 85 90 95 Tyr Tyr Thr Leu Tyr Ser Ser Val Ile Leu Phe Cys Val Phe Trp Ile 100 105 110 Pro Phe Val Tyr Phe Tyr Tyr Glu Glu Lys Asp Asp Asp Asp Thr Ser 115 120 125 Lys Cys Thr Gln Ile Lys Thr Ala Phe Lys Tyr Thr Leu Gly Phe Ala 130 135 140 Val Ile Cys Ala Leu Leu Leu Leu Val Gly Ala Phe Val Pro Leu Asn 145 150 155 160 Val Pro Asn Asn Lys Asn Ser Thr Glu Trp Glu Lys Val Lys Phe Leu 165 170 175 Phe Glu Glu Leu Gly Ser Ser His Gly Leu Ala Ala Leu Ser Phe Ser 180 185 190 Ile Ser Ser Leu Thr Leu Ile Gly Met Leu Ala Ala Ile Thr Tyr Thr 195 200 205 Ala Tyr Gly Met Ser Ala Leu Pro Leu Asn Leu Ile Lys Gly Thr Arg 210 215 220 Ser Ala Ala Tyr Glu Arg Leu Glu Asn Thr Glu Asp Ile Glu Glu Val 225 230 235 240 Glu Gln His Ile Gln Thr Ile Lys Ser Lys Ser Lys Asp Gly Arg Pro 245 250 255 Leu Pro Ala Arg Asp Lys Arg Ala Leu Lys Gln Phe Glu Glu Arg Leu 260 265 270 Arg Thr Leu Arg Lys Arg Glu Arg His Leu Glu Tyr Ile Glu Asn Ser 275 280 285 Trp Trp Thr Lys Phe Cys Gly Ala Leu Arg Pro Leu Lys Ile Ile Trp 290 295 300 Gly Ile Phe Phe Ile Leu Val Ala Leu Leu Phe Ile Ile Ser Leu Phe 305 310 315 320 Leu Ser Asn Leu Asp Lys Ala Leu His Ser Ala Gly Ile Asp Ser Gly 325 330 335 Phe Ile Ile Phe Gly Ala Asn Leu Ser Asn Pro Leu Asn Met Leu Leu 340 345 350 Pro Val Leu Gln Thr Val Phe Pro Leu Asp Tyr Ile Leu Ile Thr Ile 355 360 365 Ile Ile Met Tyr Phe Ile Phe Thr Ser Met Ala Gly Ile Arg Asn Ile 370 375 380 Gly Ile Trp Phe Phe Trp Val Arg Leu Tyr Lys Ile Arg Arg Gly Arg 385 390 395 400 Thr Arg Pro Gln Ala Leu Leu Phe Leu Cys Met Ile Leu Leu Leu Ile 405 410 415 Val Leu His Thr Ser Tyr Met Ile Tyr Ser Leu Ala Pro Gln Tyr Val 420 425 430 Met Tyr Gly Ser Gln Asn Tyr Leu Ile Glu Ser Asn Ile Thr Tyr Asp 435 440 445 Asp His Lys Asn Asn Ser Ala Phe Pro Val Pro Lys Arg Cys Asp Ala 450 455 460 Asp Ala Pro Glu Asp Gln Cys Thr Val Thr Arg Thr Tyr Leu Phe Leu 465 470 475 480 His Lys Phe Trp Phe Phe Ser Ala Tyr Tyr Phe Gly Asn Trp Ala 485,490,495 Cys Cys Lys Gly 500 505 510 Lys Lys Ser Val Ile Glu Gly Val Asp Glu Asp Asp Ser Asp Ile Ser 515,520,525 Asp Asp Glu Pro Ser Val Tyr Ser Val 530,535 <210> 6 <211> 1979 <212> DNA <213> homo sapiens <400> 6 ggcgcggctt cggcggagct ggtgatcggc tggtgcatat tggcccctt actactggct 60 atttttgcat tctgttggat atatgttcgt aaataccaaa gtcagcggga aagtgaagtt 120 gtctccacca taacggcaat tttctctg gcgattgcac ttcacatc accactctt 180 ccagtggata tattttggt ttcttacatg aaaaatcaa atgtacatt taggactgg 240 gccaatgcta atgtcagcag acagatcgag gacactgtgt tatatggtta ctacacctta 300 tattctgtta tattattctg tgtgttttc tggatccctt ttgtctactt ctactatgaa 360 gaaaaggatg atgatgatac tagtaaatgt actcaaatta aaactgcatt caagtatact 420 ttgggatttg ctgtaatttg tgcacttctt cttttagttg gagctttgt tcctctaaat 480 gttcctaata acaaaaattc tacagagtgg gaaaaagtga agttcctgtt tgaagaactt 540 ggaagtagtc atggtttagc tgcattgtca tttctatta gttctctgac cttgattgga 600 atgttggcag ctataactta cacagcctat ggcatgtctg cattaccttt aaatctaata 660 aaaggcacta gaagcgctgc ttacgaacgt ttagaaaaca ctgaagacat tgaagaagtg 720 gagcaacaca ttcaaacgat taaatcaaaa agcaaagatg gtcggcctttt gccagcaagg 780 gataaacgcg ccttaaaaca atttgaagaa aggttaagaa cacttaggaa aagagagagg 840 cacttagaat acattgaaaa cagctggtgg acaaaattttt gtggtgctct gcgtcccctg 900 aagatcattt ggggaatatt tttcatctta gttgcattgc tgtttataat ttctctcttc 960 ctgtcaaatt tggataaagc ccttcattca gctggaatag attctggtttt tataattttt 1020 ggagctaact tgagtaatcc actgaatatg cttttgcctg tactacaaac aggtttcct 1080 cttgattata ttcttataac aattattatt atgtacttta ttttacttc aatggcggga 1140 attcgaaata tcggcatatg gttcttttgg gttagactat ataaaattag aagaggtaga 1200 accaggcccc aggccctctt atttctttgc atgatacttc tgcttattgt ccttcacact 1260 agctacatga tttatagtct tgctccccaa tatgtcatgt atggaagcca aaattactta 1320 atagagagca atataactta tgatgaccat aaaaacaatt cagccttccc tgtgccaaag 1380 agatgtgatg ctgatgcccc tgaagaccaa tgtactgtta cgcggacata cctgttcctt 1440 cacaagttct ggttctttag tgctgcatac tattttggta actgggcttt tcttgtggta 1500 ttcttgattg gattaattgt atcctgttgt aaagggaaga aatcagtcat tgaaggagta 1560 gatgaagatg attcagacat aagtgatgat gagccctctg tctattctgt ttgagagcct 1620 ctgtcttagg ggttttataa tgctgactga atgtctatta tgcatttttt aaagtgttaa 1680 actaacatta ggatgaactg actagcttca tcaaaaatgg gagcatggct attaaaaaaa 1740 ctatatttt tatgttatct gaagtaacat tattgtatca tagattaaca tttaaaaattg 1800 ctgtaataat tctatgtaaa tataaaacta tggactttgt gaggaatgt ttgtggaaat 1860 cttttttctc tagtgtataa tagtgttgaa ttgattaaaa gttttccaga attaatattc 1920 cctcttgtca cttctttaaaa acataataaa tcactttac ctgtgcaaaa aaaaaaaaa 1979 <210> 7 <211> 518 <212> PRT <213> Homo sapiens <400> 7 Met Ala Gly Leu Arg Asn Glu Ser Glu Gln Glu Pro Leu Leu Gly Asp 1 5 10 15 Thr Pro Gly Ser Arg Glu Trp Asp Ile Leu Glu Thr Glu Glu His Tyr 20 25 30 Lys Ser Arg Trp Arg Ser Ile Arg Ile Leu Tyr Leu Thr Met Phe Leu 35 40 45 Ser Ser Val Gly Phe Ser Val Val Met Met Ser Ile Trp Pro Tyr Leu 50 55 60 Gln Lys Ile Asp Pro Thr Ala Asp Thr Ser Phe Leu Gly Trp Val Ile 65 70 75 80 Ala Ser Tyr Ser Leu Gly Gln Met Val Ala Ser Pro Ile Phe Gly Leu 85 90 95 Trp Ser Asn Tyr Arg Pro Arg Lys Glu Pro Leu Ile Val Ser Ile Leu 100 105 110 Ile Ser Val Ala Ala Asn Cys Leu Tyr Ala Tyr Leu His Ile Pro Ala 115 120 125 Ser His Asn Lys Tyr Tyr Met Leu Val Ala Arg Gly Leu Leu Gly Ile 130 135 140 Gly Ala Gly Asn Val Ala Val Val Arg Ser Tyr Thr Ala Gly Ala Thr 145 150 155 160 Ser Leu Gln Glu Arg Thr Ser Ser Met Ala Asn Ile Ser Met Cys Gln 165 170 175 Ala Leu Gly Phe Ile Leu Gly Pro Val Phe Gln Thr Cys Phe Thr Phe 180 185 190 Leu Gly Glu Lys Gly Val Thr Trp Asp Val Ile Lys Leu Gln Ile Asn 195 200 205 Met Tyr Thr Thr Pro Val Leu Leu Ser Ala Phe Leu Gly Ile Leu Asn 210 215 220 Ile Ile Leu Ile Leu Ala Ile Leu Arg Glu His Arg Val Asp Asp Ser 225 230 235 240 Gly Arg Gln Cys Lys Ser Ile Asn Phe Glu Glu Ala Ser Thr Asp Glu 245 250 255 Ala Gln Val Pro Gln Gly Asn Ile Asp Gln Val Ala Val Val Ala Ile 260 265 270 Asn Val Leu Phe Phe Val Thr Leu Phe Ile Phe Ala Leu Phe Glu Thr 275 280 285 Ile Ile Thr Pro Leu Thr Met Asp Met Tyr Ala Trp Thr Gln Glu Gln 290 295 300 Ala Val Leu Tyr Asn Gly Ile Ile Leu Ala Ala Leu Gly Val Glu Ala 305 310 315 320 Val Val Ile Phe Leu Gly Val Lys Leu Leu Ser Lys Lys Ile Gly Glu 325 330 335 Arg Ala Ile Leu Leu Gly Gly Leu Ile Val Val Trp Val Gly Phe Phe 340 345 350 Ile Leu Leu Pro Trp Gly Asn Gln Phe Pro Lys Ile Gln Trp Glu Asp 355 360 365 Leu His Asn Asn Ser Ile Pro Asn Thr Thr Phe Gly Glu Ile Ile Ile 370 375 380 Gly Leu Trp Lys Ser Pro Met Glu Asp Asp Asn Glu Arg Pro Thr Gly 385 390 395 400 Cys Ser Ile Glu Gln Ala Trp Cys Leu Tyr Thr Pro Val Ile His Leu 405 410 415 Ala Gln Phe Leu Thr Ser Ala Val Leu Ile Gly Leu Gly Tyr Pro Val 420 425 430 Cys Asn Leu Met Ser Tyr Thr Leu Tyr Ser Lys Ile Leu Gly Pro Lys 435 440 445 Pro Gln Gly Val Tyr Met Gly Trp Leu Thr Ala Ser Gly Ser Gly Ala 450 455 460 Arg Ile Leu Gly Pro Met Phe Ile Ser Gln Val Tyr Ala His Trp Gly 465 470 475 480 Pro Arg Trp Ala Phe Ser Leu Val Cys Gly Ile Ile Val Leu Thr Ile 485 490 495 Thr Leu Leu Gly Val Val Tyr Lys Arg Leu Ile Ala Leu Ser Val Arg 500 505 510 Tyr Gly Arg Ile Gln Glu 515 <210> 8 <211> 1909 <212> DNA <213> homo sapiens <400> 8 aggttacaag cagcagatcc caccttcagt cctggctctg acaagccctc cagcttcacg 60 ccacccggga tgggagaaag caggtgtcgc gagagttggg cgcaagacgc cttgtaggga 120 gtgtaactat ggccggcctg cggaacgaaa gtgaacagga gccgctctta ggcgacacac 180 ctggaagcag agaatgggac attttagaga ctgaagagca ttataagagc cgatggagat 240 ctattaggat tttatatctt actatgtttc tcagcagtgt agggttttct gtagtgatga 300 tgtccatatg gccatatctc caaaagattg atccgacagc tgatacaagt tttttgggct 360 gggttattgc ttcatatagt cttggccaaa tggtagcttc acctatattt ggtttatggt 420 ctaattatag accaagaaaa gagcctctta ttgtctccat cttgatttcc gtggcagcca 480 actgcctcta tgcatatctc cacatcccag cttctcataa taaatactac atgctggttg 540 ctcgtggatt gttgggaatt ggagcaggaa atgtagcagt tgttagatca tatactgctg 600 gtgctacttc ccttcaggaa agaacaagtt ccatggcaaa cataagcatg tgtcaagcat 660 taggttttat tctaggtcca gtttttcaga cttgttttac attccttgga gaaaaaggtg 720 tgacatggga tgtgattaaa ctgcagataa acatgtatac aacaccagtt ttacttagcg 780 ccttcctggg aattttaaat attattctga tccttgccat actaagagaa catcgtgtgg 840 atgactcagg aagacagtgt aaaagtatta attttgaaga agcaagtaca gatgaagctc 900 aggttcccca aggaaatatt gaccaggttg ctgttgtggc catcaatgtt ctgttttttg 960 tgactctatt tatctttgcc ctttttgaaa ccatcattac tccattaaca atggatatgt 1020 atgcctggac tcaagaacaa gctgtgttat ataatggcat aatacttgct gctcttgggg 1080 ttgaagccgt tgttattttc ttaggagtta agttgctttc caaaaagatt ggcgagcgtg 1140 ctattctact gggaggactc atcgttgtat gggttggctt ctttatcttg ttaccttggg 1200 gaaatcaatt tcccaaaata cagtgggaag atttgcacaa taattcaatc cctaatacca 1260 catttgggga aattattatt ggtctttgga agtctccaat ggaagatgac aatgaaagac 1320 caactggttg ctcgattgaa caagcctggt gcctctacac cccggtgatt catctggccc 1380 agttccttac atcagctgtg ctaataggat taggctatcc agtctgcaat cttatgtcct 1440 atactctata