Gene therapy for the treatment of solid tumors using recombinant adeno-associated virus vectors

Abstract

The use of recombinant adeno-associated virus (AAV) virions for the treatment of solid tumors is disclosed. The invention provides for the use of recombinant AAV virions to deliver an AAV vector containing a drug-susceptibility gene and a second gene capable of providing an ancillary effect to solid tumor cells. The second gene can be used to enhance the immunogenicity of the transduced tumor cell. Alternatively, the second gene can be used to provide a tumorstatic effect. The invention also provides for the use of recombinant AAV virions to deliver an interferon gene, or a tumor suppressor gene to provide a therapeutic effect in a transduced tumor cell.

1 . An adeno-associated virus (AAV) vector, comprising: 
 a first gene, comprising a drug susceptibility gene; and    a second gene encoding a cytokine, wherein the first and second genes are operably linked to control elements capable of directing the in vivo transcription and translation thereof.    
     
     
         2 . The AAV vector of  claim 1 , wherein the first gene is selected from the group consisting of a herpes simplex virus thymidine kinase (HSV-tk) gene, a cytochrome P450 gene, a human deoxycytidine kinase gene, and a bacterial cytosine deaminase gene.  
     
     
         3 . The AAV vector of  claim 2 , wherein the first gene is a herpes simplex virus thymidine kinase (HSV-tk) gene.  
     
     
         4 . The AAV vector of  claim 1 , wherein the second gene encodes a cytokine selected from the group consisting of alpha interferon (IFN-α), beta interferon (IFN-β), gamma interferon (IFN-γ), tumor necrosis factor (TNF), interleukin-2 (IL-2), lymphotoxin, interleukin-12 (IL-12) and granulocyte-macrophage colony-stimulating factor (GM-CSF).  
     
     
         5 . The AAV vector of  claim 4 , wherein the second gene encodes interleukin-2.  
     
     
         6 . An adeno-associated virus (AAV) vector, comprising: 
 a first gene, comprising a drug susceptibility gene; and    a second gene comprising a tumor suppressor gene, wherein the first gene and the second gene are operably linked to control elements capable of directing the in vivo transcription and translation thereof.    
     
     
         7 . The AAV vector of  claim 6 , wherein the second gene is selected from the group consisting of p53, RB1, WT1, NF1, VHL, and APC.  
     
     
         8 . An adeno-associated virus (AAV) vector, comprising a beta interferon (IFN-β) gene and control elements operably linked thereto, wherein said control elements are capable of directing the transcription and translation of the gene in a host cell.  
     
     
         9 . A recombinant adeno-associated virus virion (rAAV virion), comprising the vector of  claim 1 .  
     
     
         10 . A recombinant adeno-associated virus virion (rAAV virion), comprising the vector of  claim 6 .  
     
     
         11 . A recombinant adeno-associated virus virion (rAAV virion), comprising the vector of  claim 8 .  
     
     
         12 . A method of simultaneously delivering a first and a second gene to a target solid tumor cell, comprising: 
 (a) providing a recombinant adeno-associated virus virion (rAAV virion) which comprises a first gene capable of being expressed to provide the target cell with enhanced susceptibility to a selected cytotoxic agent and a second gene capable of providing an ancillary therapeutic effect, wherein said first gene and said second gene are operably linked to control elements capable of directing the in vivo transcription and translation thereof; and    (b) transducing the target solid tumor cell with the rAAV virion.    
     
     
         13 . The method of  claim 12 , wherein the first gene comprises a herpes simplex virus thymidine kinase (HSV-tk) gene.  
     
     
         14 . The method of  claim 12 , wherein expression of the second gene enhances the immunogenicity of the target cell.  
     
     
         15 . The method of  claim 14 , wherein the second gene encodes a cytokine.  
     
     
         16 . The method of  claim 15 , wherein the second gene encodes a cytokine selected from the group consisting of alpha interferon (IFN-α), beta interferon (IFN-β), gamma interferon (IFN-γ), tumor necrosis factor (TNF), interleukin-2 (IL-2), lymphotoxin, interleukin-12 (IL-12) and granulocyte-macrophage colony-stimulating factor (GM-CSF).  
     
     
         17 . The method of  claim 16 , wherein the second gene encodes interleukin-2.  
     
     
         18 . The method of  claim 16 , wherein the second gene encodes beta interferon (IFN-β).  
     
     
         19 . The method of  claim 12 , wherein the second gene is a tumor suppressor gene.  
     
