Well-tolerated and highly specific 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 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.

Claims

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.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: FIG. 1 provides an alignment of the protein sequences of (a) Cre recombinase SEQ ID NO:6; (b) Tre common consensus sequence, SEQ ID NO:7, consensus sequence of Tre recombinases specific for asymmetric target-sites within HIV-1 LTR according to WO 2011/147590; (c) Tre recombinase 3.0 consensus sequence, SEQ ID NO:8, Tre recombinases specific for SEQ ID NO:1 according to WO 2011/147590; (d) Tre recombinases 3.1 consensus sequence 85%, SEQ ID NO:9, the individual mutations in the consensus sequence versus the Cre sequence are present in 85% of all clones generated by the method of the present invention and analyzed by high-throughput sequencing (33,000 reads of unique 200 bp-sequences); (e) Tre recombinase 3.1 consensus sequence 100%, SEQ ID NO:10, consensus sequence of three Tre recombinases 3.1 selected for high specificity for SEQ ID NO:1 (no activity on loxP, loxH or loxLTR Tre 1.0 (SEQ ID NOs: 3-5) and tolerability by humans; and (f) exemplary Tre 3.1 recombinase uTre, SEQ ID NO:11, highly specific for SEQ ID NO:1 and well-tolerated by humans. The tailored recombinase according to this sequence is designated uTre, respectively universal Tre. Bold letter patches indicate conserved amino acids, variable and non-specified positions are indicated with an X. Positions of Cre mutated in SEQ ID NO: 9. 10 and/or 11 are underlined in the Cre sequence, and the position of the mutation is provided above the sequences. The Q89L exchange uniquely found in Tre3.1 is marked in italics.

(2) FIG. 2: FIG. 2 shows an exemplary evolution vector for evolutionary selecting against recombination on loxP. Corresponding vectors for selecting against recombination on loxH can easily be constructed. loxLTR comprises SEQ ID NO: 1. Tre3 evolution cycles 51-69 were performed with pEVOloxLTR-loxP and pEVOloxLTR-loxH, alternately, 100-1 g/mL of the transcription activator L-ara, including two rounds of DNA shuffling).

(3) FIG. 3: FIG. 3 shows the high specificity of uTre vs. Tre3. Tre3: Clone was isolated from Tre3.0 library cycle 43. uTre: Clone was isolated from Tre3.1 library cycle 71. Recombined product is marked with one triangle, non-recombined with two triangles. Under conditions where the tailored recombinase is expressed (induction by L-arabinose), uTre recombines loxLTR comprising SEQ ID NO: 1 in E. coli. In contrast, Tre3 recombines loxLTR and loxP and loxH, i.e., it has a relatively relaxed specificity.

(4) FIG. 4: FIG. 4A shows an exemplary lentiviral expression vector for constitutive expression of a selectable marker, EGFP, and the tre library in human cells. FIG. 4B shows the flow scheme for the cellular screening for well-tolerable highly specific Tre. The final screening for highly active Tre confirms activity in the human cells. Single clones of recombinases were selected and were subjected to further analyses.

(5) FIGS. 5A-5B show antiviral uTre activity in tissue culture in two representative cultures. PM1 T cells were transduced with vectors encoding GFP alone (Control, open circles) or encoding uTre and GFP (uTre, filled squares). Subsequently, cultures were infected with HIV-1 and viral load was monitored over time using p24 Antigen ELISA. The experiment shows that the tailored recombinase is efficient in reducing viral load. After several weeks (8 or 9 weeks), viral load is not any more detectable by a p24 antigen ELISA.

(6) FIG. 6 Pronounced antiviral uTre activity in primary human CD4+ cells derived from a HIV-infected patient is shown. Cells were transduced with a vector expressing GFP alone (Control experiment; left panel) or with a vector expressing uTre and GFP (uTre; right panel). Virus replication was monitored by HIV-1 p24 antigen release (open circles) and percent of transduced (GFP+) human CD4+ cells (filled squares) was monitored by FACS. Expression of uTre resulted in pronounced antiviral effect and protection of CD4+ cells. Of note, the decline in viral load between day 15 and day 20 in the control experiment reflects cell death due to uninhibited virus replication.

(7) FIG. 7 shows antiviral uTre activity in HIV-infected humanized mice Immunodeficient mice were engrafted with human CD34+ hematopoietic stem cells/HSC (Control), or with uTre-expressing CD34+ HSC (Animal/41 and #2). Subsequently, animals were HIV-1 infected and viral load (detected as HIV-1 RNA copies/ml; open circles) and the percentage of human CD45+CD4+ cells of all lymphocytes (filled squares) were monitored over time.