ttcaaaaatt ctaggaccaa aacctcaggg tgtatacatg ggctggttaa 1500 cagcatctgg aagtggagcc cggattcttg ggcctatgtt catcagccaa gtgtatgctc 1560 actggggacc acgatgggca ttcagcctgg tgtgtggaat aatagtgctc accatcaccc 1620 tcctgggagt ggtttacaaa agactcattg ctctttctgt aagatatggg aggattcagg 1680 aataaactag ctaagactgt gatggaaact acttgctgtg tggcacttcc tggtctaaag 1740 ctctgctaga caattgcggt gagccagtct ccaagaatca gactacagat attgcagatt 1800 ttgaagaaca agaacatatg ttgaataaca gagagaattc tacatgtcat tgtgaatagt 1860 aggttatata aaaacatact agatgataat ttcaaaaaaa aaaaaaaaa 1909 <210> 9 <211> 438 <212> PRT <213> Homo sapiens <400> 9 Met Gly Gly Cys Ala Gly Ser Arg Arg Arg Phe Ser Asp Ser Glu Gly 1 5 10 15 Glu Glu Thr Val Pro Glu Pro Arg Leu Pro Leu Leu Asp His Gln Gly 20 25 30 Ala His Trp Lys Asn Ala Val Gly Phe Trp Leu Leu Gly Leu Cys Asn 35 40 45 Asn Phe Ser Tyr Val Val Met Leu Ser Ala Ala His Asp Ile Leu Ser 50 55 60 His Lys Arg Thr Ser Gly Asn Gln Ser His Val Asp Pro Gly Pro Thr 65 70 75 80 Pro Ile Pro His Asn Ser Ser Ser Arg Phe Asp Cys Asn Ser Val Ser 85 90 95 Thr Ala Ala Val Leu Leu Ala Asp Ile Leu Pro Thr Leu Val Ile Lys 100 105 110 Leu Leu Ala Pro Leu Gly Leu His Leu Leu Pro Tyr Ser Pro Arg Val 115 120 125 Leu Val Ser Gly Ile Cys Ala Ala Gly Ser Phe Val Leu Val Ala Phe 130 135 140 Ser His Ser Val Gly Thr Ser Leu Cys Gly Val Val Phe Ala Ser Ile 145 150 155 160 Ser Ser Gly Leu Gly Glu Val Thr Phe Leu Ser Leu Thr Ala Phe Tyr 165 170 175 Pro Arg Ala Val Ile Ser Trp Trp Ser Ser Gly Thr Gly Gly Ala Gly 180 185 190 Leu Leu Gly Ala Leu Ser Tyr Leu Gly Leu Thr Gln Ala Gly Leu Ser 195 200 205 Pro Gln Gln Thr Leu Leu Ser Met Leu Gly Ile Pro Ala Leu Leu Leu 210 215 220 Ala Ser Tyr Phe Leu Leu Leu Thr Ser Pro Glu Ala Gln Asp Pro Gly 225 230 235 240 Gly Glu Glu Glu Ala Glu Ser Ala Ala Arg Gln Pro Leu Ile Arg Thr 245 250 255 Glu Ala Pro Glu Ser Lys Pro Gly Ser Ser Ser Ser Leu Ser Leu Arg 260 265 270 Glu Arg Trp Thr Val Phe Lys Gly Leu Leu Trp Tyr Ile Val Pro Leu 275 280 285 Val Val Val Tyr Phe Ala Glu Tyr Phe Ile Asn Gln Gly Leu Phe Glu 290 295 300 Leu Leu Phe Phe Trp Asn Thr Ser Leu Ser His Ala Gln Gln Tyr Arg 305 310 315 320 Trp Tyr Gln Met Leu Tyr Gln Ala Gly Val Phe Ala Ser Arg Ser Ser 325 330 335 Leu Arg Cys Cys Arg Ile Arg Phe Thr Trp Ala Leu Ala Leu Leu Gln 340 345 350 Cys Leu Asn Leu Val Phe Leu Ala Asp Val Trp Phe Gly Phe Leu 355 360 365 Pro Ser With Tyr Leu Val Phe Leu With Leu Tyr Glu Gly Leu 370 375 380 Gly Gly Ala Ala Tyr Val Asn Thr Phe His Asn Ile Ala Leu Glu Thr 385 390 395 400 Ser Asp Glu His Arg Glu Phe Ala Met Ala Ala Thr Cys Ile Ser Asp 405 410 415 Thr Leu Gly Ile Ser Leu Ser Gly Leu Leu Ala Leu Pro Leu His Asp 420 425 430 Phe and Cys Gln Ser 435 <210> 10 <211> 1689 <212> DNA <213> homo sapiens <400> 10 cccctagaca agccggagct gggaccggca atcgggct gatccttgtc acctgtcgca 60 gaccctcatc cctcccgtgg gagcccctt tggacactct atgaccctgg accctcgggg 120 gacctgaact tgatgcgatg gaggctgtg caggctcgcg gcggcgcttt tcggattccg 180 aggggagga gaccgtcccg gagccccggc tccctctgtt ggaccatcag ggcgcgcatt 240 ggaagaacgc ggtgggcttc tggctgctgg gcctttgcaa caacttctct tatgtggtga 300 tgctgagtgc cgcccacgac atccttagcc aaagaggac atcgggaaac cagagccatg 360 tggacccagg cccaacgccg atcccccaca acagctcatc acgatttgac tgcaactctg 420 tctctacggc tgctgtgctc ctggcggaca tcctccccac actcgtcatc aaattgttgg 480 ctcctcttgg ccttcacctg ctgccctaca gccccccgggt tctcgtcagt gggatttgtg 540 ctgctgggaag cttcgtcctg gttgcctttt ctcattctgt ggggaccagc ctgtgtggtg 600 tggtcttgc tagcatctca tcaggccttg gggaggtcac cttcctctcc ctcactgcct 660 tctaccccag ggccgtgatc tcctggtggt cctcagggac tggggagct gggctgctgg 720 gggccctgtc ctacctgggc ctcacccagg ccggcctctc ccctcagcag accctgctgt 780 ccatgctggg tatccctgcc ctgctgctgg ccagctattt cttgttgctc acatctcctg 840 aggcccagga ccctggaggg gaagaaag cagagagcgc agcccggcag cccctcataa 900 gaaccgaggc cccggagtcg aagccaggct ccagctccag cctctccctt cgggaaaggt 960 ggacagtatt caagggtctg ctgtggtaca ttgttccctt ggtcgtagtt tactttgccg 1020 agtatttcat taaccaggga ctttttgaac tcctcttttt ctggaacact tccctgagtc 1080 acgctcagca ataccgctgg taccagatgc tgtaccaggc tggcgtcttt gcctcccgct 1140 cttctctccg ctgctgtcgc atccgtttca cctgggccct ggccctgctg cagtgcctca 1200 acctggtgtt cctgctggca gacgtgtggt tcggctttct gccaagcatc tacctcgtct 1260 tcctgatcat tctgtatgag gggctcctgg gaggcgcagc ctacgtgaac accttccaca 1320 acatcgccct ggagaccagt gatgagcacc gggagtttgc aatggcggcc acctgcatct 1380 ctgacacact ggggatctcc ctgtcggggc tcctggcttt gcctctgcat gacttcctct 1440 gccagctctc ctgatactcg ggatcctcag gacgcaggtc acattcacct gtgggcagag 1500 ggacaggtca gacacccagg cccaccccag agaccctcca tgaactgtgc tcccagcctt 1560 cccggcaggt ctgggagtag ggaagggctg aagccttgtt tccttgcagg ggggccagcc 1620 attgtctccc acttggggag tttcttcctg gcatcatgcc ttctgaataa atgccgattt 1680 tgtccatgg 1689 <210> 11 <211> 805 <212> PRT <213> homo sapiens <400> 11 Met Ala Asn Val Ser Lys Lys Val Ser Trp Ser Gly Arg Asp Arg Asp 1 5 10 15 Asp Glu Glu Ala Ala Pro Leu Leu Arg Arg Thr Ala Arg Pro Gly Gly 20 25 30 Gly Thr Pro Leu Leu Asn Gly Ala Gly Pro Gly Ala Ala Arg Gln Ser 35 40 45 Pro Arg Ser Ala Leu Phe Arg Val Gly His Met Ser Ser Val Glu Leu 50 55 60 Asp Asp Glu Leu Leu Asp Pro Asp Met Asp Pro Pro His Pro Phe Pro 65 70 75 80 Lys Glu Ile Pro His Asn Glu Lys Leu Leu Ser Leu Lys Tyr Glu Ser 85 90 95 Leu Asp Tyr Asp Asn Ser Glu Asn Gln Leu Phe Leu Glu Glu Glu Arg 100 105 110 Arg Ile Asn His Thr Ala Phe Arg Thr Val Glu Ile Lys Arg Trp Val 115 120 125 Ile Cys Ala Leu Ile Gly Ile Leu Thr Gly Leu Val Ala Cys Phe Ile 130 135 140 Asp Ile Val Val Glu Asn Leu Ala Gly Leu Lys Tyr Arg Val Ile Lys 145 150 155 160 Gly Asn Ile Asp Lys Phe Thr Glu Lys Gly Gly Leu Ser Phe Ser Leu 165 170 175 Leu Leu Trp Ala Thr Leu Asn Ala Ala Phe Val Leu Val Gly Ser Val 180 185 190 Ile Val Ala Phe Ile Glu Pro Val Ala Ala Gly Ser Gly Ile Pro Gln 195 200 205 Ile Lys Cys Phe Leu Asn Gly Val Lys Ile Pro His Val Val Arg Leu 210 215 220 Lys Thr Leu Val Ile Lys Val Ser Gly Val Ile Leu Ser Val Val Gly 225 230 235 240 Gly Leu Ala Val Gly Lys Glu Gly Pro Met Ile His Ser Gly Ser Val 245 250 255 Ile Ala Ala Gly Ile Ser Gln Gly Arg Ser Thr Ser Leu Lys Arg Asp 260 265 270 Phe Lys Ile Phe Glu Tyr Phe Arg Arg Asp Thr Glu Lys Arg Asp Phe 275 280 285 Val Ser Ala Gly Ala Ala Ala Gly Val Ser Ala Ala Phe Gly Ala Pro 290 295 300 Val Gly Gly Val Leu Phe Ser Leu Glu Glu Gly Ala Ser Phe Trp Asn 305 310 315 320 Gln Phe Leu Thr Trp Arg Ile Phe Phe Ala Ser Met Ile Ser Thr Phe 325 330 335 Thr Leu Asn Phe Val Leu Ser Ile Tyr His Gly Asn Met Trp Asp Leu 340 345 350 Ser Ser Pro Gly Leu Ile Asn Phe Gly Arg Phe Asp Ser Glu Lys Met 355 360 365 Ala Tyr Thr Ile His Glu Ile Pro Val Phe Ile Ala Met Gly Val Val 370 375 380 Gly Gly Val Leu Gly Ala Val Phe Asn Ala Leu Asn Tyr Trp Leu Thr 385 390 395 400 Met Phe Arg Ile Arg Tyr Ile His Arg Pro Cys Leu Gln Val Ile Glu 405 410 415 Ala Val Leu Val Ala Ala Val Thr Ala Thr Val Ala Phe Val Leu Ile 420 425 430 Tyr Ser Ser Arg Asp Cys Gln Pro Leu Gln Gly Gly Ser Met Ser Tyr 435 440 445 Pro Leu Gln Leu Phe Cys Ala Asp Gly Glu Tyr Asn Ser Met Ala Ala 450 455 460 Ala Phe Phe Asn Thr Pro Glu Lys Ser Val Val Ser Leu Phe His Asp 465 470 475 480 Pro Pro Gly Ser Tyr Asn Pro Leu Thr Leu Gly Leu Phe Thr Leu Val 485 490 495 Tyr Phe Phe Leu Ala Cys Trp Thr Tyr Gly Leu Thr Val Ser Ala Gly 500 505 510 Val Phe Ile Pro Ser Leu Leu Ile Gly Ala Ala Trp Gly Arg Leu Phe 515 520 525 Gly Ile Ser Leu Ser Tyr Leu Thr Gly Ala Ala Ile Trp Ala Asp Pro 530 535 540 Gly Lys Tyr Ala Leu Met Gly Ala Ala Ala Gln Leu Gly Gly Ile Val 545 550 555 560 Arg Met Thr Leu Ser Leu Thr Val Ile Met Met Glu Ala Thr Ser Asn 565 570 575 Val Thr Tyr Gly Phe Pro Ile Met Leu Val Leu Met Thr Ala Lys Ile 580 585 590 Val Gly Asp Val Phe Ile Glu Gly Leu Tyr Asp Met His Ile Gln Leu 595 600 605 Gln Ser Val Pro Phe Leu His Trp Glu Ala Pro Val Thr Ser His Ser 610 615 620 Leu Thr Ala Arg Glu Val Met Ser Thr Pro Val Thr Cys Leu Arg Arg 625 630 635 640 Arg Glu Lys Val Gly Val Ile Val Asp Val Leu Ser Asp Thr Ala Ser 645 650 655 Asn His Asn Gly Phe Pro Val Val Glu His Ala Asp Asp Thr Gln Pro 660 665 670 Ala Arg Leu Gln Gly Leu Ile Leu Arg Ser Gln Leu Ile Val Leu Leu 675 680 685 Lys His Lys Val Phe Val Glu Arg Ser Asn Leu Gly Leu Val Gln Arg 690 695 700 Arg Leu Arg Leu Lys Asp Phe Arg Asp Ala Tyr Pro Arg Phe Pro Pro 705 710 715 720 Ile Gln Ser Ile His Val Ser Gln Asp Glu Arg Glu Cys Thr Met Asp 725 730 735 Leu Ser Glu Phe Met Asn Pro Ser Pro Tyr Thr Val Pro Gln Glu Ala 740 745 750 Ser Leu Pro Arg Val Phe Lys Leu Phe Arg Ala Leu Gly Leu Arg His 755 760 765 Leu Val Val Val Asp Asn Arg Asn Gln Val Val Gly Leu Val Thr Arg 770 775 780 Lys Asp Leu Ala Arg Tyr Arg Leu Gly Lys Arg Gly Leu Glu Glu Leu 785 790 795 800 Ser Leu Ala Gln Thr 805 <210> 12 <211> 3277 <212> DNA <213> homo sapiens <400> 12 gccggcgctt cccggccggt gtcgctccgc ggcgggccat ggccaacgtc