     
         20 . The method of  claim 19 , wherein the second gene is selected from the group consisting of p53, RB1, WT1, NF1, VHL, and APC.  
     
     
         21 . The method of  claim 12 , wherein the target solid tumor cell is transduced in vivo with the rAAV virion.  
     
     
         22 . The method of  claim 12 , wherein the target solid tumor cell is a glioma cell.  
     
     
         23 . A solid tumor cell transduced with a recombinant adeno-associated virus virion (rAAV virion) which comprises an AAV vector, said AAV vector including a first gene capable of being expressed to provide the tumor cell with enhanced susceptibility to a selected cytotoxic agent, a second gene capable of providing an ancillary therapeutic effect, and control elements operably linked to the first gene and the second gene, wherein said control elements are capable of directing the in vivo transcription and translation of the first gene and the second gene.  
     
     
         24 . The tumor cell of  claim 23 , wherein the first gene is selected from the group consisting of a herpes simplex virus thymidine kinase (HSV-tk) gene, a cytochrome P450 gene, a human deoxycytidine kinase gene, and a bacterial cytosine deaminase gene.  
     
     
         25 . The tumor cell of  claim 23 , wherein the second gene encodes a cytokine.  
     
     
         26 . The tumor cell of  claim 23 , wherein the second gene is a tumor suppressor gene.  
     
     
         27 . A method of treating neoplastic disease in a mammalian subject, comprising: 
 (a) transducing a solid tumor cell of said subject in vivo using a therapeutically effective amount of a pharmaceutical composition which comprises (i) a pharmaceutically acceptable excipient, and (ii) recombinant adeno-associated virus virions (rAAV virions), wherein said rAAV virions comprise an AAV vector having a first gene capable of being expressed to provide the transduced tumor cell with enhanced susceptibility to a selected cytotoxic agent, a second gene capable of providing an ancillary therapeutic effect, and control elements operably linked to the first gene and the second gene, wherein said control elements are capable of directing the in vivo transcription and translation of the first gene and the second gene when present in the mammalian subject; and    (b) administering a therapeutically effective amount of the selected cytotoxic agent to the mammalian subject.    
     
     
         28 . The method of  claim 27 , wherein the first gene is selected from the group consisting of a herpes simplex virus thymidine kinase (HSV-tk) gene, a cytochrome P450 gene, a human deoxycytidine kinase gene, and a bacterial cytosine deaminase gene.  
     
     
         29 . The method of  claim 27 , wherein the second gene encodes a cytokine.  
     
     
         30 . The method of  claim 27 , wherein the solid tumor cell is a glioma cell.  
     
     
         31 . A method of treating neoplastic disease in a mammalian subject, comprising transducing a solid tumor cell of said subject in vivo using a therapeutically effective amount of a pharmaceutical composition which comprises: 
 (a) a pharmaceutically acceptable excipient; and    (b) a recombinant adeno-associated virus virion (rAAV virion) comprising an AAV vector that contains a gene capable of being expressed by the transduced cell to provide either a tumorstatic or an immunogenic effect, and control elements operably linked to the gene, wherein said control elements are capable of directing the in vivo transcription and translation of the gene when present in the mammalian subject.    
     
     
         32 . The method of  claim 31 , wherein the gene encodes an interferon.  
     
     
         33 . The method of  claim 32 , wherein the interferon is selected from the group consisting of alpha interferon (IFN-α), beta interferon (IFN-β), and gamma interferon (IFN-γ).  
     
     
         34 . The method of  claim 33 , wherein the gene encodes beta interferon (IFN-β).  
     
     
         35 . The method of  claim 31 , wherein the AAV vector comprises a tumor suppressor gene.  
     
     
         36 . The method of  claim 31 , wherein the solid tumor cell is a glioma cell.  
     
     
         37 . A composition, comprising: 
 (a) a pharmaceutically acceptable excipient; and    (b) a recombinant adeno-associated virus virion (rAAV virion), wherein said rAAV virion comprises an AAV vector having (i) a first gene comprising a drug susceptibility gene, (ii) a second gene capable of being expressed to provide an ancillary therapeutic effect, and (iii) control elements capable of directing the in vivo transcription and translation of the first and second genes, wherein the first and second genes are operably linked to said control elements.    
     
     
         38 . A composition, comprising: 
 (a) a pharmaceutically acceptable excipient; and    (b) a recombinant adeno-associated virus virion (rAAV virion), wherein said rAAV virion comprises an AAV vector having a beta interferon (IFN-β) gene and control elements operably linked thereto, wherein said control elements are capable of directing the transcription and translation of the gene in a host cell.