Tailored recombinase for recombining asymmetric target sites in a plurality of retrovirus strains

Abstract

The present invention relates to a method for preparing an expression vector encoding a tailored recombinase, which tailored recombinase is capable of recombining asymmetric target sequences within the long terminal repeat (LTR) of proviral DNA of a plurality of retrovirus strains inserted into the genome of a host cell, as well as to the obtained expression vector, cells transfected with this, expressed recombinase and pharmaceutical compositions comprising the expression vector, cells and/or recombinase. Pharmaceutical compositions are useful, e.g., in treatment and/or prevention of retrovirus infection. In particular, asymmetric target sequences present in a plurality of HIV strains are disclosed, as well as tailored recombinases capable of combining these sequences (Tre 3.0 and 4.0) and expression vectors encoding them.

1 .- 17 . (canceled) 
     
     
         18 . A method for preparing an expression vector encoding a tailored recombinase, which tailored recombinase is capable of recombining asymmetric target sequences of SEQ ID NO:1 within the long terminal repeat (LTR) of proviral DNA of a plurality of HIV-1 strains, comprising the steps of
 (a) identifying SEQ ID NO:1 sequences with a homology of at least 30% to the left half-site sequence and the right half-site sequence of at least one known recombinase target site in the sequence of the LTR of proviral DNA of a plurality of retrovirus strains, wherein the homologous sequences are separated by a spacer of 5-12 nucleotides, and represent an asymmetric target sequence within the LTR of proviral DNA, and wherein the asymmetric target sequence is found in a plurality of retrovirus strains;   (b) identifying two sequences, wherein the first sequence corresponds to the sequence of the asymmetric target sequence of step (a) homologous to the left half-site of said known target site and is referred to as “half-site sequence 1”, and wherein the second sequence corresponds to the sequence of the asymmetric target sequence of step (a) homologous to the right half-site and is referred to as “half-site sequence 2”;   (c) determining the nucleotides within the sequences of step (b) deviating from the corresponding homologous left half-site and right half-site sequences of the at least one known homologous target site of step (a);   (d) generating a first subset of two target nucleic acids comprising target sequences, wherein the first target sequence is designated subsite 1 and comprises, adjacent to each other and in 5′ to 3′ order, half-site sequence 1 of step (b), the spacer sequence of the asymmetric target sequence and an inverted repeat of half-site sequence 1, and wherein the second target sequence is designated subsite 2 and comprises, adjacent to each other and in 5′ to 3′ order, an inverted repeat of half-site sequence 2, the spacer sequence of the asymmetric target sequence and half-site sequence 2 of step (b);   (e) generating a second subset of target nucleic acids comprising modified target sequences on the basis of the target sequences in the first subset of step (d),
 wherein, in sequences based on subsite 1, in the left half-site sequence, a portion of the nucleotides deviating from the corresponding homologous half-site sequence of the at least one known target-site of step (a) is replaced by the native nucleotides found in said known target-site, until said half-site sequence contains one, two or three nucleotides deviating from said known target site, wherein the right half-site of said modified target sequence is formed by an inverted repeat of said modified left half-site sequence, which is separated from said modified left half-site sequence by the spacer sequence of the asymmetric target sequence, and 
 wherein, in sequences based on subsite 2, in the right half-site sequence, a portion of the nucleotides deviating from the corresponding homologous half-site sequence of the at least one known target-site of step (a) is replaced by the native nucleotides found in said known target-site, until said half-site sequence contains one, two or three nucleotides deviating from said known target site, wherein the left half-site of said modified target sequence is formed by an inverted repeat of said modified right half-site sequence, which is separated from said modified right half-site sequence by the spacer sequence of the asymmetric target sequence, 
 such that in all modified half-site sequences originating from one target sequence of the first subset of step (d) taken together, all deviating nucleotides can be found, whereas none of said modified half-site sequences alone comprises all deviating nucleotides, 
   (f) separately applying molecular directed evolution on at least one recombinase recognising a known homologous target site according to step (a) using each nucleic acid of the second subset obtained in step (e) as a substrate;   (g) shuffling the recombinase libraries evolved in step (f), wherein all recombinase libraries evolved on sequences based on subsite 1 are combined and shuffled, and wherein all recombinase libraries evolved on sequences based on subsite 2 are combined and shuffled;   (h) applying molecular directed evolution on the shuffled libraries obtained in step (g) using each nucleic acid of the subset according to step (d) as a substrate;   (i) shuffling the recombinase libraries evolved in step (h);   (j) applying molecular directed evolution on the shuffled library obtained in step (g) using a nucleic acid comprising the asymmetric target sequence of step (a) as a substrate, until at least one recombinase is obtained that is active on the asymmetric target sequence within the LTR of the proviral DNA of step (a);   (k) isolating the nucleic acid encoding the least one recombinase obtained in step (j) from the library; and   (l) optionally, cloning the nucleic acid obtained in step (k) into a suitable expression vector.   
     