tctaagaagg tgtcctggtc cggccgggac cgggacgacg aggaggcggc gccgctgctg cggaggacgg 120 cgcggcccgg cggggggacg ccgctgctga acggggctgg gcccggggct gcgcgccagt 180 caccacgttc tgcgcttttc cgagtcggac atatgagcag cgtggagctg gatgatgac ttttggaccc ggatatggac cctccacatc ccttccccaa ggagatccca cacaacgaga agctcctgtc cctcaagtat gagagcttgg actatgacaa cagtgagaac cagctgttcc tggaggagga gcggcggatc aatcacacgg ccttccggac ggtggagatc aagcgctggg 420 tcatctgcgc cctcattggg atcctcacgg gcctcgtggc ctgcttcatt gacatcgtgg 480 tggaaaacct ggctggcctc aagtacaggg tcatcaaggg caatatcgac aagttcacag 540 agaagggcgg actgtccttc tccctgttgc tgtgggccac gctgaacgcc gccttcgtgc 600 tcgtgggctc tgtgattgtg gctttcatag agccggtggc tgctggcagc ggaatccccc 660 agatcaagtg cttcctcaac ggggtgaaga tcccccacgt ggtgcggctc aagacgttgg 720 tgatcaaagt gtccggtgtg atcctgtccg tggtcggggg cctggccgtg ggaaaggaag 780 ggccgatgat ccactcaggt tcagtgattg ccgccgggat ctctcaggga aggtcaacgt 840 cactgaaacg agatttcaag atcttcgagt acttccgcag agacacagag aagcgggact 900 tcgtctccgc aggggctgcg gccggagtgt cagcggcgtt tggagccccc gtgggtgggg 960 tcctgttcag cttggaggag ggtgcgtcct tctggaacca gttcctgacc tggaggatct 1020 tctttgcttc catgatctcc acgttcaccc tgaattttgt tctgagcatt taccacggga 1080 acatgtggga cctgtccagc ccaggcctca tcaacttcgg aaggtttgac tcggagaaaa 1140 tggcctacac gatccacgag atcccggtct tcatcgccat gggcgtggtg ggcggtgtgc 1200 ttggagctgt gttcaatgcc ttgaactact ggctgaccat gtttcgaatc aggtacatcc 1260 accggccctg cctgcaggtg attgaggccg tgctggtggc cgccgtcacg gccacagttg 1320 ccttcgtgct gatctactcg tcgcgggatt gccagcccct gcaggggggc tccatgtcct 1380 acccgctgca gctcttttgt gcagatggcg agtacaactc catggctgcg gccttcttca 1440 acaccccgga gaagagcgtg gtgagcctct tccacgaccc gccaggctcc tacaaccccc 1500 tgaccctcgg cctgttcacg ctggtctact tcttcctggc ctgctggacc tacgggctca 1560 cggtgtctgc cggggtcttc atcccgtccc tgctcatcgg ggctgcctgg ggccggctct 1620 ttgggatctc cctgtcctac ctcacggggg cggcgatctg ggcggacccc ggcaaatacg 1680 ccctgatggg agctgctgcc cagctgggcg ggattgtgcg gatgacactg agcctgaccg 1740 tcatcatgat ggaggccacc agcaacgtga cctacggctt ccccatcatg ctggtgctca 1800 tgaccgccaa gatcgtgggc gacgtcttca ttgagggcct gtacgacatg cacattcagc 1860 tgcagagtgt gcccttcctg cactgggagg ccccggtcac ctcacactca ctcactgcca 1920 gggaggtgat gagcacacca gtgacctgcc tgaggcggcg tgagaaggtc ggcgtcattg 1980 tggacgtgct gagcgacacg gcgtccaatc acaacggctt ccccgtggtg gagcatgccg 2040 atgacaccca gcctgcccgg ctccagggcc tgatcctgcg ctcccagctc atcgttctcc 2100 taaagcacaa ggtgtttgtg gagcggtcca acctgggcct ggtacagcgg cgcctgaggc 2160 tgaaggactt ccgagacgcc tacccgcgct tcccacccat ccagtccatc cacgtgtccc 2220 aggacgagcg ggagtgcacc atggacctct ccgagttcat gaacccctcc ccctacacgg 2280 tgccccagga ggcgtcgctc ccacgggtgt tcaagctgtt ccgggccctg ggcctgcggc 2340 acctggtggt ggtggacaac cgcaatcagg ttgtcgggtt ggtgaccagg aaggacctcg 2400 ccaggtaccg cctgggaaag agaggcttgg aggagctctc gctggcccag acgtgaggcc 2460 cagccctgcc cataatgggc actggcgctg gcaccccggc ccttctgcat ttcctcccgg 2520 agtcactggt ttctcggccc aaaccatgct ccccagcagt ggcaatggcg agcaccctgc 2580 agctgggcgg gcaggcggca ggcgcggaac tgaccctctc gcgggactga ccctgttgtg 2640 ggcagtggtc tccccccttg gcgcctcctt gcgcaggccc agcctccact ctcctcgtct 2700 aggtttcttt acctccaggg atcagctgtg tgtgtgtgac ctccctaccg ggctatcggc 2760 ctcttgggag ccagcggcag ggccggcacc tgcgtgcctg tgcccgtgtg cgtgagacag 2820 agcccttgcc cctgctgctg ccccgagggc tgccctgccc tggaagggcc cctctgcctc 2880 cacaccagtg gagtcttcga gacttgggag ctgcttggcc tcattttcag ccatgagcag 2940 acggcctgtg gtccctgggc ctgaggcacg gactcgtagc accagggttt ggaggctgcg 3000 accgccccgg agagcagctt cacactggcg ccacagagga gccccacgtg cactccccgg 3060 cctgcatccg gcttgggtac acaggcccag aggactgggg tgactcacgg gccctgtgct 3120 gtgatgttga gagctgagaa aaacctccaa ggccctgagc cccatgccca gccctgcctt 3180 ggtcccccaa tccccagagc ttggagtctg ggccccacac ccagccctgc cttggtccct 3240 gagcctcaaa gcgtggaatt gctgccctgt ggacact 3277 <210> 13 <211> 334 <212> PRT <213> Homo sapiens <400> 13 Met Glu Pro Gly Pro Thr Ala Ala Gln Arg Arg Cys Ser Leu Pro Pro 1 5 10 15 Trp Leu Pro Leu Gly Leu Leu Leu Trp Ser Gly Leu Ala Leu Gly Ala 20 25 30 Leu Pro Phe Gly Ser Ser Pro His Arg Val Phe His Asp Leu Leu Ser 35 40 45 Glu Gln Gln Leu Leu Glu Val Glu Asp Leu Ser Leu Ser Leu Leu Gln 50 55 60 Gly Gly Gly Leu Gly Pro Leu Ser Leu Pro Pro Asp Leu Pro Asp Leu 65 70 75 80 Asp Pro Glu Cys Arg Glu Leu Leu Leu Asp Phe Ala Asn Ser Ser Ala 85 90 95 Glu Leu Thr Gly Cys Leu Val Arg Ser Ala Arg Pro Val Arg Leu Cys 100 105 110 Gln Thr Cys Tyr Pro Leu Phe Gln Gln Val Val Ser Lys Met Asp Asn 115 120 125 Ile Ser Arg Ala Ala Gly Asn Thr Ser Glu Ser Gln Ser Cys Ala Arg 130 135 140 Ser Leu Leu Met Ala Asp Arg Met Gln Ile Val Val Ile Leu Ser Glu 145 150 155 160 Phe Phe Asn Thr Thr Trp Gln Glu Ala Asn Cys Ala Asn Cys Leu Thr 165 170 175 Asn Asn Ser Glu Glu Leu Ser Asn Ser Thr Val Tyr Phe Leu Asn Leu 180 185 190 Phe Asn His Thr Leu Thr Cys Phe Glu His Asn Leu Gln Gly Asn Ala 195 200 205 His Ser Leu Leu Gln Thr Lys Asn Tyr Ser Glu Val Cys Lys Asn Cys 210 215 220 Arg Glu Ala Tyr Lys Thr Leu Ser Ser Leu Tyr Ser Glu Met Gln Lys 225 230 235 240 Met Asn Glu Leu Glu Asn Lys Ala Glu Pro Gly Thr His Leu Cys Ile 245 250 255 Asp Val Glu Asp Ala Met Asn Ile Thr Arg Lys Leu Trp Ser Arg Thr 260 265 270 Phe Asn Cys Ser Val Pro Cys Ser Asp Thr Val Pro Val Ile Ala Val 275 280 285 Ser Val Phe Ile Leu Phe Leu Pro Val Val Phe Tyr Leu Ser Ser Phe 290 295 300 Leu His Ser Glu Gln Lys Lys Arg Lys Leu Ile Leu Pro Lys Arg Leu 305 310 315 320 Lys Ser Ser Thr Ser Phe Ala Asn Ile Gln Glu Asn Ser Asn 325 330 <210> 14 <211> 2869 <212> DNA <213> homo sapiens <400> 14 ggctgtccgc ggtgccggct gggggcggag aggcggcggt gggctccctg gggtgtgtga 60 gccggtgat ggagccgggc ccgacagccg cgcagcggag gtgttcgttg ccgccgtggc 120 tgccgctggg gctgctgctg tggtcggggc tgccctggg cgcgctcccc ttcggcagca 180 gtccgcacag ggtcttccac gacctcctgt cggagcagca gttgctggag gtggaggact 240 tgtccctgtc cctcctgcag ggtggagggc tggggcctct gtcgctgcc ccggacctgc 300 cggatctgga tcctgagtgc cgggagctcc tgctggactt cgccaacagc agcgcagagc 360 tgacagggtg tctggtgcgc agcgcccggc ccgtgcgcct ctgtcagacc tgctaccccc 420 tcttccaaca gtcgtcagc aagatggaca acatcagccg agccgcgggg atacttcag 480 agagtcagag ttgtgccaga agtctcttaa tggcagatag atgcaaata gttgtgattc 540 tctcagaatt ttttaatacc acatggcagg aggcaattg tgcaattgt ttacaaca 600 660 cctgctttga acatacctt cagggaatg cacatagtct tttacagaca aaaaattatt 720 cagaagtag caaaactgc cgtgaagcat aaaactct gagtagtctg tacagtgaaa 780 tgcaaaaat gatgaactt gagaatagg ctgaacctgg aacacattta tgcattgatg 840 tggaagatgc aatgaacatc actcgaaaac tatggattcg aactttcac tgttcagtcc 900 cttgcagtga cacagtgcct gtaattgctg ttctgtgtt cattctctt ctacctgttg 960 tcttctacct tagtagcttt cttcactcag agcaaagaa acgcaaacct attctcccca 1020 aacgtctcaa gtccagtacc agttttgcaa atattcagga aaattcaac tgagacctac 1080 aaaatggaga attgacatat cacgtgaatg aatggtggaa gandacaactt ggtttcagaa 1140 agaagataa ctgtgatttg acagtcaag ctcttaagaa atacaggac ttcagatcca 1200 tttttaaata agaatttcg atttttcttt ccttttccac ttcttctaa cagatttgga 1260 tatttttaat ttccaggcat agcagtgtta tctattttaa tgtgtatttg tcacaataac 1320 agaacatgca agaacaatca ttttatt tttaggcat tgattacta tttagactt 1380 ctggtatctt cttactaca taagtatctc aagtagaaaa gttttgaa actacattt 1440 aaaattaat cagttacagt aagactttg aaaaagaat gtactgtta ggtagct 1500 taattacccc ccattgcagt attattgtta tatatatagt taatatgttg tacatcacaa 1560 taatatataa ttcagtctct agttcccta gagtcatttt tgaaccact gattgcaac 1620 ctccctgaca atttttaaa gtagtaagcc acatcatt tatcttgta aaagattta 1680 tggtaactgg tttcttactt gactttatta atagtattt tacatcttat tttgcctttt 1740 atttcattag taatttaaaa atcactggat tgctttatta tattcaggc atatggatt 1800 atttttac caggattg catcgtgaat taattaagt tattttggca tttatattt 1860 attactactt taaatcaaat gtagcattat cacactgtat ttaaattgtc atttttaaa 1920 ggaatatttt cttcttaaga tatatagagg attttggaga agagagacag gaggggtaaa 1980 accagcttaa ggttcagcga gcagaaaggg acctgagagg atgctcactg taagactgtt 2040 ggacagtggt gtgtattgag gggatgaatc ggaacgatag tctcatgcag aaaatagtga 2100 gattaagatc atccttattg tttctaaatt atttcaatca gatgaaagtg atacgattga 2160 aatgaaatca catagttcgt gctcagaaat tctattttgg tatgtttgta ttagccttta 2220 gaaaaaacac tccgtttcag aattgttcac agttttattt cttaggtttt tagagttcag 2280 gatttcattt attaatttct tcttgctttt ttggtggaaa taggctttgt tgtaaacatt 2340 aagaatataa aatctcctct atatagaaac aagaattttg ttaaaaagag aatttgaatc 2400 ccttcctata ctataaaatg ctctatagg agacaaagtg tttctttttt cttttatgtt 2460 tactgtttat gtggagtgaa atataaggct cttggatgta taacatactc aaaagctgtt 2520 acactttctc tgatctgctg tgatccactg aaaatgtgct ggggtttgtt ctgctgtcac 2580 tgttatgct gctggaactt agcactgtct tgatttgaag catatgattg agagccattt 2640 gaagcaatct tcattaatgc agataaaaca agtttacatg tgcagagtta gaaaatgaca 2700 tgttcaattc tgtaagtggt gactttttga gcacctttca gtattatgta tttgtaaaaa 2760 ccattgtttt tggatataaa gctaataagc actttaaaaa aaaaaaaaa aaaaaaaaa 2820 2869 <210> 15 <211> 580 <212> PRT <213> homo