Targeting Pseudotyped Retroviral Vectors

Abstract

The present invention relates to retroviral vectors, particularly lentiviral vectors, pseudotyped with Sindbis envelope and targeted to specific cell types via a targeting moiety linked to the envelope.

1 . A pseudotyped, targeted retroviral vector comprising:
 a) a mutated Sindbis envelope comprising Sindbis envelope proteins E1, E2, and E3, wherein at least one of E1, E2, or E3 is mutated as compared to a wild-type sequence;   b) a targeting moiety linked to the Sindbis envelope.   
     
     
         2 . The vector of  claim 1 , further comprising a retroviral-based nucleic acid genome. 
     
     
         3 . The vector of  claim 1 , wherein the vector nucleic acid comprises a heterologous gene operably linked to a promoter. 
     
     
         4 . The vector of  claim 3 , wherein the promoter is a tissue-specific promoter. 
     
     
         5 . The vector of  claim 4 , wherein the tissue-specific promoter is a PSE-BC promoter. 
     
     
         6 . The vector of  claim 1 , wherein the targeting moiety specifically binds to a target protein selected from the group consisting of P-glycoprotein, Her2/Neu, erythropoietin (EPO), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGF-R), cadherin, carcinoembryonic antigen (CEA), CD4, CD8, CD19, CD20, CD33, CD34, CD45, CD117 (c-kit), CD133, HLA-A, HLA-B, HLA-C, chemokine receptor 5 (CCR5), stem cell marker ABCG2 transporter, ovarian cancer antigen CA125, an integrin, prostate specific antigen (PSA), prostate stem cell antigen (PSCA), dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), thyroglobulin, granulocyte-macrophage colony stimulating factor (GM CSF), myogenic differentiation promoting factor-1 (MyoD-1), Leu-7 (CD57), LeuM-1, cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67 (Ki-67), HIV gp120, and transferrin receptor. 
     
     
         7 . The vector of  claim 6 , wherein the targeting moiety is an antibody. 
     
     
         8 . The vector of  claim 7 , wherein the targeting moiety is an antibody directed against prostate stem cell antigen (PSCA). 
     
     
         9 . The vector of  claim 7 , wherein the targeting moiety is an antibody directed against P-glycoprotein (P-gp). 
     
     
         10 . The vector of  claim 7 , wherein the targeting moiety is an antibody directed against a transferrin receptor. 
     
     
         11 . The vector of  claim 1 , wherein the targeting moiety is covalently linked to the Sindbis envelope. 
     
     
         12 . The vector of  claim 1 , wherein the targeting moiety is non-covalently linked to the Sindbis envelope. 
     
     
         13 . The vector of  claim 1 , wherein the targeting moiety is covalently linked to the E2 or the E3 protein of the Sindbis envelope. 
     
     
         14 . The vector of  claim 1 , wherein the targeting moiety is non-covalently linked to the E2 or the E3 protein of the Sindbis envelope. 
     
     
         15 . The vector of  claim 14 , wherein the targeting moiety is non-covalently linked to the E2 protein Sindbis envelope via the ZZ domain of protein A. 
     
     
         16 . The vector of  claim 1 , wherein E2 protein is mutated at one amino acid position. 
     
     
         17 . The vector of  claim 1 , wherein E2 protein is mutated at two or more amino acid positions. 
     
     
         18 . The vector of  claim 1 , wherein E3 protein is mutated at one amino acid position. 
     
     
         19 . The vector of  claim 1 , wherein E3 protein is mutated at two or more amino acid positions. 
     
     
         20 . The vector of  claim 1 , wherein the mutated Sindbis envelope is encoded by a sequence listed in Table 1. 
     
     
         21 . The vector of  claim 1 , wherein the mutated Sindbis envelope is encoded by m168 which has a mutation in Sindbis virus envelope protein E2. 
     
     
         22 . The vector of  claim 21 , further comprising a mutation in Sindbis envelope protein E1. 
     
     
         23 . A packaging system comprising a cell comprising nucleic acids encoding the pseudotyped, targeted retroviral vector of  claim 1 . 
     
     
         24 . The packaging system of  claim 23 , wherein the vector further comprises a retroviral-based nucleic acid genome. 
     
     
         25 . The packaging system of  claim 24 , wherein the Sinbis envelope proteins E1, E2, and E3 and the retroviral-based nucleic acid genome are encoded on the same nucleic acid. 
     
     
         26 . The packaging cell of  claim 24 , wherein the Sinbis envelope proteins E1, E2, and E3 and the retroviral-based nucleic acid genome are encoded on separate nucleic acids. 
     
     
         27 . An expression vector comprising a nucleic acid encoding Sindbis envelope proteins E1, E2, and E3, wherein at least one of E, E2, or E3 is mutated as compared to a wild-type sequence. 
     
     
         28 . A method of making the pseudotyped, targeted retroviral vector of  claim 1 , the method comprising the step of expressing in a cell a nucleic acid comprising Sindbis envelope proteins E1, E2, and E3. 
     
     
         29 . The method of  claim 28 , wherein the vector further comprises a retroviral-based nucleic acid genome. 
     
     
         30 . The method of  claim 29 , wherein the Sindbis envelope proteins E1, E2, and E3 and the retroviral based nucleic acid genome are encoded on the same nucleic acid. 
     
     
         31 . The method of  claim 29 , wherein the Sindbis envelope proteins E1, E2, and E3 and the retroviral based nucleic acid genome are encoded on separate nucleic acids. 
     
     
         32 . The method of  claim 28 , further comprising the step of isolating a virus particle from the cell. 
     
     
         33 . A method of transducing cells with a heterologous gene, the method comprising the step of contacting the cell with the pseudotyped, targeted retroviral vector of  claim 1 . 
     
     
         34 . The method of  claim 33 , wherein the cells are in a subject and the vector is administered intravenously. 
     
     
         35 . The method of  claim 33 , wherein the cells are transduced ex vivo. 
     
     
         36 . The method of  claim 33 , wherein the cells are transduced in vivo. 
     
     
         37 . The method of  claim 33 , wherein the cells are transduced in vitro. 
     
     
         38 . A method of treating or preventing a disease state, the method comprising the step of contacting a cell with the pseudotyped, targeted retroviral vector of  claim 1 . 
     
     
         39 . The method of  claim 38 , wherein the cell is contacted in vivo. 
     
     
         40 . The method of  claim 38 , wherein the cell is contacted ex vivo. 
     
     
         41 . The method of  claim 38 , wherein the cell is contacted in vitro. 
     
     
         42 . A method of diagnosing a disease state, the method comprising the step of contacting a cell with the pseudotyped, targeted retroviral vector of  claim 1 . 
     
     
         43 . The method of  claim 42 , wherein the cell is contacted in vivo. 
     
     
         44 . The method of  claim 42 , wherein the cell is contacted ex vivo. 
     
     
         45 . The method of  claim 42 , wherein the cell is contacted in vitro. 
     
     
         46 . A method of delivering a pseudotyped, targeted retroviral vector across the blood brain barrier in a subject, the method comprising the step of contacting a cell with the pseudotyped, targeted retroviral vector of  claim 10 .