Method of targeted gene delivery using viral vectors

Abstract

Methods and compositions are provided for delivering a polynucleotide encoding a gene of interest to a target cell using a virus. The virus envelope comprises a cell-specific binding determinant that recognizes and binds to a component on the target cell surface, leading to endocytosis of the virus. A separate fusogenic molecule is also present on the envelope and facilitates delivery of the polynucleotide across the membrane and into the cytosol of the target cell. The methods and related compositions can be used for treating patients having suffering from a wide range of conditions, including infection, such as HIV; cancers, such as non-Hodgkin's lymphoma and breast cancer; and hematological disorders, such as severe combined immunodeficiency.

1 . A method of delivering a polynucleotide to a target cell, the method comprising: 
 infecting the target cell with a recombinant retrovirus,    wherein the recombinant retrovirus comprises the polynucleotide to be delivered, the R and U5 sequences from a 5′ lentiviral long terminal repeat (LTR), a self-inactivating lentiviral 3′ LTR, a fusogenic molecule, and a cell-specific binding determinant.    
   
   
       2 . The method of  claim 1 , wherein the fusogenic molecule is pH sensitive.  
   
   
       3 . The method of  claim 1 , wherein the fusogenic molecule is a class I fusogen.  
   
   
       4 . The method of  claim 1 , wherein the fusogenic molecule is a class II fusogen.  
   
   
       5 . The method of  claim 1 , wherein the fusogenic molecule is viral glycoprotein.  
   
   
       6 . The method of  claim 5 , wherein the fusogenic molecule has a reduced binding ability.  
   
   
       7 . The method of  claim 5 , wherein the fusogenic molecule comprises a viral glycoprotein derived from one of the group of viruses consisting of: Lassa fever virus, tick-borne encephalitis virus, Dengue virus, Hepatitis B virus, Rabies virus, Semliki Forest virus, Ross River virus, Aura virus, Borna disease virus, Hantaan virus, and SARS-CoV virus.  
   
   
       8 . The method of  claim 1 , wherein the cell-specific binding determinant comprises a protein.  
   
   
       9 . The method of  claim 1 , wherein the cell-specific binding determinant comprises an antibody.  
   
   
       10 . The method of  claim 9 , wherein the antibody comprises the light and heavy constant chain regions of human IgG1.  
   
   
       11 . The method of  claim 11 , wherein the cell-specific binding determinant further comprises immunoglobulin alpha and immunoglobulin beta.  
   
   
       12 . The method of  claim 9 , wherein the antibody is a single chain antibody.  
   
   
       13 . The method of  claim 12 , wherein the single chain antibody is fused with a transmembrane domain from another protein.  
   
   
       14 . The method of  claim 9 , wherein the antibody is selected from the group consisting of an anti-CD20 antibody, an anti-CD34 antibody and an anti-DEC-205 antibody.  
   
   
       15 . The method of  claim 1 , wherein the 5′ LTR sequences are from HIV.  
   
   
       16 . The method of  claim 1  wherein the self-inactivating 3′ LTR comprises a U3 element with a deletion of its enhancer sequence.  
   
   
       17 . The method of  claim 16 , wherein the self-inactivating 3′ LTR is a modified HIV 3′ LTR.  
   
   
       18 . The method of  claim 1 , wherein the recombinant retrovirus is pseudotyped.  
   
   
       19 . The method of  claim 1 , wherein the polynucleotide is a gene of interest.  
   
   
       20 . The method of  claim 1 , wherein the target cell is a cancer cell.  
   
   
       21 . The method of  claim 1 , further comprising the steps of: 
 transfecting a packaging cell line with a vector comprising the polynucleotide to be delivered, the R and U5 sequences and the self-inactivating lentiviral 3′ LTR, a vector comprising a gene encoding the fusogenic molecule, and a vector comprising a gene encoding the cell-specific binding determinant; and    recovering recombinant retrovirus from the packaging cell line.    
   
   
       22 . The method of  claim 21 , wherein said packaging cell line is a 293 cell line.  
   
   
       23 . A recombinant retrovirus comprising: 
 a nucleic acid having the R and U5 sequences from a 5′ lentiviral long terminal repeat (LTR);    a self-inactivating lentiviral 3′ LTR; a    a fusogenic molecule, and a membrane-bound cell-specific binding determinant.    
   
   
       24 . The recombinant retrovirus of  claim 23 , wherein the fusogenic molecule is a viral glycoprotein.  
   
   
       25 . The recombinant retrovirus of  claim 23 , wherein the fusogenic molecule is pH sensitive.  
   
   
       26 . The recombinant retrovirus of  claim 23 , wherein the fusogenic molecule is mutant hemagglutinin.  
   
   
       27 . The recombinant retrovirus of  claim 23 , wherein the fusogenic molecule is SIN.  
   
   
       28 . The recombinant retrovirus of  claim 23 , wherein the cell-specific binding determinant comprises a protein.  
   
   
       29 . The recombinant retrovirus of  claim 23 , wherein the cell-specific binding determinant comprises an antibody.  
   
   
       30 . The recombinant retrovirus of  claim 29 , wherein the antibody is human IgG 1 .  
   
   
       31 . The recombinant retrovirus of  claim 30 , wherein the cell-specific binding determinant further comprises immunoglobulin alpha and immunoglobulin beta.  
   
   
       32 . The recombinant retrovirus of  claim 29 , wherein the antibody is a single chain antibody.  
   
   
       33 . The recombinant retrovirus of  claim 29 , wherein the viral construct further comprises at least one gene of interest.  
   
   
       34 . The recombinant retrovirus of  claim 33 , wherein the gene of interest is a reporter gene.  
   
   
       35 . The method of  claim 33 , wherein the gene of interest encodes an siRNA molecule.