sapiens <400> 15 Put Thr Ala Pro Ala Gly Pro Arg Gly Ser Glu Thr Glu Arg Leu Leu 1 5 10 15 Thr Pro Asn Pro Gly Tyr Gly Thr Gln Ala Gly Pro Ser Pro Ala Pro 20 25 30 Pro Thr Pro Pro Glu Glu Glu Asp Leu Arg Arg Arg Leu Lys Tyr Phe 35 40 45 Phe Met Ser Pro Cys Asp Lys Phe Arg Ala Lys Gly Arg Lys Pro Cys 50 55 60 Lys Leu Met Leu Gln Val Val Lys Ile Leu Val Val Thr Val Gln Leu 65 70 75 80 Ile Leu Phe Gly Leu Ser Asn Gln Leu Ala Val Thr Phe Arg Glu Glu 85 90 95 Asn Thr Ile Ala Phe Arg His Leu Phe Leu Leu Gly Tyr Ser Asp Gly 100 105 110 Ala Asp Asp Thr Phe Ala Ala Tyr Thr Arg Glu Gln Leu Tyr Gln Ala 115 120 125 Ile Phe His Ala Val Asp Gln Tyr Leu Ala Leu Pro Asp Val Ser Leu 130 135 140 Gly Arg Tyr Ala Tyr Val Arg Gly Gly Gly Asp Pro Trp Thr Asn Gly 145 150 155 160 Ser Gly Leu Ala Leu Cys Gln Arg Tyr Tyr His Arg Gly His Val Asp 165 170 175 Pro Ala Asn Asp Thr Phe Asp Ile Asp Pro Met Val Val Thr Asp Cys 180 185 190 Ile Gln Val Asp Pro Pro Glu Arg Pro Pro Pro Pro Pro Ser Asp Asp 195 200 205 Leu Thr Leu Leu Glu Ser Ser Ser Ser Tyr Lys Asn Leu Thr Leu Lys 210 215 220 Phe His Lys Leu Val Asn Val Thr Ile His Phe Arg Leu Lys Thr Ile 225 230 235 240 Asn Leu Gln Ser Leu Ile Asn Asn Glu Ile Pro Asp Cys Tyr Thr Phe 245 250 255 Ser Val Leu Ile Thr Phe Asp Asn Lys Ala His Ser Gly Arg Ile Pro 260 265 270 Ile Ser Leu Glu Thr Gln Ala His Ile Gln Glu Cys Lys His Pro Ser 275 280 285 Val Phe Gln His Gly Asp Asn Ser Phe Arg Leu Leu Phe Asp Val Val 290 295 300 Val Ile Leu Thr Cys Ser Leu Ser Phe Leu Leu Cys Ala Arg Ser Leu 305 310 315 320 Leu Arg Gly Phe Leu Leu Gln Asn Glu Phe Val Gly Phe Met Trp Arg 325 330 335 Gln Arg Gly Arg Val Ile Ser Leu Trp Glu Arg Leu Glu Phe Val Asn 340 345 350 Gly Trp Tyr Ile Leu Leu Val Thr Ser Asp Val Leu Thr Ile Ser Gly 355 360 365 Thr Ile Met Lys Ile Gly Ile Glu Ala Lys Asn Leu Ala Ser Tyr Asp 370 375 380 Val Cys Ser Ile Leu Leu Gly Thr Ser Thr Leu Leu Val Trp Val Gly 385 390 395 400 Val Ile Arg Tyr Leu Thr Phe Phe His Asn Tyr Asn Ile Leu Ile Ala 405 410 415 Thr Leu Arg Val Ala Leu Pro Ser Val Met Arg Phe Cys Cys Cys Val 420 425 430 Ala Val Ile Tyr Leu Gly Tyr Cys Phe Cys Gly Trp Ile Val Leu Gly 435 440 445 Pro Tyr His Val Lys Phe Arg Ser Leu Ser Met Val Ser Glu Cys Leu 450 455 460 Phe Ser Leu Ile Asn Gly Asp Asp Met Phe Val Thr Phe Ala Ala Met 465 470 475 480 Gln Ala Gln Gln Gly Arg Serves Leu Val Trp Leu Phe Serves Gln Leu 485,490,495 Tyr Leu Tyr Ser Phe Ile Ser Leu Phe Ile Tyr Met Val Leu Ser Leu 500 505 510 Phe Ile Ala Leu Ile Thr Gly Ala Tyr Asp Thr Ile Lys His Pro Gly 515,520,525 Gly Ala Gly Ala Glu Glu Ser Glu Leu Gln Ala Tyr Ile Ala Gln Cys 530 535 540 Gln Asp Ser Pro Thr Ser Gly Lys Phe Arg Arg Gly Ser Gly Ser Ala 545,550,555,560 Cys Ser Leu Leu Cys Cys Cys Gly Arg Asp Pro Ser Glu Glu His Ser 565,570,575 Leu Leu Val Asn 580 <210> 16 <211> 2051 <212> DNA <213> homo sapiens <400> 16 agatcagctg atgccggagg gtttgaagcc gcgccgcgag ggagcgaggt cgcagtgaca 60 gcggcgggcg atcggaccca ggctgccccg cgtacccgc ctgcgtcccg cgctcccgcc 120 ccagcatgac agccccggcg ggtccgcgcg gctcagagac cgagcggctt ctgaccccca 180 accccgggta tgggacccag gcggggcctt caccggcccc tccgacaccc ccagaagagg 240 aagaccttcg ccgtcgtctc aatactttt tcatgagtcc ctgcgacaag ttcgagcca 300 agggccgcaa gccctgcaag ctgatgctgc aagtggtcaa gatcctggtg gtcacggtgc 360 agctcatcct gtttgggctc agtaatcagc tggctgtgac attccgggaa gagaacacca 420 tcgccttccg acacctcttc ctgctgggct actcggacgg agcggatgac accttcgcag 480 cctacacgcg ggagcagctg taccaggcca tcttccatgc tgtggaccag tacctggcgt 540 tgcctgacgt gtcactgggc cggtatgcgt atgtccgtgg tgggggtgac ccttggacca 600 atggctcagg gcttgctctc tgccagcggt actaccaccg aggccacgtg gacccggcca 660 acgacacatt tgacattgat ccgatggtgg ttactgactg catccaggtg gatccccccg 720 agcggccccc tccgcccccc agcgacgatc tcaccctctt ggaaagcagc tccagttaca 780 agaacctcac gctcaaattc cacaagctgg tcaatgtcac catccacttc cggctgaaga 840 ccattaacct ccagagcctc atcaataatg agatcccgga ctgctatacc ttcagcgtcc 900 tgatcacgtt tgacaacaaa gcacacagtg ggcggatccc catcagcctg gagacccagg 960 cccacatcca ggagtgtaag caccccagtg tcttccagca cggagacaac agcttccggc 1020 tcctgtttga cgtggtggtc atcctcacct gctccctgtc cttcctcctc tgcgcccgct 1080 cactccttcg aggcttcctg ctgcagaacg agtttgtggg gttcatgtgg cggcagcggg 1140 gacgggtcat cagcctgtgg gagcggctgg aatttgtcaa tggctggtac atcctgctcg 1200 tcaccagcga tgtgctcacc atctcgggca ccatcatgaa gatcggcatc gaggccaaga 1260 acttggcgag ctacgacgtc tgcagcatcc tcctgggcac ctcgacgctg ctggtgtggg 1320 tgggcgtgat ccgctacctg accttcttcc acaactacaa tatcctcatc gccacactgc 1380 gggtggccct gcccagcgtc atgcgcttct gctgctgcgt ggctgtcatc tacctgggct 1440 actgcttctg tggctggatc gtgctggggc cctatcatgt gaagttccgc tcactctcca 1500 tggtgtctga gtgcctgttc tcgctcatca atggggacga catgtttgtg acgttcgccg 1560 ccatgcaggc gcagcagggc cgcagcagcc tggtgtggct cttctcccag ctctaccttt 1620 actccttcat cagcctcttc atctacatgg tgctcagcct cttcatcgcg ctcatcaccg 1680 gcgcctacga caccatcaag catcccggcg gcgcaggcgc agaggagagc gagctgcagg 1740 cctacatcgc acagtgccag gacagcccca cctccggcaa gttccgccgc gggagcggct 1800 cggcctgcag ccttctctgc tgctgcggaa gggacccctc ggaggagcat tcgctgctgg 1860 tgaattgatt cgacctgact gccgttggac cgtaggccct ggactgcaga gacccccgcc 1920 cccgaccccg cttatttatt tgtagggttt gcttttaagg atcggctccc tgtcgcgccc 1980 gaggagggcc tggacctttc gtgtcggacc cttggggggcg gggagactgg gtggggaggg 2040 tgttgaataa a 2051 <210> 17 <211> 663 <212> PRT <213> Homo sapiens <400> 17 Met Thr Gly Path Arg Path Ser Path Path Glu Gln Arg Arg Path Gly Arg 1 5 10 15 Ser Gly Gln Ala Arg Ala Ala Glu Arg Ala Ala Gly Met Ser Gly Ala 20 25 30 Gly Arg Ala Leu Ala Ala Leu Leu Leu Ala Ser Val Leu Ser Ala 35 40 45 Ala Leu Leu Ala Pro Gly Gly Ser Ser Gly Arg Asp Ala Gln Ala Ala 50 55 60 Pro Pro Arg Asp Leu Asp Lys Lys Arg His Ala Glu Leu Lys Met Asp 65 70 75 80 Gln Ala Leu Leu Leu Ile His Asn Glu Leu Leu Trp Thr Asn Leu Thr 85 90 95 Val Tyr Trp Lys Ser Glu Cys Cys Tyr His Cys Leu Phe Gln Val Leu 100 105 110 Val Asn Val Pro Gln Ser Pro Lys Ala Gly Lys Pro Ser Ala Ala Ala 115 120 125 Ala Ser Val Ser Thr Gln His Gly Ser Ile Leu Gln Leu Asn Asp Thr 130 135 140 Leu Glu Glu Lys Glu Val Cys Arg Leu Glu Tyr Arg Phe Gly Glu Phe 145 150 155 160 Gly Asn Tyr Ser Leu Leu Val Lys Asn Ile His Asn Gly Val Ser Glu 165 170 175 Ile Ala Cys Asp Leu Ala Val Asn Glu Asp Pro Val Asp Ser Asn Leu 180 185 190 Pro Val Ser Ile Ala Phe Leu Ile Gly Leu Ala Val Ile Ile Val Ile 195 200 205 Ser Phe Leu Arg Leu Leu Leu Ser Leu Asp Asp Phe Asn Asn Trp Ile 210 215 220 Ser Lys Ala Ile Ser Ser Arg Glu Thr Asp Arg Leu Ile Asn Ser Glu 225 230 235 240 Leu Gly Ser Pro Ser Arg Thr Asp Pro Leu Asp Gly Asp Val Gln Pro 245 250 255 Ala Thr Trp Arg Leu Ser Ala Leu Pro Pro Arg Leu Arg Ser Val Asp 260 265 270 Thr Phe Arg Gly Ile Ala Leu Ile Leu Met Val Phe Val Asn Tyr Gly 275 280 285 Gly Gly Lys Tyr Trp Tyr Phe Lys His Ala Ser Trp Asn Gly Leu Thr 290 295 300 Val Ala Asp Leu Val Phe Pro Trp Phe Val Phe Ile Met Gly Ser Ser 305 310 315 320 Ile Phe Leu Ser Met Thr Ser Ile Leu Gln Arg Gly Cys Ser Lys Phe 325 330 335 Arg Leu Leu Gly Lys Ile Ala Trp Arg Ser Phe Leu Leu Ile Cys Ile 340 345 350 Gly Ile Ile Ile Val Asn Pro Asn Tyr Cys Leu Gly Pro Leu Ser Trp 355 360 365 Asp Lys Val Arg Ile Pro Gly Val Leu Gln Arg Leu Gly Val Thr Tyr 370 375 380 Phe Val Val Ala Val Leu Glu Leu Leu Phe Ala Lys Pro Val Pro Glu 385 390 395 400 His Cys Ala Ser Glu Arg Ser Cys Leu Ser Leu Arg Asp Ile Thr Ser 405 410 415 Ser Trp Pro Gln Trp Leu Leu Ile Leu Val Leu Glu Gly Leu Trp Leu 420 425 430 Gly Leu Thr Phe Leu Leu Pro Val Pro Gly Cys Pro Thr Gly Tyr Leu 435 440 445 Gly Pro Gly Gly Ile Gly Asp Phe Gly Lys Tyr Pro Asn Cys Thr Gly 450 455 460 Gly Ala Ala Gly Tyr Ile Asp Arg Leu Leu Leu Gly Asp Asp His Leu 465 470 475 480 Tyr Gln His Pro Ser Ser Ala Val Leu Tyr His Thr Glu Val Ala Tyr 485 490 495 Asp Pro Glu Gly Ile Leu Gly Thr Ile Asn Ser Ile Val Met Ala Phe 500 505 510 Leu Gly Val Gln Ala Gly Lys Ile Leu Leu Tyr Tyr Lys Ala Arg Thr 515 520 525 Lys Asp Ile Leu Ile Arg Phe Thr Ala Trp Cys Cys Ile Leu Gly Leu 530 535 540 Ile Ser Val Ala Leu Thr Lys Val Ser Glu Asn Glu Gly Phe Ile Pro 545 550 555 560 Val Asn Lys Asn Leu Trp Ser Leu Ser Tyr Val Thr Thr Leu Ser Ser 565,570,575 Phe Ala Phe Phe Ile Leu Leu Val Leu Tyr Pro Val Val Asp Val Lys 580,585,590 Gly Leu Trp Thr Gly Thr Pro Phe Phe Tyr Pro Gly Met Asn Ser Ile 595,600,605 Leu Val Tyr Val Gly His Glu Val Phe Glu Asn Tyr Phe Pro Phe Gln 610 615 620 Trp Lys Leu Lys Asp Asn Gln Ser His Lys Glu His Leu Thr Gln Asn 625 630 635 640 There Are Thr There Are Trp Trp There Are Two Tyr There Are Two Tyr Arg 645,650,655 Lys Lys Phe Trp Lys Lyle 660 <210> 18 <211> 5228 <212> DNA <213> homo sapiens <400> 18 agggcggggc gcagcggggca ggcaagggcg gccgagcggg cggcggggcat gagcggggcg 60 ggcagggcgc tggccgcgct gctgctggcc gcgtccgtgc tgagcgccgc gctgctggcc 120 cccggcggct cttcggggcg cgatgcccag