Gene therapy vectors and cytosine deaminases

Abstract

This disclosure provides modified cytosine deaminases(CDs). The disclosure further relates to cells and vector expressing or comprising such modified CDs and methods of using such modified CDs in the treatment of disease and disorders.

1 . A recombinant replication competent retrovirus (RCR) comprising:
 a retroviral GAG protein;   a retroviral POL protein;   a retroviral envelope;   a retroviral polynucleotide comprising Long-Terminal Repeat (LTR) sequences at the 3′ end of the retroviral polynucleotide, a promoter sequence at the 5′ end of the retroviral polynucleotide, said promoter being suitable for expression in a mammalian cell, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain;   a cassette comprising an internal ribosome entry site (IRES) operably linked to a human codon optimized nucleic acid encoding a polypeptide having cytosine deaminase activity and a nucleic acid comprising an siRNA, wherein the cassette is positioned 5′ to the 3′ LTR and 3′ to the env nucleic acid domain encoding the retroviral envelope; and   cis-acting sequences necessary for reverse transcription, packaging and integration in a target cell.   
     
     
         2 . The RCR of  claim 1 , wherein the retroviral polynucleotide is derived from murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV), Feline leukemia Virus or Gibbon ape leukemia virus (GALV). 
     
     
         3 . The RCR of  claim 2 , wherein the MLV is an amphotropic MLV. 
     
     
         4 . The RCR of  claim 1 , wherein the retrovirus is a gammaretrovirus. 
     
     
         5 . The RCR of  claim 1 , wherein the target cell is a cancer cell. 
     
     
         6 . The RCR of  claim 5 , wherein the cancer is selected from the group consisting of lung cancer, colon-rectum cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, stomach cancer and ovarian cancer, rheumatoid arthritis or other auto-immune disease. 
     
     
         7 . The RCR according to  claim 1 , wherein the promoter sequence comprises a tissue-specific promoter sequence. 
     
     
         8 - 12 . (canceled) 
     
     
         13 . The RCR of  claim 1 , wherein the gag nucleic acid domain is derived from a gammaretrovirus. 
     
     
         14 . (canceled) 
     
     
         15 . The RCR of  claim 1 , wherein the pol nucleic acid domain of the polynucleotide is derived from a gammaretrovirus. 
     
     
         16 . (canceled) 
     
     
         17 . The RCR of  claim 1 , wherein the env nucleic acid domain encodes an amphotrophic envelope. 
     
     
         18 . The RCR of  claim 1 , wherein the IRES is derived from an encephalomyocarditis virus. 
     
     
         19 - 20 . (canceled) 
     
     
         21 . The retrovirus of  claim 1 , wherein the 3′ LTR is derived from a gammaretrovirus. 
     
     
         22 . The retrovirus of  claim 21 , wherein the 3′ LTR comprises a U3-R-U5 domain. 
     
     
         23 . (canceled) 
     
     
         24 . A method of treating a cell proliferative disorder in a subject comprising contacting the subject with a retrovirus of  claim 1 , and contacting the subject with 5-fluorocytosine. 
     
     
         25 . (canceled)