     
         19 . The method according to  claim 18 , wherein the at least one known recombinase whose target sequence is used in step (a) and upon which molecular directed evolution is applied in step (f) is part of a recombinase library. 
     
     
         20 . The method according to  claim 18 , wherein the molecular directed evolution employed is substrate-linked protein evolution. 
     
     
         21 . The method according to  claim 18 , wherein the expression vector in step (l) is selected from the group consisting of retroviral vectors, lentiviral vectors, spumavirus vectors and adenoviral vectors. 
     
     
         22 . The method according to  claim 18 , further comprising the step of preparing the expression vector as a pharmaceutical composition. 
     
     
         23 . A method for preparing a tailored recombinase, comprising steps (a) to (l) according to  claim 18  and the further step of expressing the tailored recombinase from the nucleic acid encoding the recombinase inserted into the expression vector in a suitable host cell, wherein the recombinase is optionally expressed as a fusion polypeptide comprising the amino acid sequence of the tailored recombinase. 
     
     
         24 . The method according to  claim 23 , further comprising the step of preparing the tailored recombinase as a pharmaceutical composition. 
     
     
         25 . A method for preparing a transformed cell, comprising steps (a) to (l) according to  claim 18  and the further step of introducing the expression vector in vitro into a cell. 
     
     
         26 . The method according to  claim 25 , wherein the cell is an adult stem cell. 
     
     
         27 . The method according to  claim 25 , further comprising the step of preparing the transformed cell as a pharmaceutical composition. 
     
     
         28 . A nucleic acid encoding a tailored recombinase, which tailored recombinase is capable of recombining asymmetric target sequences of SEQ ID NO:1 within the long terminal repeat (LTR) of proviral DNA of a plurality of HIV-1 strains, wherein the amino acid sequence of the tailored recombinase has at least 99% sequence identity to a sequence according to SEQ ID NO: 38, and wherein said tailored recombinase comprises defined amino acid exchanges P12S, P15L, M44V, K86N, G93A, A175S, ad P307A as compared to the Cre sequence of SEQ ID NO:36. 
     
     
         29 . A nucleic acid according to  claim 28 , wherein the amino acid sequence of the tailored recombinase comprises SEQ ID NO: 38. 
     
     
         30 . The nucleic acid according to  claim 28 , wherein the amino acid sequence of the tailored recombinase is selected from the group consisting of SEQ ID NOS: 40-64. 
     
     
         31 . The nucleic acid according to  claim 28 , wherein the amino acid sequence of the tailored recombinase is selected from a combination of sequences from the group consisting of SEQ ID NO: 40-64. 
     
     
         32 . The nucleic acid according to  claim 28 , wherein amino acid exchanges at undefined positions with regard to the sequence of Cre recombinase specified in SEQ ID NO:36 are conservative substitutions. 
     
     
         33 . The nucleic acid according to  claim 28 , wherein the nucleic acid is an expression vector. 
     
     
         34 . The nucleic acid according to  claim 28 , wherein the amino acid sequence of the tailored recombinase comprises the sequence according to SEQ ID NO: 39. 
     
     
         35 . A pharmaceutical composition comprising a nucleic acid of  claim 28 , a tailored recombinase encoded by a nucleic acid of  claim 28 , and/or a transformed cell comprising a nucleic acid of  claim 28 . 
     
     
         36 . A tailored recombinase encoded by a nucleic acid of  claim 28 , which is optionally expressed as a fusion protein. 
     
     
         37 . A transformed cell comprising a nucleic acid of  claim 28 . 
     
     
         38 . The transformed cell according to  claim 37 , wherein the cell is a stem cell from the hematopoietic lineage. 
     