gccgcgccgc cacgagactt agacaaaaaa 180 agacatgcag agctgaagat ggatcaggct ttgctactca tccataatga acttctctgg 240 accaacttga ccgtctactg gaaatctgaa tgctgtttatc actgcttgtt tcaggttctg 300 gtaaacgttc ctcagagtcc aaaagcaggg aagcctagtg ctgcagctgc ctctgtcagc 360 acccagcacg gatctatcct gcagctgaac gacaccttgg aagagaaaga agtttgtagg 420 ttggaataca gatttggaga atttggaaac tattctctct tggtaaagaa catccataat 480 ggagttagtg aaattgcctg tgacctggct gtgaacgagg atccagttga tagtaacctt 540 cctgtgagca ttgcattcct tattggtctt gctgtcatca ttgtgatatc ctttctgagg 600 660 actgatcgcc tcatcaattc tgagctggga tctcccagca ggacagaccc tctcgatggt 720 gatgttcagc cagcaacgtg gcgtctatct gccctgccgc cccgctctccg cagcgtggac 780 accttcaggg ggattgctct tatactcatg gtctttgtca attatggagg aggaaaatat 840 tggtacttca aacatgcaag ttggaatggg ctgacagtgg ctgacctcgt gttcccgtgg 900 tttgtattta ttatgggatc ttccattttt ctatcgatga cttctatact gcaacggggg 960 tgttcaaaat tcagattgct ggggaagatt gcatggagga gtttcctgtt aatctgcata 1020 ggaattatca ttgtgaatcc caattattgc cttggtccat tgtcttggga caaggtgcgc 1080 attcctggtg tgctgcagcg attgggagtg acatactttg tggttgctgt gttggagctc 1140 ctctttgcta aacctgtgcc tgaacattgt gcctcggaga ggagctgcct ttctcttcga 1200 gacatcacgt ccagctggcc ccagtggctg ctcatcctgg tgctggaagg cctgtggctg 1260 ggcttgacat tcctcctgcc agtccctggg tgccctactg gttatcttgg tcctgggggc 1320 attggagatt ttggcaagta tccaaattgc actggaggag ctgcaggcta catcgaccgc 1380 ctgctgctgg gagacgatca cctttaccag cacccatctt ctgctgtact ttaccacacc 1440 gaggtggcct atgaccccga gggcatcctg ggcaccatca actccatcgt gatggccttt 1500 ttaggagttc aggcaggaaa aatactattg tattacaagg ctcggaccaa agacatcctg 1560 attcgattca ctgcttggtg ttgtattctt gggctcattt ctgttgctct gacgaaggtt 1620 tctgaaaatg aaggctttat tccagtaaac aaaaatctct ggtccctttc gtatgtcact 1680 acgctcagtt cttttgcctt cttcatcctg ctggtcctgt acccagttgt ggatgtgaag 1740 gggctgtgga caggaacccc attcttttat ccaggaatga attccattct ggtatatgtc 1800 ggccacgagg tgtttgagaa ctacttcccc tttcagtgga agctgaagga caaccagtcc 1860 cacaaggagc acctgactca gaacatcgtc gccactgccc tctgggtgct cattgcctac 1920 atcctctata gaaagaagat tttttggaaa atctgatggc tcccactgag atgtgctgct 1980 ggaagactct agtaggcctg cagggaggac tgaagcagcc tttgttaaag ggaagcattc 2040 attaggaaat tgactggctg cgtgtttaca gactctgggg gaagacactg atgtcctcaa 2100 actggttaac tgtgacacgg ctcgccagaa ctctgcctgt ctatttgtga cttacagatt 2160 tgaaatgtaa ttgtcttttt tcctccatct tctgtggaaa tggatgtctt tggaacttca 2220 ttccgaggag ataagcttta actttccaaa agggaattgc catgggtgtt tttcttctgt 2280 ggtgagtgaa acaatctgag gtctggttct tgctgacctt gttgccctgc aaacttcctt 2340 tccacgtgta cgcgcacacc aacacgaaat gccatcactc ctactgcggc tgctatgaag 2400 cttactggtt gtgatgtgtt ataatttagt ctgttttttt gattgaatgc agtttaatgt 2460 ttccagaaag ccaaagtaat tttcttttca gatatgcaag gctttggtgg gtccaaaaaa 2520 tgtctatcac aagccatttt ttccttttcc tctctcgaaa agttaaaata tctatgtgtt 2580 attcccaaac cctcttacct atgtatctgc ctgtctgtcc atcatcttc ttcctcccta 2640 tctctgtgta tctggatggc agccgctgcc caggggagtg gctgtgggga gggcaggtac 2700 tgtctttgcc tgtgggtcca gctgagccat ccctgctggg tgatgctggg caagaccctt 2760 ggcccgtctg ggccttggct tcctcacttg tgaaatgagc gggaagatga ctctcagttc 2820 cttccacctc ttagacatgg tgaggtaaca gacatcaaaa gctttctga aatcttcaga 2880 agaaatagtt ccattacaga aaactcttca aaataatag tagtgaaaac ttttaaaaac 2940 tctcattgga gtaagtcttt tcaagatgat cctccacaat ggaggcagcg ttcctacttg 3000 tcatcacaca gctgaagaca ttgtttctta ggtgtgaaat cggggacaaa ggacaaacag 3060 agacacacgg cattgttcat gggaggcatc gtcaccctcc tgggtgttct gtgggaattt 3120 cctgtgtgag gaaaacgtgg ccacagggtt gtgctgtacc cacccttccc cggcgagatg 3180 gccctcggcc tgtgccgctg cttccaccct cgccactcca tggcagcttt tggtctgttt 3240 ccggctctgc cctctgccct gaactctcat ccggcttgta cctgcctgct ggacccctcc 3300 acctggaggc cagcccatgt ctcaggccca gccctagcct cttctcctca aattctaagt 3360 gttttctctt taggtttccc tggctttgtg aatggatcat gtgtctctag gtataaacct 3420 gacatcatct ttccacccgg cttacctcca ccagatctcc ccagttctgt ctccatcttc 3480 tacctgcagc tgctctgttc tcatggtcac tgctgcatca ctgagtctgg acccttgtta 3540 tcattttcaa actggcctcc ttccctcgtt ccccacttct taaagtcacc tgtccattgc 3600 caccagatta agctttctcc agccagatca cctctctctg agaaacctcc attgacatgg 3660 aaacaccatt gtctggcaca catactcaca tactcacctt cccgtcttga tccccacaca 3720 tctttccagc ctcccctccc actccactcc ctgctctctc ctccacctcc ccatcctctt 3780 gtctcccctc ccctctgaat ccagcccagc ggggcttctc ctgcctccat cacatcacag 3840 aagtacctcc tgcttctggt tttaattaga gccttccccg attacatttt cctctgaatt 3900 ttttcctatc tacatttgat ctgtcatgtt taaaccccct acttctaagg gaacttctct 3960 aatctcttat cctcatcccc aaatagtgtt ttcttctctct gggttcttat aatgttggta 4020 tcaatctcac agcatttagt gcttcctgcc tggtgtgaca gttacctgtg tgcatgtgca 4080 atttctaatt tcccacgcta gactgtgagc ttcctaaggc aagaatcatg ccttgttggt 4140 ttctgtattc ctcatggtgc caaacacagt gccttctaca ttgcaggcgc tgaataaaca 4200 tttttaaagc aaaatgatgt ggatttttaa aataaatatt taagtgctgg taagatgagc 4260 atgtatccgg ggtgcccatg aaatgttctt ggggccgtgt ggggacagtc gtcattcctc 4320 ctcctgccac ccttttcttt cagtgagtca ctgtggatgg tcccagctgt gtcatcccaa 4380 agttcagcag ggaaagctga gctggggcctc tccaggtgag ttttctagaa gcatttctca 4440 aactgtgggt tacatcaact tgggtgtctt gagctgtaag gaaggaactc cggagtcagc 4500 tgggctacag gggagcttct ctaagtcctg cgggaggcca gacccagcct gagcttgctg 4560 ttagctagcg gaggcagctg ctggtggccc aggtgctcga caccaggcat cccctctcct 4620 cccacgaagg gtgtgccata atccccttca acaggaaatg cttcccagaa gcctctcagc 4680 agcctcccct cctgtcctat cagctagaag cgcctcgctt gtcccaagac cagcagggac 4740 agggaactgt ccgagcccgt ggctgtgtgg aggaagcga cccccagcac aagattggtt 4800 tcctttggga agggaagagg gagtgtgttg gggtaagggg tagagcagag gaatggtcag 4860 ggggcaacaa ccgctgacag ctgcaacagg tgcatggcat ctcacaggga ggcagggagg 4920 tgcgagctcc tagtaatgg agcaaaaaaaaa ttctattctg tagaatgggg agaaaaatg 4980 tgacatttta atttttttt gcatttatt tcctaattcc tacttaaagt gaatatactg 5040 ccgctgtaga tcataaaaatg tatcttttcc atggccaaca aggggcatct tttataaatg 5100 cataataacc cagtttgtat caaagggtat cgacttaagt gaaatttcaa catgctgtta 5160 ctttttcctt ttaatgtaat tctgttttcc aaaataatgg gggagaaaa tggaaaaaaa 5220 aaaaaaaa 5228 <210> 19 <211> 1278 <212> PRT <213> homo sapiens <400> 19 Met Thr Ala Arg Gly Leu Ala Leu Gly Leu Leu Leu Leu Leu Leu Cys 1 5 10 15 Pro Ala Gln Val Phe Ser Gln Ser Cys Val Trp Tyr Gly Glu Cys Gly 20 25 30 Ile Ala Tyr Gly Asp Lys Arg Tyr Asn Cys Glu Tyr Ser Gly Pro Pro 35 40 45 Lys Pro Leu Pro Lys Asp Gly Tyr Asp Leu Val Gln Glu Leu Cys Pro 50 55 60 Gly Phe Phe Phe Gly Asn Val Ser Leu Cys Cys Asp Val Arg Gln Leu 65 70 75 80 Gln Thr Leu Lys Asp Asn Leu Gln Leu Pro Leu Gln Phe Leu Ser Arg 85 90 95 Cys Pro Ser Cys Phe Tyr Asn Leu Leu Asn Leu Phe Cys Glu Leu Thr 100 105 110 Cys Ser Pro Arg Gln Ser Gln Phe Leu Asn Val Thr Ala Thr Glu Asp 115 120 125 Tyr Val Asp Pro Val Thr Asn Gln Thr Lys Thr Asn Val Lys Glu Leu 130 135 140 Gln Tyr Tyr Val Gly Gln Ser Phe Ala Asn Ala Met Tyr Asn Ala Cys 145 150 155 160 Arg Asp Val Glu Ala Pro Ser Ser Asn Asp Lys Ala Leu Gly Leu Leu 165 170 175 Cys Gly Lys Asp Ala Asp Ala Cys Asn Ala Thr Asn Trp Ile Glu Tyr 180 185 190 Met Phe Asn Lys Asp Asn Gly Gln Ala Pro Phe Thr Ile Thr Pro Val 195 200 205 Phe Ser Asp Phe Pro Val His Gly Met Glu Pro Met Asn Asn Ala Thr 210 215 220 Lys Gly Cys Asp Glu Ser Val Asp Glu Val Thr Ala Pro Cys Ser Cys 225 230 235 240 Gln Asp Cys Ser Ile Val Cys Gly Pro Lys Pro Gln Pro Pro Pro Pro 245 250 255 Pro Ala Pro Trp Thr Ile Leu Gly Leu Asp Ala Met Tyr Val Ile Met 260 265 270 Trp Ile Thr Tyr Met Ala Phe Leu Leu Val Phe Phe Gly Ala Phe Phe 275 280 285 Ala Val Trp Cys Tyr Arg Lys Arg Tyr Phe Val Ser Glu Tyr Thr Pro 290 295 300 Ile Asp Ser Asn Ile Ala Phe Ser Val Asn Ala Ser Asp Lys Gly Glu 305 310 315 320 Ala Ser Cys Cys Asp Pro Val Ser Ala Ala Phe Glu Gly Cys Leu Arg 325 330 335 Arg Leu Phe Thr Arg Trp Gly Ser Phe Cys Val Arg Asn Pro Gly Cys 340 345 350 Val Ile Phe Phe Ser Leu Val Phe Ile Thr Ala Cys Ser Ser Gly Leu 355 360 365 Val Phe Val Arg Val Thr Thr Asn Pro Val Asp Leu Trp Ser Ala Pro 370 375 380 Ser Ser Gln Ala Arg Leu Glu Lys Glu Tyr Phe Asp Gln His Phe Gly 385 390 395 400 Pro Phe Phe Arg Thr Glu Gln Leu Ile Ile Arg Ala Pro Leu Thr Asp 405 410 415 Lys His Ile Tyr Gln Pro Tyr Pro Ser Gly Ala Asp Val Pro Phe Gly 420 425 430 Pro Pro Leu Asp Ile Gln Ile Leu His Gln Val Leu Asp Leu Gln Ile 435 440 445 Ala Ile Glu Asn Ile Thr Ala Ser Tyr Asp Asn Glu Thr Val Thr Leu 450 455 460 Gln Asp Ile Cys Leu Ala Pro Leu Ser Pro Tyr Asn Thr Asn Cys Thr 465 470 475 480 Ile Leu Ser Val Leu Asn Tyr Phe Gln Asn Ser His Ser Val Leu Asp 485 490 495 His Lys Lys Gly Asp Asp Phe Phe Val Tyr Ala Asp Tyr His Thr His 500 505 510 Phe Leu Tyr Cys Val Arg Ala Pro Ala Ser Leu Asn Asp Thr Ser Leu 515 520 525 Leu His Asp Pro Cys Leu Gly Thr Phe Gly Gly Pro Val Phe Pro Trp 530 535 540 Leu Val Leu Gly Gly Tyr Asp Asp Gln Asn Tyr Asn Asn Ala Thr Ala 545 550 555 560 Leu Val Ile Thr Phe Pro Val Asn Asn Tyr Tyr Asn Asp Thr Glu