Thymidine kinase gene

Abstract

Nucleic acid sequences encoding improved Herpes Simplex Virus Thymidine Kinases are provided, including their use in diagnostic and therapeutic applications. The thymidine kinases may be mutated using conservative mutations, non-conservative mutations, or both. Also provided are gene therapeutic systems, including viral and retroviral particles.

1 .- 67 . (canceled) 
     
     
         68 . A polynucleotide encoding a mutated form of thymidine kinase from a human herpes simplex virus type 1 (HSV1-TK) and at least one additional suicide gene, wherein the encoded HSV1-TK comprises at least two mutated amino acids in a nuclear localization sequence (NLS) relative to a wild-type HSV1-TK amino acid sequence, wherein the NLS comprises amino acid residues 1-33 of SEQ ID NO: 2, and wherein the combination of the mutated HSV1-TK and the at least one additional suicide gene increases cell kill activity relative to a wild-type HSV1-TK. 
     
     
         69 . The polynucleotide according to  claim 68 , wherein the at least two mutated amino acids in the NLS correspond to at least two amino acid residues at positions 25, 26, 32, and 33 of SEQ ID NO: 2, wherein each amino acid residue is independently mutated to an amino acid chosen from the group consisting of glycine, serine, cysteine, glutamic acid, and aspartic acid. 
     
     
         70 . The polynucleotide according to  claim 69 , wherein the encoded mutated HSV1-TK further comprises a mutation at amino acid residue positions 167 or 168 of SEQ ID NO: 2, or a combination thereof, to a polar, non-polar, acidic, or basic amino acid. 
     
     
         71 . The polynucleotide according to  claim 68 , wherein the encoded mutated HSV1-TK further comprises a nuclear export signal sequence comprising LQKKLEELELDG (SEQ ID NO: 24). 
     
     
         72 . The polynucleotide according to  claim 68 , wherein the encoded mutated HSV1-TK exhibits a reduced amount of thymidine kinase activity as compared to wild-type HSV1-TK. 
     
     
         73 . The polynucleotide according to  claim 68 , wherein the at least one additional suicide gene comprises a cytosine deaminase, a VSV-tk, an IL-2, a nitroreductase (NR), a carboxylesterase, a beta-glucuronidase, a cytochrome p450, a beta-galactosidase, a diphtheria toxin A-chain (DT-A), a carboxypeptide G2 (CPG2), a purine nucleoside phosphorylase (PNP), or a deoxycytidine kinase (dCK). 
     
     
         74 . The polynucleotide according to  claim 73 , wherein the at least one additional suicide gene is the nitroreductase (NR). 
     
     
         75 . A retroviral vector comprising the polynucleotide of  claim 68  encoding the mutated HSV1-TK and the at least one additional suicide gene. 
     
     
         76 . The retroviral vector of  claim 75 , further comprising a polynucleotide encoding for a PiT-2 or PiT-1 polypeptide. 
     
     
         77 . A method of treating cancer in a patient in need thereof, the method comprising transducing a cell of interest from the patient by contacting the cell of interest with a retroviral vector of  claim 75 , the contacting of the cell of interest with the retroviral vector occurs in vitro or in vivo, followed by administration of a nucleoside analogue or a prodrug thereof and at least one additional prodrug to the patient in need thereof, wherein the retroviral vector transduces the cell of interest for inducing cell kill activity, thereby treating the cancer in the patient in need thereof. 
     
     
         78 . The method of  claim 77 , wherein the retroviral vector is administered intravenously, intramuscularly, subcutaneously, intra-arterially, intra-hepatic arterially, intra-thecally, intra-peritoneally and/or intra-tumorally. 
     
     
         79 . The method of  claim 77 , wherein the cell of interest is transduced with the retroviral vector in vitro, wherein the cell of interest is subsequently administered to the patient in need thereof. 
     
     
         80 . The method of  claim 77 , wherein at least 1×10 5  TVP of retroviral vector is administered cumulatively to the subject in need thereof. 
     
     
         81 . The method of  claim 77 , wherein the nucleoside analogue is ganciclovir. 
     
     
         82 . The method of  claim 77 , wherein the at least one additional suicide gene is nitroreductase, and the at least one additional prodrug is CB1954. 
     
     
         83 . A method of inducing cell kill activity in a cancer cell of interest in a patient, the method comprising:
 a. contacting the cancer cell with a retroviral vector of  claim 75 ;   b. transducing the cancer cell with the polynucleotide encoding the mutated HSV1-TK; and   c. contacting the cancer cell with a nucleoside analogue or a prodrug thereof, thereby inducing cell kill activity in the cancer cell.   
     
     
         84 . The method of  claim 83 , wherein the nucleoside analogue is ganciclovir. 
     
     
         85 . The method of  claim 83 , wherein the cancer cell of interest is transduced in vivo in the patient by administering the retroviral vector to the patient, wherein the cancer cell is contacted with the retroviral vector in the patient. 
     
     
         86 . The method of  claim 83 , wherein the cancer cell in (b) is transduced in vitro by contacting the cancer cell obtained from the patient with the retroviral vector, wherein the contacting of the cancer cell with the retroviral vector occurs outside the patient, and wherein the cancer cell contacted with the retroviral vector is subsequently administered to the patient in need thereof. 
     
     
         87 . The method of  claim 83 , wherein the at least one additional suicide gene is nitroreductase, and the at least one additional prodrug is CB1954.