EXAMPLES

Example 1

(8) Materials and methods as described in WO 2008/083931, WO 2011/147590 and BUCHHOLZ & STEWART, 2001 are used, if not specified otherwise. Tailored recombinases capable of recombining asymmetric target sequences in a plurality of different HIV-1 strains were prepared as described in WO 2011/147590. The resulting tre libraries were employed in further experiments.

Example 2

(9) To enhance uTre specificity, additional evolution cycles selecting against recombination activity on loxP and loxH were performed. For this purpose the evolved Tre library obtained from evolution cycle 50 was cloned into an evolution vector containing the two loxLTR sites (SEQ ID NO: 1) intertwined with two loxP sites or two loxH sites, respectively. An exemplary vector is shown in FIG. 2. Upon induction of recombinase expression, recombination on loxLTR resulted in the removal of the only present NdeI site whereas recombination on loxP or loxH did not. Plasmid DNA isolated after each evolution cycle was digested with NdeI and the recombinase coding sequences that had successfully recombined loxLTR rather than loxP or loxH were amplified by PCR and subcloned back into the evolution vector for the next evolution cycle. A total of 19 additional Tre3 evolution cycles, including two rounds of DNA shuffling, were performed, alternately selecting against recombination on loxP and loxH.

Example 3

(10) To screen for uTre-recombinases with significantly diminished cell toxicity (i.e. cytopathicity), the tre libraries were ligated into a lentiviral vector that constituively expresses EGFP from an internal SFFV LTR promoter and the tre library from the constitutive EF1alpha promoter (FIG. 4A). Transduction of Jurkat T cells allowed the sequential sorting (by FACS) of high GFP-expressing cells and subsequent isolation of non-toxic uTre clones (FIG. 4B). For this, cell sorts on the transduced T cell cultures were performed at day 3, day 10 and day 24 post transduction with increased stringency on EGFP expression. After another week of culturing, the remaining tre library was isolated and selected clones were analyzed with respect to enhanced Tre activity.

Example 4

(11) To analyze uTre activity in cell lines, cultures of PM-1 T cells were transduced with ASLV-derived retroviral vectors expressing either uTre and GFP, or GFP alone (negative control vector). Of note, GFP expression allowed the tracking of transduced cells. At 10 days post transduction, cells were infected with HIV-1.sub.Bal. The effect of uTre expression on HIV-1 replication was monitored by weekly ELISA measurements of the amount of viral p24 antigen in the culture supernatants. As shown (FIG. 5), p24 release decreased remarkably in the uTre-transduced cultures, whereas it stays stable or even increases in the control cultures (expressing GFP alone).

Example 5

(12) Analysis of uTre activity in primary CD4+ cells derived from an HIV-1-infected patient. CD4+ cells were stimulated with CD3/CD28 magnetic beads for 48 h. Subsequently, cells were transduced with lentiviral vectors either expressing GFP alone (serving as negative control) or expressing uTre together with GFP. Cells were cultured in the presence of 100 IU of IL2 for 20 days. Viral loads (measured by p24 antigen ELISA) and human transduced CD4+ cell counts (analyzed by FACS) were monitored at the indicated days post transduction. As shown in FIG. 6, uTre expression results in pronounced antiviral effect (indicated by open circles) and protection of CD4+ cells (indicated by filled squares). In contrast, decline in viral load between day 15 and day 20 in the control experiment reflects cell death due to uninhibited virus replication.

Example 6

(13) Analysis of uTre activity in vivo. Immunodeficient NOG mice (NOD.Cg-Prkdc.sup.scidIL2rg.sup.tmlWjlSzJ) were engrafted with human CD34+ hematopoietic stem cells/HSC (Control), or with uTre-expressing CD34+ HSC. Subsequently, animals were infected with HIV-1.sub.Bal and viral load (detected by ultrasensitive PCR-based assay) and percent human CD45+CD4+ cells (analyzed by FACS) were monitored over time. As shown (FIG. 7) uTre expression resulted in significant antiviral activities in vivo.