Lys 565 570 575 Leu Gln Arg Ala Gln Ala Trp Glu Lys Glu Phe Ile Asn Phe Val Lys 580 585 590 Asn Tyr Lys Asn Pro Asn Leu Thr Ile Ser Phe Thr Ala Glu Arg Ser 595 600 605 Ile Glu Asp Glu Leu Asn Arg Glu Ser Asp Ser Asp Val Phe Thr Val 610 615 620 Val Ile Ser Tyr Ala Ile Met Phe Leu Tyr Ile Ser Leu Ala Leu Gly 625 630 635 640 His Ile Lys Ser Cys Arg Arg Leu Leu Val Asp Ser Lys Val Ser Leu 645 650 655 Gly Ile Ala Gly Ile Leu Ile Val Leu Ser Ser Val Ala Cys Ser Leu 660 665 670 Gly Val Phe Ser Tyr Ile Gly Leu Pro Leu Thr Leu Ile Val Ile Glu 675 680 685 Val Ile Pro Phe Leu Val Leu Ala Val Gly Val Asp Asn Ile Phe Ile 690 695 700 Leu Val Gln Ala Tyr Gln Arg Asp Glu Arg Leu Gln Gly Glu Thr Leu 705 710 715 720 Asp Gln Gln Leu Gly Arg Val Leu Gly Glu Val Ala Pro Ser Met Phe 725 730 735 Leu Ser Ser Phe Ser Glu Thr Val Ala Phe Phe Leu Gly Ala Leu Ser 740 745 750 Val Met Pro Ala Val His Thr Phe Ser Leu Phe Ala Gly Leu Ala Val 755 760 765 Phe Ile Asp Phe Leu Leu Gln Ile Thr Cys Phe Val Ser Leu Leu Gly 770 775 780 Leu Asp Ile Lys Arg Gln Glu Lys Asn Arg Leu Asp Ile Phe Cys Cys 785 790 795 800 Val Arg Gly Ala Glu Asp Gly Thr Ser Val Gln Ala Ser Glu Ser Cys 805 810 815 Leu Phe Arg Phe Phe Lys Asn Ser Tyr Ser Pro Leu Leu Leu Lys Asp 820 825 830 Trp Met Arg Pro Ile Val Ile Ala Ile Phe Val Gly Val Leu Ser Phe 835 840 845 Ser Ile Ala Val Leu Asn Lys Val Asp Ile Gly Leu Asp Gln Ser Leu 850 855 860 Ser Met Pro Asp Asp Ser Tyr Met Val Asp Tyr Phe Lys Ser Ile Ser 865 870 875 880 Gln Tyr Leu His Ala Gly Pro Pro Val Tyr Phe Val Leu Glu Glu Gly 885 890 895 His Asp Tyr Thr Ser Ser Lys Gly Gln Asn Met Val Cys Gly Gly Met 900 905 910 Gly Cys Asn Asn Asp Ser Leu Val Gln Gln Ile Phe Asn Ala Ala Gln 915 920 925 Leu Asp Asn Tyr Thr Arg Ile Gly Phe Ala Pro Ser Ser Trp Ile Asp 930 935 940 Asp Tyr Phe Asp Trp Val Lys Pro Gln Ser Ser Cys Cys Arg Val Asp 945 950 955 960 Asn Ile Thr Asp Gln Phe Cys Asn Ala Ser Val Val Asp Pro Ala Cys 965 970 975 Val Arg Cys Arg Pro Leu Thr Pro Glu Gly Lys Gln Arg Pro Gln Gly 980 985 990 Gly Asp Phe Met Arg Phe Leu Pro Met Phe Leu Ser Asp Asn Pro Asn 995 1000 1005 Pro Lys Cys Gly Lys Gly Gly His Ala Ala Tyr Ser Ser Ala Val 1010 1015 1020 Asn Ile Leu Leu Gly His Gly Thr Arg Val Gly Ala Thr Tyr Phe 1025 1030 1035 Met Thr Tyr His Thr Val Leu Gln Thr Ser Ala Asp Phe Ile Asp 1040 1045 1050 Ala Leu Lys Lys Ala Arg Leu Ile Ala Ser Asn Val Thr Glu Thr 1055 1060 1065 Met Gly Ile Asn Gly Ser Ala Tyr Arg Val Phe Pro Tyr Ser Val 1070 1075 1080 Phe Tyr Val Phe Tyr Glu Gln Tyr Leu Thr Ile Ile Asp Asp Thr 1085 1090 1095 Ile Phe Asn Leu Gly Val Ser Leu Gly Ala Ile Phe Leu Val Thr 1100 1105 1110 Met Val Leu Leu Gly Cys Glu Leu Trp Ser Ala Val Ile Met Cys 1115 1120 1125 Ala Thr Ile Ala Met Val Leu Val Asn Met Phe Gly Val Met Trp 1130 1135 1140 Leu Trp Gly Ile Ser Leu Asn Ala Val Ser Leu Val Asn Leu Val 1145 1150 1155 Met Ser Cys Gly Ile Ser Val Glu Phe Cys Ser His Ile Thr Arg 1160 1165 1170 Ala Phe Thr Val Ser Met Lys Gly Ser Arg Val Glu Arg Ala Glu 1175 1180 1185 Glu Ala Leu Ala His Met Gly Ser Ser Val Phe Ser Gly Ile Thr 1190 1195 1200 Leu Thr Lys Phe Gly Gly Ile Val Val Leu Ala Phe Ala Lys Ser 1205 1210 1215 Gln Ile Phe Gln Ile Phe Tyr Phe Arg Met Tyr Leu Ala Met Val 1220 1225 1230 Leu Leu Gly Ala Thr His Gly Leu Ile Phe Leu Pro Val Leu Leu 1235 1240 1245 Ser Tyr Ile Gly Pro Ser Val Asn Lys Ala Lys Ser Cys Ala Thr 1250 1255 1260 Glu Glu Arg Tyr Lys Gly Thr Glu Arg Glu Arg Leu Leu Asn Phe 1265 1270 1275 <210> 20 <211> 4673 <212> DNA <213> homo sapiens <400> 20 tttgctcctg ctcctccgct cctcctgcgc ggggtgctga aacagcccgg ggaagtagag 60 ccgcctccgg gggacccac cagccgaacg ccgccggcgt cagcagcctt gcgcggccac 120 agcatgaccg ctcgcggcct ggccttgc ctcctcctgc tgctactgtg tccagcgcag 180 gtgttttcac agtcctgtgt ttggtatgga gagtgtggaa ttgcatatgg ggacaagagg 240 tacaattgcg atattctgg cccaccaaaa ccattgccaa aggatggata tgacttagtg 300 caggaactct gtccaggatt cttctttggc aatgtcagtc tctgttgtga tgttcggcag 360 cttcagacac taaaagacaa cctgcagctg cctctacagt ttctgtccag atgtccatcc 420 tgtttttata acctactgaa cctgttttgt gagctgacat gtagccctcg acagagtcag 480 tttttgaatg ttacagctac tgaagattat gttgatcctg ttacaaacca gacgaaaaca 540 aatgtgaaag agttacaata ctacgtcgga cagagttttg ccaatgcaat gtacaatgcc 600 tgccgggatg tggaggcccc ctcaagtaat gacaaggccc tgggactcct gtgtgggaag 660 gacgctgacg cctgtaatgc caccaactgg attgaataca tgttcaataa ggacaatgga 720 caggcacctt ttaccatcac tcctgtgttt tcagattttc cagtccatgg gatggagccc 780 atgaacaatg ccaccaaagg ctgtgacgag tctgtggatg aggtcacagc accatgtagc 840 tgccaagact gctctattgt ctgtggcccc aagccccagc ccccacctcc tcctgctccc 900 tggacgatcc ttggcttgga cgccatgtat gtcatcatgt ggatcaccta catggcgttt 960 ttgcttgtgt tttttggagc attttttgca gtgtggtgct acagaaaacg gtattttgtc 1020 tccgagtaca ctcccatcga tagcaatata gctttttctg ttaatgcaag tgacaaagga 1080 gaggcgtcct gctgtgaccc tgtcagcgca gcatttgagg gctgcttgag gcggctgttc 1140 acacgctggg ggtctttctg cgtccgaaac cctggctgtg tcatttctt ctcgctggtc 1200 ttcattactg cgtgttcgtc aggcctggtg tttgtccggg tcacaaccaa tccagttgac 1260 ctctggtcag cccccagcag ccaggctcgc ctggaaaaag agtactttga ccagcacttt 1320 gggcctttct tccggacgga gcagctcatc atccgggccc ctctcactga caaacacatt 1380 taccagccat acccttcggg agctgatgta ccctttggac ctccgcttga catacagata 1440 ctgcaccagg ttcttgactt acaatagcc atcgaaaaca ttactgcctc ttatgacaat 1500 gagactgtga cacttcaaga catctgcttg gcccctcttt caccgtataa cacgaactgc 1560 accattttga gtgtgttaaa ttacttccag aacagccatt ccgtgctgga ccacaagaaa 1620 ggggacgact tctttgtgta tgccgattac cacacgcact ttctgtactg cgtacgggct 1680 cctgcctc tgaatgatac aagtttgctc catgaccctt gtctgggtac gtttggtgga 1740 ccagtgttcc cgtggcttgt gttgggaggc tatgatgatc aaaactacaa taacgccact 1800 gcccttgtga ttaccttccc tgtcaataat tactataatg atacagagaa gctccagagg 1860 gcccaggcct gggaaaaaga gtttattaat tttgtgaaaa actacaagaa tcccaatctg 1920 accatttcct tcactgctga acgaagtatt gaagatgaac taaatcgtga aagtgacagt 1980 gatgtcttca ccgttgtaat tagctatgcc atcatgtttc tatatatttc cctagccttg 2040 gggcacatca aaagctgtcg caggcttctg gtggattcga aggtctcact aggcatcgcg 2100 ggcatcttga tcgtgctgag ctcggtggct tgctccttgg gtgtcttcag ctacattggg 2160 ttgcccttga ccctcattgt gattgaagtc atcccgttcc tggtgctggc tgttggagtg 2220 gacaacatct tcattctggt gcaggcctac cagagagatg aacgtcttca aggggaaacc 2280 ctggatcagc agctgggcag ggtcctagga gaagtggctc ccagtatgtt cctgtcatcc 2340 tttctgaga ctgtagcatt tttcttagga gcattgtccg tgatgccagc cgtgcacacc 2400 ttctctctct ttgcgggatt ggcagtcttc attgactttc ttctgcagat tacctgtttc 2460 gtgagtctct tggggttaga cattaaacgt caagagaaaa atcggctaga catcttttgc 2520 tgtgtcagag gtgctgaaga tggaacaagc gtccaggcct cagagagctg tttgttcgc 2580 ttcttcaaaa actcctattc tccacttctg ctaaaggact ggatgagacc aattgtgata 2640 gcaatatttg tgggtgttct gtcattcagc atcgcagtcc tgaacaaagt agatattgga 2700 ttggatcagt ctctttcgat gccagatgac tcctacatgg tggattattt caaatccatc 2760 agtcagtacc tgcatgcggg tccgcctgtg tactttgtcc tggaggaagg gcacgactac 2820 acttcttcca aggggcagaa catggtgtgc ggcggcatgg gctgcaacaa tgattccctg 2880 gtgcagcaga tatttaacgc ggcgcagctg gacaactata cccgaatagg cttcgccccc 2940 tcgtcctgga tcgacgatta ttcgactgg gtgaagccac agtcgtcttg ctgtcgagtg 3000 gacaatatca ctgaccagtt ctgcaatgct tcagtggttg accctgcctg cgtcgctgc 3060 aggcctctga ctccggaagg caaacagagg cctcaggggg gagacttcat gagattcctg 3120 cccatgttcc ttcggataa ccctaacccc aagtgtggca aagggggaca tgctgcctat 3180 agttctgcag ttaacatcct ccttggccat ggcaccaggg tcggagccac gtacttcatg 3240 acctaccaca ccgtgctgca gacctctgct gactttattg acgctctga gaaagcccga cttatagcca gtaatgtcac cgaaaccatg ggcattaacg gcagtgccta ccgagtattt ccttacagtg tgttttatgt cttctacgaa cagtacctga ccatcattga cgacactatc ttcaacctcg gtgtgtccct gggcgcgata tttctggtga ccatggtcct cctgggctgt 3480 gagctctggt ctgcagtcat catgtgtgcc accatcgcca tggtcttggt caacatgttt 3540 ggagttatgt ggctctgggg catcagtctg aacgctgtat ccttggtcaa cctggtgatg 3600 agctgtggca tctccgtgga gttctgcagc cacataacca gagcgttcac ggtgagcatg aaaggcagcc gcgtggagcg cgcggagag gcacttgccc acatgggcag ctccgtgttc 3720. agtggatca cacttacaaa atttggaggg attgtggtgt tggcttttgc caatctcaa attttccaga tattctactt caggatgtat ttggccatgg tcttactggg agccactcac ggattaatat ttctccctgt cttactcagt tacatagggc catcagtaaa taaagccaaa agttgtgcca ctgaagagcg atacaaagga acagagcgcg aacggcttct aaatttctag ccctctcgca gggcatcctg actgaactgt gtctaagggt cggtcggttt accactggac 4020 gggtgctgca tcggcaaggc caagttgaac accggatggt gccaaccatc ggttgtttgg cagcagcttt gaacgtagcg cctgtgaact caggaatgca cagttgactt gggaagcagt attack ctggaggca ccacaggaca ctaaacttct cccagcctct tcaggaaga aacctcattc tttggcaagc aggaggtgac actagatggc tgtgaatgtg atccgctcac tgacactctg taaggccaa tcaatgcact gtctgtcctc tcctttttag gagtaagcca 4380. tcccacaagt tctataccat atttttagtg acagttgagg ttgtagatac actttataac attttatagt ttaagagct ttattaatgc aataattaatta ctttgtacac atttttatat aaaaaaacag caagtgattt cagaatgttg tagcctcat taggcttgg tctccaaaaa tctgtttga