Well-tolerated and highly specific tailored recombinase for recombining asymmetric target sites in a plurality of retrovirus strains
The present invention relates to a method for preparing an expression vector encoding a well-tolerated and highly specific 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 which may be inserted into the genome of a host cell, as well as to the obtained expression vector, cells transfected with these, 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, HIV infection. In particular, the invention relates to well-tolerated and highly specific tailored recombinases capable of combining asymmetric target sequences in a more than 90% of HIV-strains, thereby excising the HIV-1 sequences, and expression vectors encoding them.
1. A nucleic acid encoding a tailored recombinase, which tailored recombinase is capable of recombining the asymmetric target sequence 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 95% sequence identity to SEQ ID NO:10, wherein said tailored recombinase comprises all of the following defined amino acid exchanges as compared to SEQ ID NO:6: V7L, P12S, P15L, M30V, H40R, M44V, S51T, Y77H, K86N, Q89L, G93A, S108G, C155G, A175S, A249V, R259D, E262R, T268A, D278G, P307A, N317T, and I320S.
2. The nucleic acid according to claim 1 , wherein the tailored recombinase comprises the amino acid sequence of SEQ ID NO:10.
3. The nucleic acid according to claim 1 , wherein the tailored recombinase comprises the amino acid sequence of SEQ ID NO:11, 12, or 13.
4. The nucleic acid of claim 1 , wherein the tailored recombinase does not recombine loxP (SEQ ID NO:4) or loxH (SEQ ID NO:5) sequences with detectable activity.
5. A transformed cell comprising the nucleic acid of claim 1 .
6. A pharmaceutical composition comprising the nucleic acid of claim 1 .
7. The pharmaceutical composition of claim 6 , wherein the pharmaceutical composition is for use in treatment or prevention of retrovirus infection in a subject, wherein the retrovirus is HIV, and wherein the pharmaceutical composition is optionally formulated for administration to a subject, if 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.
8. A method for preparing a tailored recombinase, comprising: expressing the tailored recombinase from the nucleic acid of claim 1 inserted into an 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.
9. A method for preparing a transformed cell, comprising: introducing an expression vector that comprises the nucleic acid of claim 1 into a cell in vitro.
10. A pharmaceutical composition comprising a transformed cell according to claim 5 .
11. The nucleic acid of claim 1 , wherein the amino acid sequence of the tailored recombinase has at least 99% sequence identity to SEQ ID NO:10.
12. The method of claim 9 , wherein the cell is an adult stem cell.
13. The transformed cell of claim 5 , where the cell is a stem cell from the hematopoietic lineage.
14. A tailored recombinase encoded by the nucleic acid of claim 1 .
15. The tailored recombinase of claim 14 , wherein the tailored recombinase is expressed as a fusion protein.
16. A pharmaceutical composition comprising a tailored recombinase according to claim 14 .
17. The pharmaceutical composition of claim 7 , wherein the retrovirus is HIV-1.