REFERENCE LIST

(14) Abremski K, Hoess R H, Sternberg N (1983) Studies on the properties of PI site-specific recombination: evidence for topologically unlinked products following recombination. Cell 32, 1301-1311. Abremski K, Hoess R (1983) Bacteriophage PI site-specific recombination. Purification and properties of the Cre recombinase protein. J Biol. Chem. 259, 1509-1514. Adachi A, Gendelman H E, Koenig S, Folks T, Willey R, Rabson A, Martin M A (1986) Production of acquired immunodeficiency syndrome-associated retrovirus in human and non-human cells transfected with an infectious molecular clone. J Virol. 59, 284-291. Alper H, Fischer C, Nevoigt E, Stephanopoulos G (2006) Tuning genetic control through promoter engineering Proc. Natl. Acad. Sci. USA 102, 12678-12683. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D. J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic acids research, 25, 3389-3402. Beyer W R, Westphal M, Ostertag W, von Laer D (2002)Oncoretrovirus and lentivirus vectors pseudotyped with lymphocytic choriomeningitis virus glycoprotein: generation, concentration and broad host range. J Virol. 76, 1488-1495. Blackard J T, Renjifo B R, Mwakagile D, Montano M A, Fawzi W W, Essex M (1999) Transmission of human immunodeficiency type 1 viruses with intersubtype recombinant long terminal repeat sequences. Virology 254, 220-225. Bloom J D, Meyer M M, Meinhold P, Otey C R, MacMillan D, Arnold F H (2005) Evolving strategies for enzyme engineering. Curr. Opin. Struct. Biol. 15, 447-452. Buchholz F, Ringrose L, Angrand P O, Rossi F, Stewart A F (1996) Different thermostabilities of FLP and Cre recombinases: implications for applied site-specific recombination. Nucl. Acids Res. 24, 4256-4262. Buchholz F, Angrand P O, Stewart A F (1998) Improved properties of FLP recombinase evolved by cycling mutagenesis. Nat. Biotechnol. 16, 657-662. Buchholz F, Stewart A F (2001) Alteration of Cre recombinase site specificity by substrate-linked protein evolution. Nat. Biotechnol. 19, 1047-1052. Chiu Y L, Soros V B, Kreisberg J F, Stopak K, Yonemoto W, Greene W C (2005) Cellular APOBEC3G restricts HIV-1 infection in resting CD4+ T cells. Nature 435, 108-114 Chun T-W, Engel D, Berrey M M, Shea T, Corey L, Fauci A S (1998) Early establishment of a pool of latently infected, resting CD4+ T cells during primary HIV-1 infection. Proc. Natl. Acad. Sci. USA 95, 8869-8873. Coates C J, Kaminski J M, Summers J B, Segal D J, Miller A D, Kolb A F (2005) Site-directed genome modification: derivatives of DNA-modifying enzymes as targeting tools. Trends Biotechnol. 23, 407-419. Collins C H, Yokobayashi Y, Umeno D, Arnold F H, (2003) Engineering proteins that bind, move, make and break DNA. Curr. Opin. Biotechnol. 14, 665. Combes P, Till R, Bee S, Smith M C (2002) The streptomyces genome contains multiple pseudo-attB sites for the (phi)C31-encoded site-specific recombination system. J Bacteriol. 184, 5746-5752. Crameri A, Raillard S A, Bermudez E, Stemmer W P (1998) DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391, 288-291. Derossi D, Joliot A H, Chassaing G, Prochiantz A (1994) The third helix of the Antennapedia homeodomain translocates through biological membranes. J Biol Chem. 269, 10444-10450. Derossi D, Calvet S, Trembleau A, Chassaing G, Prochiantz A (1996) Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J Biol Chem 271, 18188-18193. Donovan, P. J., Gearhart, J. (2001) The end of the beginning for pluripotent stem cells. Nature 414, 92-97. Donzella G A, Schols D, Lin S W, Este J A, Nagashima K A, Maddon P J, Allaway G P, Sakmar T P, Henson G, De Clercq E, Moore J P (1998) AMD3100, a small molecule inhibitor of HIV-1 entry via the CXCR4 co-receptor. Nature Medicine 4, 72-77. Dybul M, Fauci A S, Bartlett J G, Kaplan J E, Pau A K (2002) Guidelines for using antiretroviral agents among HIV-infected adults and adolescents. Annals of Internal Medicine 137, 381-433. Eddy, S. R. (1998) Profile