Variants of thymidine kinase, related nucleic acids sequences and their use in genic therapy

Abstract

The present invention relates to a nucleic acid sequence characterized in that it is derived from the wild nucleic acid sequence coding for a thymidine kinase, said nucleic acid sequence having at least one mutation in the region corresponding to the ATP binding site and conveniently a second mutation in the N-terminal region and/or C-terminal region. It also relates to variants of the wild thymidine kinase and their use in genic therapy.

1 . Nucleic acid sequence coding for a thymidine kinase, characterized in that it possesses, relative to the wild-type sequence, at least one mutation in the region corresponding to the ATP-binding site combined with at least one mutation in the N-terminal and/or C-terminal region.  
     
     
         2 . Nucleic acid sequence, characterized in that it is derived from the sequence coding for a wild-type thymidine kinase, the said sequence possessing at least one mutation in the region corresponding to the ATP-binding site and at least one mutation in the N-terminal and/or C-terminal region.  
     
     
         3 . Nucleic acid sequence according to  claim 2 , characterized in that it is derived from the sequence coding for herpes simplex virus type 1 thymidine kinase.  
     
     
         4 . Nucleic acid sequence according to  claim 3 , characterized in that the said sequence comprises at least one substitution of a guanine at position 180 by an adenine (G180A).  
     
     
         5 . Nucleic acid sequence according to  claim 3  or  4 , characterized in that the said sequence comprises at least one substitution of a guanine at position 180 by an adenine (G180A) and at least one substitution of the guanine at position 16 by an adenine (G16A).  
     
     
         6 . Nucleic acid sequence according to  claim 3  or  4 , characterized in that the said sequence comprises at least one substitution of a guanine at position 180 by an adenine (G180A) and at least a double substitution of the guanines at position 28 and 30 by adenines (G28A and G30A).  
     
     
         7 . Nucleic acid sequences according to one of  claims 4  to  6 , characterized in that the said sequence comprises, in addition, at least one mutation in its C-terminal region.  
     
     
         8 . Nucleic acid sequence according to one of claims  4 ,  6  and  7 , characterized in that it possesses at least one substitution of a guanine at position 180 by an adenine (G180A), at least a double substitution of the guanines at position 28 and 30 by adenines (G28A and G30A) a double substitution of the cystosines at position 591 and 892 by thymines (C591T and C892T) and a double substitution of the guanines at position 1010 and 1011 by adenines (G1010A and G1011A).  
     
     
         9 . Nucleic acid sequence coding for a thymidine kinase variant, characterized in that it is chosen from: 
 (a) the sequence SEQ ID No. 3 or a portion of the latter carrying the (G180A) mutation or one of their complementary strand,    (b) the sequences SEQ ID No. 6 and SEQ ID No. 7 or a portion of these sequences carrying the (G180A) mutation and, respectively, the G16A mutation and the (G28A; G30A) double mutation or one of their complementary strand,    (c) the sequence SEQ ID No. 8 or a portion of the latter carrying the (G180A) mutation the (G28A: G30A) double mutation, and the (C591T; C892T; G1010A; G1011A) quadruple mutation or one of their complementary strand    (d) any sequence hybridizing with the sequences (a), (b) and/or (c) and coding for a thymidine kinase variant and as defined in  claim 1  or  2 ,    (e) the variants of (a), (b), (c) and (d) resulting from the degeneracy of the genetic code.    
     
     
         10 . Nucleic acid sequence coding for a variant of a wild-type thymidine kinase, capable of being obtained by mutagenesis, site-directed or otherwise, of a sequence according to one of claims  1  to 9.  
     
     
         11 . Nucleic acid sequence according to one of the preceding claims, characterized in that it can be of eukaryotic, bacterial, viral, synthetic or semi-synthetic origin.  
     
     
         12 . Variant of a wild-type thymidine kinase, capable of being expressed from a nucleic acid sequence according to any one of  claims 1  to  11 .  
     
     
         13 . Variant of a thymidine kinase, comprising at least one mutation in the region corresponding to the ATP-binding site combined with at least one mutation in its N-terminal and/or C-terminal region.  
     
     
         14 . Variant according to  claim 13 , characterized in that the region involved in the region corresponding to the ATP-binding site is represented by the consensus GXXXXGK(T/S).  
     
     
         15 . Variant according to  claim 14 , characterized in that it is preferably the motif GPHGMGKT.  
     
     
         16 . Variant according to  claim 15 , characterized in that it comprises at least one substitution at position 60 of a methionine by an isoleucine.  
     
     
         17 . Variant of a wild-type thymidine kinase, characterized in that it is the mutant 1537:E4.  
     
     
         18 . Variant according to one of  claims 13  to  16 , characterized in that the mutation in the N-terminal region lies between amino acids 1 and 20 of the said region.  
     
     
         19 . Variant according to  claim 18 , characterized in that this mutation lies between amino acids 1 and 15 of the N-terminal region.  
     
