Cocal vesiculovirus envelope pseudotyped retroviral vectors

Abstract

Provided herein are Cocal vesiculovirus envelope pseudotyped retroviral vectors that exhibit high titers, broad species and cell-type tropism, and improved serum stability. Disclosed Cocal vesiculovirus envelope pseudotyped retroviral vectors may be suitably employed for gene therapy applications and, in particular, for the ex vivo and in vivo delivery of a gene of interest to a wide variety of target cells.

1 . A Cocal vesiculovirus envelope pseudotyped retroviral vector particle, comprising a retroviral Gag protein, a retroviral Pol protein, and a Cocal vesiculovirus envelope protein. 
     
     
         2 . The Cocal vesiculovirus envelope pseudotyped retroviral vector particle of  claim 1  wherein said retroviral Gag protein and said retroviral Pol protein are derived from a retrovirus selected from the group consisting of a lentivirus, an alpharetrovirus, a betaretrovirus, a gammaretrovirus, a deltaretrovirus, and an epsilonretrovirus. 
     
     
         3 . The Cocal vesiculovirus envelope pseudotyped retroviral vector particle of  claim 1  further comprising one or more retroviral accessory protein. 
     
     
         4 . The Cocal vesiculovirus envelope pseudotyped retroviral vector particle of  claim 1  wherein said Cocal vesiculovirus envelope protein is encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1. 
     
     
         5 . The Cocal vesiculovirus envelope pseudotyped retroviral vector particle of  claim 1  wherein said Cocal vesiculovirus envelope protein is encoded by a polynucleotide comprising a nucleotide sequence that is human codon-optimized. 
     
     
         6 . The Cocal vesiculovirus envelope pseudotyped retroviral vector particle of  claim 5  wherein said Cocal vesiculovirus envelope protein is encoded by a polynucleotide comprising human codon-optimized nucleotide sequence of SEQ ID NO: 3. 
     
     
         7 . The Cocal vesiculovirus envelope pseudotyped retroviral vector particle of  claim 6  wherein said Cocal vesiculovirus envelope protein comprises the amino acid sequence of SEQ ID NO: 4. 
     
     
         8 . A polynucleotide comprising a human codon-optimized nucleotide sequence encoding a Cocal vesiculovirus envelope protein. 
     
     
         9 . The polynucleotide of  claim 8  wherein said human codon-optimized nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 3. 
     
     
         10 . The polynucleotide of  claim 8  wherein said human codon-optimized nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 4. 
     
     
         11 . A plasmid vector for the expression of a Cocal vesiculovirus envelope protein, said vector comprising a polynucleotide encoding a Cocal vesiculovirus envelope protein wherein said polynucleotide is under the transcriptional control of a eukaryotic transcriptional promoter. 
     
     
         12 . The plasmid vector of  claim 11  wherein said polynucleotide encoding a Cocal vesiculovirus envelope protein comprises the nucleotide sequence of SEQ ID NO: 1. 
     
     
         13 . The plasmid vector of  claim 11  wherein said polynucleotide encoding a Cocal vesiculovirus envelope protein is human codon-optimized. 
     
     
         14 . The plasmid vector of  claim 13  wherein said polynucleotide encoding a Cocal vesiculovirus envelope protein comprises the nucleotide sequence of SEQ ID NO: 3. 
     
     
         15 . The plasmid vector of  claim 13  wherein said Cocal vesiculovirus envelope protein comprises the amino acid sequence of SEQ ID NO: 4. 
     
     
         16 . The plasmid vector of  claim 13  wherein said plasmid vector comprises the nucleotide sequence of SEQ ID NO: 5. 
     
     
         17 . The plasmid vector of  claim 16  wherein said plasmid vector is pMD2.CocalG (SEQ ID NO: 5). 
     
     
         18 . An isolated Cocal vesiculovirus envelope protein encoded by a human codon-optimized nucleotide sequence. 
     
     
         19 . The isolated Cocal vesiculovirus envelope protein of  claim 18  wherein said human codon-optimized nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 3. 
     
     
         20 . The isolated Cocal vesiculovirus envelope protein of  claim 18  wherein said human codon-optimized nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 4. 
     
     
         21 . A method for generating a Cocal envelope pseudotyped retroviral vector particle, said method comprising the step of transfecting a cell with a retroviral vector plasmid, a retroviral helper plasmid, and a plasmid vector for the expression of a Cocal vesiculovirus envelope protein. 
     
     
         22 . The method of  claim 21  wherein said retroviral vector plasmid and said retroviral helper plasmid are derived from a retrovirus selected from the group consisting of a lentivirus, an alpharetrovirus, a betaretrovirus, a gammaretrovirus, a deltaretrovirus, and an epsilonretrovirus. 
     
     
         23 . The method of  claim 22  wherein said plasmid vector for the expression of a Cocal vesiculovirus envelope protein comprises the nucleotide sequence of SEQ ID NO: 1. 
     