aaaagcaaca tgttcttcac agtgttcccc tagaagga gagatttaat 4620. tgccagttag atgtggcatg aaatgaggga caaagaaagc atctcgtag tgtgtctact gggttttaac ttattttct ttataaaat acacttgtttt cctaaaaaa aaa 4673 <210> 21 <211> 410 <212> PRT <213> homo sapiens <400> 21 Met Val Cys Phe Arg Leu Phe Pro Val Pro Gly Ser Gly Leu Val Leu 1 5 10 15 Val Cys Leu Val Leu Gly Wing Val Arg Ser Tyr Wing Leu Glu Leu Asn 20 25 30 Thr Asp Ser Glu Asn Has Thr Cys Leu Tyr Having Lys Trp Gln Met 35 40 45 Asn Phe Thr Val Arg Tyr Glu Thr Asn Lys Thr Tyr Lys Thr Val 50 55 60 Thr Ile Ser Asp His Gly Thr Val Thr Tyr Asn Gly Ser Ile Cys Gly 65 70 75 80 Asp Asp Gln Asn Gly Pro Lys Ile Ala Val Gln Phe Gly Pro Gly Phe 85 90 95 Ser Trp Ile Ala Asn Phe Thr Lys Ala Ala Ser Thr Tyr Ser Ile Asp 100 105 110 Ser Val Ser Phe Ser Tyr Asn Thr Gly Asp Asn Thr Thr Phe Pro Asp 115 120 125 Ala Glu Asp Lys Gly Ile Leu Thr Val Asp Glu Leu Leu Ala Ile Arg 130 135 140 Ile Pro Leu Asn Asp Leu Phe Arg Cys Asn Ser Leu Ser Thr Leu Glu 145 150 155 160 Lys Asn Asp Val Val Gln His Tyr Trp Asp Val Leu Val Gln Ala Phe 165 170 175 Val Gln Asn Gly Thr Val Ser Thr Asn Glu Phe Leu Cys Asp Lys Asp 180 185 190 Lys Thr Ser Thr Val Ala Pro Thr Ile His Thr Thr Val Pro Ser Pro 195 200 205 Thr Thr Thr Pro Thr Pro Lys Glu Lys Pro Glu Ala Gly Thr Tyr Ser 210 215 220 Val Asn Asn Gly Asn Asp Thr Cys Leu Leu Ala Thr Met Gly Leu Gln 225 230 235 240 Leu Asn Ile Thr Gln Asp Lys Val Ala Ser Val Ile Asn Ile Asn Pro 245 250 255 Asn Thr Thr His Ser Thr Gly Ser Cys Arg Ser His Thr Ala Leu Leu 260 265 270 Arg Leu Asn Ser Ser Thr Ile Lys Tyr Leu Asp Phe Val Phe Ala Val 275 280 285 Lys Asn Glu Asn Arg Phe Tyr Leu Lys Glu Val Asn Ile Ser Met Tyr 290 295 300 Leu Val Asn Gly Ser Val Phe Ser Ile Ala Asn Asn Asn Leu Ser Tyr 305 310 315 320 Trp Asp Ala Pro Leu Gly Ser Ser Tyr Met Cys Asn Lys Glu Gln Thr 325 330 335 Val Ser Val Ser Gly Ala Phe Gln Ile Asn Thr Phe Asp Leu Arg Val 340 345 350 Gln Pro Phe Asn Val Thr Gln Gly Lys Tyr Ser Thr Ala Gln Asp Cys 355 360 365 Ser Ala Asp Asp Asp Asn Phe Leu Val Pro Ile Ala Val Gly Ala Ala 370 375 380 Leu Ala Gly Val Leu Ile Leu Val Leu Leu Ala Tyr Phe Ile Gly Leu 385 390 395 400 Lys His His His Ala Gly Tyr Glu Gln Phe 405 410 <210> 22 <211> 1868 <212> DNA <213> homo sapiens <400> 22 ccgattcctg gcttttgcaa ggctgtggtc ggtggtcatc agtgctcttg acccaggtcc 60 agcgagcctt ttccctggtg ttgcagctgt tgttgtaccg ccgccgtcgc cgccgtcgcc 120 gcctgctctg cggggtcatg gtgtgcttcc gcctcttccc ggttccgggc tcagggctcg 180 ttctggtctg cctagtcctg ggagctgtgc ggtcttatgc attggaactt aatttgacag 240 attcagaaaa tgccacttgc ctttatgcaa aatggcagat gaatttcaca gtacgctatg 300 aaactacaaa taaaacttat aaaactgtaa ccatttcaga ccatggcact gtgacatata 360 atggaagcat ttgtggggat gatcagaatg gtcccaaaat agcagtgcag ttcggacctg 420 gcttttcctg gattgcgaat tttaccaagg cagcatctac ttattcaatt gacagcgtct 480 cattttccta caacactggt gataacacaa catttcctga tgctgaagat aaaggaattc 540 ttactgttga tgaacttttg gccatcagaa ttccattgaa tgaccttttt agatgcaata 600 gtttatcaac tttggaaaag aatgatgttg tccaacacta ctgggatgtt cttgtacaag 660 cttttgtcca aaatggcaca gtgagcacaa atgagttcct gtgtgataaa gacaaaactt 720 caacagtggc acccaccata cacaccactg tgccatctcc tactacaaca cctactccaa 780 aggaaaaacc agaagctgga acctattcag ttaataatgg caatgatact tgtctgctgg 840 ctaccatggg gctgcagctg aacatcactc aggataggt tgcttcagtt attacatca 900 acccatac aactcactcc acaggcagct gccgttctca cactgctcta cttagactca 960 atagcagcac cattaagtat ctagactttg tctttgctgt gaaaaatgaa aaccgatttt 1020 atctgaagga agtgaacatc agcatgtatt tggttaatgg ctccgttttc agcattgcaa 1080 aacaatct cagctactgg gatgcccccc tgggagttc ttatatgtgc aacaaagagc 1140 agactgtttc agtgtctgga gcatttcaga taaatacctt tgatctagg gttcagcctt 1200 tcaatgtgac ahaggaag tattctacag ctcaagactg cagtgcagat gacgacaact 1260 tccttgtgcc catagcggtg ggagctgcct tggcaggagt acttattcta gtgttgctgg 1320 cttattttat tggtctcaag caccatcatg ctggatatga gcaatttag aatctgcaac 1380 ctgattgatt ataaaaat acatgcaat aaagattt tcttacctct cagttgttga 1440 aacactttgc ttctttaaat tgatatgttg aaactttaat tctttatca atcccagcat 1500 tttgagatca gtctttatta aaaacctg ttctctttaa tcagcttaa atccaagtg 1560 tcatatttac tggtcctgga gandaacttg ttcaaagaa catcaacgtg caatgtttta 1620 aggtctatct taagaagccc tggccaattt ttgatcctaa ccttgaagta tgccttgaac 1680 ttattacat ggccattata agataaaat atgtagttgt gtcttaatgg aattaataaa 1740 tgtcattca ctactgtgt tctgttttca atgtataagg actatagtga tttaactca 1800 tcaatgtgcc ttgcataaa gttgattaaa taaatattga tgtgtataa atgcccatca 1860 gatatgct 1868 <210> 23 <211> 410 <212> PRT <213> homo sapiens <400> 23 Met Val Cys Phe Arg Leu Phe Pro Val Pro Gly Ser Gly Leu Val Leu 1 5 10 15 Val Cys Leu Val Leu Gly Wing Val Arg Ser Tyr Wing Leu Glu Leu Asn 20 25 30 Thr Asp Ser Glu Asn Has Thr Cys Leu Tyr Having Lys Trp Gln Met 35 40 45 Asn Phe Thr Val Arg Tyr Glu Thr Thr Asn Lys Thr Tyr Lys Thr Val 50 55 60 Thr Ile Ser Asp His Gly Thr Val Thr Tyr Asn Gly Ser Ile Cys Gly 65 70 75 80 Asp Asp Gln Asn Gly Pro Lys Ile Ala Val Gln Phe Gly Pro Gly Phe 85 90 95 Ser Trp Ile Ala Asn Phe Thr Lys Ala Ala Ser Thr Tyr Ser Ile Asp 100 105 110 Ser Val Ser Phe Ser Tyr Asn Thr Gly Asp Asn Thr Thr Phe Pro Asp 115 120 125 Ala Glu Asp Lys Gly Ile Leu Thr Val Asp Glu Leu Leu Ala Ile Arg 130 135 140 Ile Pro Leu Asn Asp Leu Phe Arg Cys Asn Ser Leu Ser Thr Leu Glu 145 150 155 160 Lys Asn Asp Val Val Gln His Tyr Trp Asp Val Leu Val Gln Ala Phe 165 170 175 Val Gln Asn Gly Thr Val Ser Thr Asn Glu Phe Leu Cys Asp Lys Asp 180 185 190 Lys Thr Ser Thr Val Ala Pro Thr Ile His Thr Thr Val Pro Ser Pro 195 200 205 Thr Thr Thr Pro Thr Pro Lys Glu Lys Pro Glu Ala Gly Thr Tyr Ser 210 215 220 Val Asn Asn Gly Asn Asp Thr Cys Leu Leu Ala Thr Met Gly Leu Gln 225 230 235 240 Leu Asn Ile Thr Gln Asp Lys Val Ala Ser Val Ile Asn Ile Asn Pro 245 250 255 Asn Thr Thr His Ser Thr Gly Ser Cys Arg Ser His Thr Ala Leu Leu 260 265 270 Arg Leu Asn Ser Ser Thr Ile Lys Tyr Leu Asp Phe Val Phe Ala Val 275 280 285 Lys Asn Glu Asn Arg Phe Tyr Leu Lys Glu Val Asn Ile Ser Met Tyr 290 295 300 Leu Val Asn Gly Ser Val Phe Ser Ile Ala Asn Asn Asn Leu Ser Tyr 305 310 315 320 Trp Asp Ala Pro Leu Gly Ser Ser Tyr Met Cys Asn Lys Glu Gln Thr 325 330 335 Val Ser Val Ser Gly Ala Phe Gln Ile Asn Thr Phe Asp Leu Arg Val 340 345 350 Gln Pro Phe Asn Val Thr Gln Gly Lys Tyr Ser Thr Ala Gln Glu Cys 355 360 365 Sister Leu Asp Asp Asp Thr Ile Leu Ile Pro Ile Ile Val Gly Ala Gly 370 375 380 Leu Ser Gly Leu Ile Ile Val Ile Val Ile Ala Tyr Val Ile Gly Arg 385,390,395,400 Arg Lys Ser Tyr Ala Gly Tyr Gln Thr Leu 405 410 <210> 24 <211> 4006 <212> DNA <213> homo sapiens <400> 24 ccgattcctg gcttttgcaa ggctgtggtc ggtggtcatc agtgctcttg acccaggtcc 60 agcgagcctt ttccctggtg ttgcagctgt tgttgtaccg ccgccgtcgc cgccgtcgcc 120 gcctgctctg cggggtcatg gtgtgcttc gcctcttccc ggttccgggc tcagggctcg 180 ttctggtctg cctagtcctg ggagctgtgc ggtcttatgc attggaactt aatttgacag 240 attcagaaaa tgccacttgc ctttatgcaa aatggcagat gaatttcaca gtacgctatg 300 aaactacaaa taaaacttat aaaactgtaa ccatttcaga ccatggcact gtgacatata 360 atggaagcat ttgtggggat gatcagaatg gtcccaaaat agcagtgcag ttcggacctg 420 gctttttcctg gattgcgaat tttaccagg cagcatctac ttcatt gagcgtct 480 cattttccta cacactggt gataacaca cattttcctga tgctgaat aaggaattc 540 ttactgttga tgaactttg gccatcagaa ttccattgaa tgacttttt agatgcaata 600 gtttatcaac tttggaaag aatgatgttg tccacacta ctgggatgtt ctgtacaag 660 cttttgtcca aaatggcaca gtgagcacaa atgagttcct gtgtgataaa gataaactt 720 caacagtggc acccaccata cacaccactg tgccatctcc tactacaaca cctactccaa 780 aggaaaaacc agaagctgga acctattcag ttaataatgg caatgatact tgtctgctgg 840 ctaccatggg gctgcagctg aacatcactc aggataggt tgcttcagtt attacatca 900 acccatac aactcactcc acaggcagct gccgttctca cactgctcta cttagactca 960 atagcagcac cattaagtat ctagactttg tctttgctgt gaaaaatgaa aaccgatttt 1020 atctgaagga agtgaacatc agcatgtatt tggttaatgg ctccgttttc agcattgcaa 1080 aacaatct cagctactgg gatgcccccc tgggagttc ttatatgtgc aacaaagagc 1140 agactgtttc agtgtctgga gcatttcaga taaatacctt tgatctaagg gttcagcctt 1200 tcaatgtgac acaaggaaag tattctacag cccaagagtg ttcgctggat gatgacacca 1260 ttctaatccc aattatagtt ggtgctggtc tttcaggctt gattatcgtt atagtgattg 1320 cttacgtaat tggcagaaga aaaagttatg ctggatatca gactctgtaa cactaatcaa 1380 tacgtgatct ctgttacaaa agaaaaaagc aagtacaagt tccaacatgc aatactggtc 1440 aacttaaggt atatttagtt gcagtccagc tctttagaat gggtggtatg ggggatttca 1500 aacttaaaca aaaaactatc aactacaaat tagttgcctg actttggttt ttccaaccaa 1560 ggaatttaaa actgttattt ttacagcaaa agatgtgcaa aatcactgga ttataagttc 1620 tattttactg tcttgaatta gtatttcagt gttttcattt tagacattca gactaaaaat 1680 acaccgttta gaaaaaacaa tttttgaaaa agagattttt tttccctgca ggtagttgag 1740 ttgaacaaca tgttctaccg tggatttgta cttgctcctt ttgctctttt tgtgtgtgtg 1800 tgtgtgtgtg tgtgtgtgtg tgtgattttt gtttgcaggt taacttagct actttggcat 1860 tgctgcatat tgaccttg agagatataa tagtagatt gacaggggc tgttattatt 1920 