     
         20 . Variant according to  claim 19 , characterized in that this mutation lies between amino acids 1 and 10 of the N-terminal region.  
     
     
         21 . Variant according to  claim 20 , characterized in that this mutation lies between amino acids 5 and 10 of the N-terminal region.  
     
     
         22 . Variant of herpes simplex virus type 1 thymidine kinase comprising at least one substitution at position 60 of a methionine by an isoleucine and a substitution at position 10 of an alanine by a threonine.  
     
     
         23 . Variant of herpes simplex virus type 1 thymidine kinase comprising at least one substitution at position 60 of a methionine by an isoleucine and a substitution at position 6 of a glycine by a serine.  
     
     
         24 . Variant of a wild-type thymidine kinase, characterized in that it is the mutant 2-865:H12.  
     
     
         25 . Variant of a wild-type thymidine kinase, characterized in that it is the mutant 2-3361:D3.  
     
     
         26 . Variant according to  claims 14  to  23 , comprising, in addition, at least one mutation in the C-terminal region.  
     
     
         27 . Variant according to  claim 26 , characterized in that this mutation lies between amino acids 320 and 350 of the C-terminal region.  
     
     
         28 . Variant according to  claim 27 , characterized in that this mutation lies between amino acids 325 and 345 of the C-terminal region.  
     
     
         29 . Variant according to  claim 28 , characterized in that this mutation lies between amino acids 330 and 343 of the C-terminal region.  
     
     
         30 . Variant according to  claim 29 , characterized in that this mutation lies between amino acids 335 and 340 of the C-terminal region.  
     
     
         31 . Variant of herpes simplex virus type 1 thymidine kinase comprising at least one substitution at position 60 of a methionine by an isoleucine, a substitution at position 10 of an alanine by a threonine and a substitution at position 337 of an arginine by a glutamine.  
     
     
         32 . Variant of a wild-type thymidine kinase, characterized in that it is the mutant 3-4216:H2.  
     
     
         33 . Variant of a wild-type thymidine kinase, characterized in that it displays the following kinetic properties: 
 a substantial or even complete decrease in the inhibition of the activity of phosphorylation of ganciclovir contrary to the wild-type enzyme for which the inhibition is very marked at and above 15 μM;    an increase by at least a factor of 2 to 2.5 in the initial rate of phosphorylation of GCV at and above 15-20 μM, relative to the wild-type enzyme and    a Kcat/Km ratio for thymidine which is reduced by a factor of 1 to 6 relative to that of the wild-type enzyme.    
     
     
         34 . Variant of a thymidine kinase according to  claims 13  to  25 , characterized in that it displays at least one of the following kinetic performance features: 
 a significant decrease in the inhibition of the phosphorylation of ganciclovir or of the nucleoside analogue at high concentrations of ganciclovir or of nucleoside analogue;  
 a rate of phosphorylation of ganciclovir or of nucleoside analogue, which is at least tripled, and/or  
 a Kcat/Km ratio comes out unchanged either for thymidine reduced by a factor greater than or equal to 5 relative to that of the wild-type enzyme.  
 
     
     
         35 . Variant of a thymidine kinase according to  claims 26  to  32 , characterized in that it displays at least one of the following kinetic performance features: 
 an absence of inhibition of the phosphorylation of ganciclovir or of the nucleoside analogue at high concentrations of ganciclovir or of nucleoside analogue,  
 an increase by a factor greater than 3.5 in the initial rate of phosphorylation of GCV at and above 15-20 μM, relative to the wild-type enzyme.  
 a Kcat/Km ratio for thymidine which is decreased by a factor of 4, relative to that of the wild-type enzyme.  
 
     
     
         36 . Expression cassette comprising a nucleic acid according to one of  claims 1  to  11 , a promoter permitting its expression and a transcription termination signal.  
     
     
         37 . Vector comprising a nucleic acid according to one of  claims 1  to  11  or a cassette according to  claim 36 .  
     
     
         38 . Vector according to according to  claim 37 , characterized in that it is a viral vector.  
     
     
         39 . Vector according to  claim 38 , characterized in that it is a defective recombinant adenovirus.  
     
     
         40 . Vector according to  claim 38 , characterized in that it is a defective recombinant retrovirus.  
     
     
         41 . Vector according to  claim 38 , characterized in that it is a defective recombinant AAV.  
     
     
         42 . Vector according to  claim 38 , characterized in that it is a defective recombinant HSV.  
     
     
         43 . Vector according to  claim 37 , characterized in that it is a chemical or biochemical vector.  
     
     
         44 . Pharmaceutical composition comprising a nucleic acid according to any one of  claims 1  to  11 , and/or a vector according to one of  claims 37  to  43 .  
     
     
         45 . Pharmaceutical composition comprising a variant according to one of  claims 12  to  35 .  
     
     
         46 . Pharmaceutical composition according to either of claims  45  and  46 , for the treatment of hyperproliferative disorders.