     
         39 . A nucleic acid of  claim 28  as obtainable from a method that comprises:
 (a) identifying sequences with a homology of at least 30% to the left half-site sequence and the right half-site sequence of at least one known recombinase target site in the sequence of the LTR of proviral DNA of a plurality of HIV strains, wherein the homologous sequences are separated by a spacer of 5-12 nucleotides, and represent an asymmetric target sequence within the LTR of proviral DNA, and wherein the asymmetric target sequence is SEQ ID NO:1 and is found in a plurality of HIV strains; 
 (b) identifying two sequences, wherein the first sequence corresponds to the sequence of the asymmetric target sequence of step (a) homologous to the left half-site of said known target site (half-site sequence 1), and wherein the second sequence corresponds to the sequence of the asymmetric target sequence of step (a) homologous to the right half-site (half-site sequence 2); 
 (c) determining the nucleotides within the sequences of step (b) deviating from the corresponding homologous left half-site and right half-site sequences of the at least one known homologous target site of step (a); 
 (d) generating a first subset of two target nucleic acids comprising target sequences, wherein the first target sequence is designated subsite 1 and comprises, adjacent to each other and in 5′ to 3′ order, half-site sequence 1 of step (b), the spacer sequence of the asymmetric target sequence and an inverted repeat of half-site sequence 1, and wherein the second target sequence is designated subsite 2 and comprises, adjacent to each other and in 5′ to 3′ order, an inverted repeat of half-site sequence 2, the spacer sequence of the asymmetric target sequence and half-site sequence 2 of step (b); 
 (e) generating a second subset of target nucleic acids comprising modified target sequences on the basis of the target sequences in the first subset of step (d),
 wherein, in sequences based on subsite 1, in the left half-site sequence, a portion of the nucleotides deviating from the corresponding homologous half-site sequence of the at least one known target-site of step (a) is replaced by the native nucleotides found in said known target-site, until said half-site sequence contains one, two or three nucleotides deviating from said known target site, wherein the right half-site of said modified target sequence is formed by an inverted repeat of said modified left half-site sequence, which is separated from said modified left half-site sequence by the spacer sequence of the asymmetric target sequence, and 
 wherein, in sequences based on subsite 2, in the right half-site sequence, a portion of the nucleotides deviating from the corresponding homologous half-site sequence of the at least one known target-site of step (a) is replaced by the native nucleotides found in said known target-site, until said half-site sequence contains one, two or three nucleotides deviating from said known target site, wherein the left half-site of said modified target sequence is formed by an inverted repeat of said modified right half-site sequence, which is separated from said modified right half-site sequence by the spacer sequence of the asymmetric target sequence, 
 such that in all modified half-site sequences originating from one target sequence of the first subset of step (d) taken together, all deviating nucleotides can be found, whereas none of said modified half-site sequences alone comprises all deviating nucleotides, 
 
 (f) separately applying molecular directed evolution on at least one recombinase recognising a known homologous target site according to step (a) using each nucleic acid of the second subset obtained in step (e) as a substrate to form recombinase libraries; 
 (g) shuffling the recombinase libraries evolved in step (f), wherein all recombinase libraries evolved on sequences based on subsite 1 are combined and shuffled, and wherein all recombinase libraries evolved on sequences based on subsite 2 are combined and shuffled; 
 (h) applying molecular directed evolution on the shuffled libraries obtained in step (g) using each nucleic acid of the subset according to step (d) as a substrate to form recombinase libraries; 
 (i) shuffling the recombinase libraries evolved in step (h); 
 (j) applying molecular directed evolution on the shuffled libraries obtained in step (g) using a nucleic acid comprising the asymmetric target sequence of step (a) as a substrate, until at least one recombinase is obtained that is active on the asymmetric target sequence within the LTR of the proviral DNA of step (a); and 
 (k) isolating the nucleic acid encoding the least one recombinase obtained in step (j) from the library. 
 
     
     
         40 . A tailored recombinase encoded by a nucleic acid of  claim 39 , which is optionally expressed as a fusion protein. 
     
     
         41 . A transformed cell comprising a nucleic acid of  claim 39 . 
     
     
         42 . The transformed cell according to  claim 41 , wherein the cell is a stem cell from the hematopoietic lineage. 
     
     
         43 . A pharmaceutical composition comprising a nucleic acid of  claim 39 , a tailored recombinase encoded by a nucleic acid of  claim 39 , and/or a transformed cell comprising a nucleic acid of  claim 39 . 
     
     
         44 . A method for treating or preventing retrovirus infection in a subject, comprising administering to a subject a pharmaceutical composition according to  claim 35 , wherein proviral DNA found in a sample obtained from the subject comprises the asymmetric target sequence on which the recombinase has been selected. 
     
     
         45 . A method for treating or preventing retrovirus infection in a subject, comprising administering to a subject a pharmaceutical composition according to  claim 43 , wherein proviral DNA found in a sample obtained from the subject comprises the asymmetric target sequence identified in step (a) on which the recombinase has been selected. 
     
     
         46 . A nucleic acid encoding a tailored recombinase, which tailored recombinase is capable of recombining asymmetric target sequences of SEQ ID NO:1 within the long terminal repeat (LTR) of proviral DNA of a plurality of HIV-1 strains, wherein the amino acid sequence of the tailored recombinase has at least 97% sequence identity to a sequence according to SEQ ID NO: 39, and wherein said tailored recombinase comprises the defined amino acid exchanges P12S, P15L, M44V, K86N, G93A, A175S, ad P307A as compared to the Cre sequence of SEQ ID NO:36.