     
         24 . The method of  claim 22  wherein said plasmid vector for the expression of a Cocal vesiculovirus envelope protein comprises a polynucleotide encoding a Cocal vesiculovirus envelope protein that is human codon-optimized. 
     
     
         25 . The method of  claim 24  wherein said human codon-optimized polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3. 
     
     
         26 . The method of  claim 25  wherein said Cocal vesiculovirus envelope protein comprises the amino acid sequence of SEQ ID NO: 4. 
     
     
         27 . The method of  claim 26  wherein said plasmid vector comprises the nucleotide sequence of SEQ ID NO: 5. 
     
     
         28 . The method of  claim 27  wherein said plasmid vector is pMD2.CocalG (SEQ ID NO: 5). 
     
     
         29 . A method for delivering a gene of interest to a target cell, said method comprising the step of contacting said target cell with a Cocal vesiculovirus envelope pseudotyped retroviral vector particle. 
     
     
         30 . The method of  claim 29  wherein said Cocal vesiculovirus envelope pseudotyped retroviral vector particle is selected from the group consisting of a lentiviral vector particle, an alpharetroviral vector particle, a betaretroviral vector particle, a gammaretroviral vector particle, a deltaretroviral vector particle, and an epsilonretroviral vector particle. 
     
     
         31 . The method of  claim 29  wherein said Cocal vesiculovirus envelope pseudotyped retroviral vector particle comprises a Cocal vesiculovirus envelope protein encoded by the nucleotide sequence of SEQ ID NO: 1. 
     
     
         32 . The method of  claim 29  wherein said Cocal vesiculovirus envelope pseudotyped retroviral vector particle comprises a Cocal vesiculovirus envelope protein comprising the amino acid sequence of SEQ ID NO: 2. 
     
     
         33 . The method of  claim 29  wherein said Cocal vesiculovirus envelope pseudotyped retroviral vector particle comprises a Cocal vesiculovirus envelope protein encoded by the nucleotide sequence of SEQ ID NO: 3. 
     
     
         34 . The method of  claim 29  wherein said Cocal vesiculovirus envelope pseudotyped retroviral vector particle comprises a Cocal vesiculovirus envelope protein comprising the amino acid sequence of SEQ ID NO: 4. 
     
     
         35 . A method for delivering a gene of interest into a hematopoietic system of a patient, said method comprising the steps of:
 (a) harvesting a CD34 +  hematopoietic stem cell (HSC) from said patient;   (b) transducing said HSC ex vivo with a Cocal vesiculovirus envelope pseudotyped retroviral vector particle comprising said gene of interest; and   (c) introducing said ex vivo transduced HSC into said patient under conditions that allow reconstitution of said patient's hematopoietic system.   
     
     
         36 . A method for identifying a patient susceptible to treatment with a Cocal vesiculovirus envelope pseudotyped retroviral vector particle; said method comprising the steps of:
 (a) isolating a serum sample from said patient;   (b) contacting said Cocal vesiculovirus envelope pseudotyped retroviral vector particle with said serum sample; and   (c) testing said Cocal vesiculovirus envelope pseudotyped retroviral vector particle for serum inactivation.   
     
     
         37 . A method for delivering a gene of interest to a target cell, said method comprising the step of contacting said target cell with a Cocal vesiculovirus envelope pseudotyped retroviral vector particle comprising said gene of interest. 
     
     
         38 . The method of  claim 37  wherein said target cell is a cell line or a primary cell. 
     
     
         39 . The method of  claim 38  wherein said target cell is a primary cell selected from the group consisting of a fibroblast, a lymphocyte, an epithelial cell, an endothelial cell, a stromal cell, an osteosarcoma cell, a leukemia cell, a stem cell, and a hematopoietic progenitor cell. 
     
     
         40 . The method of  claim 39  wherein said stem cell is a pluripotent stem cell. 
     
     
         41 . The method of  claim 37  wherein said cell is contacted ex vivo with said Cocal vesiculovirus envelope pseudotyped retroviral vector particle. 
     
     
         42 . The method of  claim 37  wherein said cell is contacted in vivo with said Cocal vesiculovirus envelope pseudotyped retroviral vector particle. 
     
     
         43 . A method for delivering a gene of interest into a cell from a patient, said method comprising the steps of:
 (a) harvesting said cell from said patient;   (b) transducing said cell ex vivo with a Cocal vesiculovirus envelope pseudotyped retroviral vector particle comprising said gene of interest; and   (c) introducing said ex vivo transduced cell into said patient.   
     
     
         44 . The method of  claim 43  wherein said cell is selected from the group consisting of a fibroblast, a lymphocyte, an epithelial cell, an endothelial cell, a stromal cell, an osteosarcoma cell, a leukemia cell, a stem cell, and a hematopoietic progenitor cell. 
     
     
         45 . The method of  claim 44  wherein said stem cell is a pluripotent stem cell.