atgttcttag caataaatgc ttttctaatg ccttttgaat acatttgtat ttatgtggct 1980 gtaatgacaa aagatacaa agctttttaa aatttagagt aggtattaat cttattgtttt 2040 aatcttttt ttaaaaaaac tggatatttc aatcttta attgcaat atagactat 2100 tccaactggg cattcaatc catttttag gtgctttaga gatatgct tgccagtgcc 2160 aattgagggc attagtactt tgtgctcata aattggcctc tgtagcagt actaaaatta 2220 atgcagattt ctctttagcc ttccacatt tcttgttgat atgcagatt tttattt 2280 tctttttctt aagaatgcc agtgtgtcct agaactaga taacgaagtg cacttacact 2340 tataaaataa cttgcatcta gggctggggt gcggctcac gcctgtaatc ccagcacttt 2400 gggaggccga agtgggtgga tggtgagg ccaggagttt gagaccagcc tggcacat 2460 ggtgaaaccc catcttac agaaatacaa aaaattagct gggcatggtg gtgggcgcct 2520 gtaatcccag ttactcggga ggctgaggca ggagaatcac ttgaacccgg gaggcagagg 2580 ttgcggtgag ccaagagcgc accattgcac tccagccttg ggcgacaaaa acgaaactcc atcttcaaaa caaacaaaaa caaacaaaac aaacaaaac aacttgcatc ttaaccaaaaa gtcttggttt tatcttaatc cattaaaagt tggtcttgtt tccagcttgc attgattgct acaacatcac taatttggct ttcacattta aatggttctg tgctaatcaa aactttcgtt gttattattc gttatggtag aatcattttt aattcacgtg ctttgtgttc agttttgtgg tctgagat gtaccaattg tcaaattacc gtgtaccacc taatgtttat aggagaaagc aaaatacatc agcttggtag ttaacacatc aaatatttct tgctgcttct aggagaactt ttttggtgtg tgttggaatg gctgagcaaa tattaaaatt gttaatatgc agccatatat ggaaggttcc tgtggggttg ttttttcgtg tttttttttt ttgtggtggg attatgtgcc 3120 tcccattcac tagaaaatga gaaaattgtc tgggttccaa aatattgaca ttgaatggat caatacacac acacagacat fathers fathers fathers agttgcatgc ctagcatggg ctagcatggg ctagcatggg ctagcatggg ctagcatcg tcacttaaat ttgttctttg tttagcctga aaacctttat ggctcaagat cagattcctg 3360 actaacccct ctcttagagc tacagcgagc tgcattacca gcttaaaaca cttcttaggg 3420 attaaatata gatgtaattt ttcaaaatcg tttttaattt aaactgtgtt ttagtgtaaa 3480 attgttaacc ttgtaagatg gataatgtgt ataagaatgt aggccttaac tatttcacat 3540 gagtcaaaac aaagcagctt taaaaaaata attggaagca caatgcatgg cactgactga 3600 atgctgttaa tatttctaaa agtttctaca ttcagattat atgcctgatt catagtaaaa 3660 tacctctaat aaacactgtt ttatagaaaa cctgacttca gtgaatattt ttgtatttta 3720 catgggccag tttatatact gctatttaca ctattatttc ctatagctac atgttctttg 3780 taccttttgt agttttattt gtattactag attcatacct tgatggtaac gctctatctg 3840 gttttgggtg tttttcatgt tttagcattt gtataaagaa actggtccat gtaaatactt 3900 tccatgtttt ttcttcaaat gtttaaacca ctagttgatg tatggtatct ttagatattt 3960 gcctgtctgt ttgctcaaaa ttgcttctaa aacaataaag attctt 4006 <210> 25 <211> 411 <212> PRT <213> homo sapiens <400> 25 Met Val Cys Phe Arg Leu Phe Pro Val Pro Gly Ser Gly Leu Val Leu 1 5 10 15 Val Cys Leu Val Leu Gly Ala Val Arg Ser Tyr Ala Leu Glu Leu Asn 20 25 30 Leu Thr Asp Ser Glu Asn Ala Thr Cys Leu Tyr Ala Lys Trp Gln Met 35 40 45 Asn Phe Thr Val Arg Tyr Glu Thr Thr Asn Lys Thr Tyr Lys Thr Val 50 55 60 Thr Ile Ser Asp His Gly Thr Val Thr Tyr Asn Gly Ser Ile Cys Gly 65 70 75 80 Asp Asp Gln Asn Gly Pro Lys Ile Ala Val Gln Phe Gly Pro Gly Phe 85 90 95 Ser Trp Ile Ala Asn Phe Thr Lys Ala Ala Ser Thr Tyr Ser Ile Asp 100 105 110 Ser Val Ser Phe Ser Tyr Asn Thr Gly Asp Asn Thr Thr Phe Pro Asp 115 120 125 Ala Glu Asp Lys Gly Ile Leu Thr Val Asp Glu Leu Leu Ala Ile Arg 130 135 140 Ile Pro Leu Asn Asp Leu Phe Arg Cys Asn Ser Leu Ser Thr Leu Glu 145 150 155 160 Lys Asn Asp Val Val Gln His Tyr Trp Asp Val Leu Val Gln Ala Phe 165 170 175 Val Gln Asn Gly Thr Val Ser Thr Asn Glu Phe Leu Cys Asp Lys Asp 180 185 190 Lys Thr Ser Thr Val Ala Pro Thr Ile His Thr Thr Val Pro Ser Pro 195 200 205 Thr Thr Thr Pro Thr Pro Lys Glu Lys Pro Glu Ala Gly Thr Tyr Ser 210 215 220 Val Asn Asn Gly Asn Asp Thr Cys Leu Leu Ala Thr Met Gly Leu Gln 225 230 235 240 Leu Asn Ile Thr Gln Asp Lys Val Ala Ser Val Ile Asn Ile Asn Pro 245 250 255 Asn Thr Thr His Ser Thr Gly Ser Cys Arg Ser His Thr Ala Leu Leu 260 265 270 Arg Leu Asn Ser Ser Thr Ile Lys Tyr Leu Asp Phe Val Phe Ala Val 275 280 285 Lys Asn Glu Asn Arg Phe Tyr Leu Lys Glu Val Asn Ile Ser Met Tyr 290 295 300 Leu Val Asn Gly Ser Val Phe Ser Ile Ala Asn Asn Asn Leu Ser Tyr 305 310 315 320 Trp Asp Ala Pro Leu Gly Ser Ser Tyr Met Cys Asn Lys Glu Gln Thr 325 330 335 Val Ser Val Ser Gly Ala Phe Gln Ile Asn Thr Phe Asp Leu Arg Val 340 345 350 Gln Pro Phe Asn Val Thr Gln Gly Lys Tyr Ser Thr Ala Glu Glu Cys 355 360 365 Ser Ala Asp Ser Asp Leu Asn Phe Leu Ile Pro Val Ala Val Gly Val 370 375 380 Only Leu Gly Leu Ile Ile Val Val Where I Will Be Tyr With Gly 385 390 395 400 Arg Arg Lys Ser Arg Thr Gly Tyr Gln Ser Val 405 410 <210> 26 <211> 1236 <212> DNA <213> homo sapiens <400> 26 atggtgtgct tccgctctt cccggttccg ggctcaggc tcgttctggt ctgcctagtc 60 ctgggagctg tgcggtctta tgcattggaa cttaatttga cagattcaga aaatgccact 120 tgcctttatg caaaatggca gatgaatttc acagtacgct atgaaactac aaataaaact 180 tataaaactg taaccatttc agaccatggc actgtgacat ataatggaag catttgtggg 240 gatgatcaga atggtcccaa aatagcagtg cagttcggac ctggcttttc ctggattgcg 300 aattttacca aggcagcatc tacttattca attgacagcg tctcattttc ctacaacact 360 ggtgataaca caacatttcc tgatgctgaa gataaaggaa ttcttactgt tgatgaactt 420 ttggccatca gaattccatt gaatgacctt tttagatgca atagtttatc aactttggaa 480 aagaatgatg ttgtccaaca ctactgggat gttcttgtac aagcttttgt ccaaaatggc 540 acagtgagca caaatgagtt cctgtgtgat aaagacaaaa cttcaacagt ggcacccacc 600 atacacacca ctgtgccatc tcctactaca acacctactc caaaggaaaa accagaagct 660 ggaacctatt cagttaataa tggcaatgat acttgtctgc tggctaccat ggggctgcag 720 ctgaacatca ctcaggataa ggttgcttca gttattaaca tcaaccccaa tacaactcac 780 tccacaggca gctgccgttc tcacactgct ctacttagac tcaatagcag caccattaag 840 tatctagact ttgtctttgc tgtgaaaaat gaaaaccgat tttatctgaa ggaagtgaac 900 atcagcatgt atttggttaa tggctccgtt ttcagcattg caaataacaa tctcagctac 960 tgggatgccc ccctgggaag ttcttatatg tgcaacaaag agcagactgt ttcagtgtct 1020 ggagcatttc agataaatac ctttgatcta agggttcagc ctttcaatgt gacacaagga 1080 aagtattcta cagctgaaga atgttctgct gactctgacc tcaactttct tattcctgtt 1140 gcagtgggtg tggccttggg cttccttata attgttgtct ttatctctta tatgattgga 1200 agaaggaaaa gtcgtactgg ttatcagtct gtgtaa 1236 <210> 27 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 27 His His His His His His 1 5

Claims

1. A pharmaceutical composition for treating a disease in a subject, comprising hematopoietic stem cells and / or hematopoietic progenitor cells, a vector comprising a promoter and a polynucleotide encoding a functional protein is introduced into hematopoietic stem cells and / or hematopoietic progenitor cells derived from the subject; and the hematopoietic stem cells and hematopoietic progenitor cells are transplanted into the subject to treat the disease; the disease is a cystinosis and the protein comprises CTNS, or the disease is Salla disease and the protein is SLC17A5, or the disease is an infantile sialic acid storage disorder and the protein is SLC17A5, or the disease is cobalaminopathies type F and the protein is LMBRD1, or the disease is late-onset childhood neuronal ceroid lipofuscinosis and the protein is MFSD8, or the disease is juvenile neuronal ceroid lipofuscinosis and the protein is CLN3, or the disease is malignant infantile osteopetrosis and the protein is CLCN7 or OSTM1, or the disease is mucolipidosis IV and the protein is MCOLN1, or the disease is mucopolysaccharidosis type IIC and the protein is HGSNAT, or the disease is Niemann-Pick disease type C and the protein is NPC1, Medicine.

2. The pharmaceutical according to claim 1, wherein the introduction comprises contacting the vector with hematopoietic stem cells and hematopoietic progenitor cells and expressing the protein.

3. The pharmaceutical according to claim 1 or 2, wherein the vector is a viral vector selected from the group consisting of a lentiviral vector, an adenoviral vector, and an AAV vector.

4. The pharmaceutical described in claim 3, wherein the vector is a self-inactivating (SIN) lentiviral vector.

5. (i) the subject is a mammal, and optionally, the subject is a human; (ii) the transfer is performed ex vivo; and / or (iii) hematopoietic stem and progenitor cells are isolated from the subject's bone marrow; The pharmaceutical composition according to any one of claims 1 to 4.

6. A pharmaceutical described in any one of claims 1 to 5, wherein the hematopoietic stem cells and hematopoietic progenitor cells are CD34+ cells.

7. (A) the subject's autophagic flux is improved after treatment; (B) the dosage is between 1.0×10 6 and 5.0×10 6 cells / kg, optionally the dosage is 2.5×10 6 cells / kg administered as a single dose; or (C) Administration is intravenous; The pharmaceutical composition according to claim 6.

8. The pharmaceutical composition of claim 1, wherein the dosage of hematopoietic stem cells and hematopoietic progenitor cells is 1.0×10 6 to 5.0×10 6 cells / kg, optionally the dosage is 2.5×10 6 cells / kg administered as a single dose.

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