hidden Markov models. Bioinformatics (Oxford, England), 14, 755-763. Edelman G M, Meech R, Owens G C, Jones F S (2000) Synthetic promoter elements obtained by nucleotide sequence variation and selection for activity. Proc. Natl. Acad. Sci. USA 97, 3038-3043. Elliott G, O'Hare P., Intercellular trafficking and protein delivery by a herpesvirus structural protein, Cell. 1997 Jan. 24; 88(2):223-33 Emerman M, Malim M H (1998) HIV-1 regulatory/accessory genes: keys to unraveling viral and host cell biology. Science 280, 1880-1884. Fawell S, Seery J, Daikh Y, Moore C, Chen L L, Pepinsky B, Barsoum J., Tat-mediated delivery of heterologous proteins into cells. Proc Natl Acad Sci USA. 1994 Jan. 18; 91(2):664-8. Finzi D, Hemankova M, Pierson T, Carruth L M, Buck C, Chaisson R E, Quinn T C, Chadwick K, Margolick J, Brookmeyer R, Gallant J, Markowitz M, Ho D D, Richman D D, Siliciano R F (1997) Identification of a reservoir for HIV-1 in patients on highly active antiretro viral therapy. Science 278, 1295-1300. Flowers C C, Woffendin C, Petryniak J, Yang S, Nabel G J (1997) Inhibition of recombinant human immunodeficiency virus type 1 replication by a site-specific recombinase. J. Virol. 71, 2685-2692. Gulick R M, Mellors J W, Havlir D, Eron J J, Gonzalez C, McMahon D, Richman D D, Valentine F T, Jonas L, Meibohm A, Emini E A, Chodakewitz J A (1997) Treatment with indinavir, zidovudine, and lamivudine in adults with human immunodeficiency virus infection and prior antiretroviral therapy. N Engl. J. Med. 337, 734-739. Guzman L M, Belin D, Carson M J, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. Ill, 4121-4130. Hartenbach S, Fussenegger M (2006) A novel synthetic mammalian promoter derived from an internal ribosome entry site. Biotechnology and Bioengineering 95, 547-559. Hauber I, Bevec D, Heukeshoven J, Kratzer F, Horn F, Choidas A, Harrer T, Hauber J (2005) Identification of cellular deoxyhypusine synthase as a novel target for antiretroviral therapy. J. Clin. Invest. 115, 76-85. Hazuda D J, Young S D, Guare J P, Anthony N J, Gomez R P, Wai J S, Vacca J P, Handt L, Motzel S L, Klein H J, Dornadula G, Danovich R M, Witmer M V, Wilson K A, Tussey L, Schleif W A, Gabryelski L S, Jin L, Miller M D, Casimiro D R, Emini E A, Shiver J W (2004) Integrase inhibitors and cellular immunity suppress retroviral replication in rhesus macaques. Science 305, 528-532. Hoess R H, Abremski K (1985) Mechanism of strand cleavage and exchange in the Cre-lox site-specific recombination system. J. Mol. Biol. 181, 351-362. Johannes T W, Zhao H (2006) Directed evolution of enzymes and biosynthetic pathways. Curr. Opin. Microbiol. 9, 261-267. Krasnow M A, Cozzarelli N R (1983) Site-specific relaxation and recombination by the Tn3 resolvase: Recognition of the DNA path between oriented res sites. Cell 32, 1313-1324. Kulkosky J, Bray S (2006) HAART-persistent HIV-1 latent reservoirs: their origin, mechanisms of stability and potential strategies for eradication. Cuff. HIV Res. 4, 199-208. Lalezari J P, Henry K, O'Hearn M, Montaner J S, Piliero P J, Trottier B, Walmsley S, Cohen C, Kuritzkes D R, Eron Jr. J J, Chung J, DeMasi R, Donatacci L, Drobnes C, Delehanty J, Salgo M (2003) Enfuvirtide, an HIV-1 fusion inhibitor, for drug-resistant HIV infection in North and South America. N. Engl. J. Med. 348, 2175-2185. Lee Y S, Park J S (1998) A novel mutant lox? containing part of long terminal repeat of HIV-1 in spacer region: presentation of possible target site for antiviral strategy using site-specific recombinase. Biochem. Biophys. Res. Comm. 253, 588-593. Lee Y S, Kim S T, Kim G W, Lee M, Park J S (2000) An engineered lox sequence containing part of a long terminal repeat of HIV-1 permits Cre recombinase-mediated DNA excision. Biochem. Cell Biol. 78, 653-658. Lehrman G, Hogue I B, Palmer S, Jennings C, Spina C A, Wiegand A, Landay A L, Coombs R W, Richman D D, Mellors J W, Coffin J M, Bosch R J, Margolis D M (2005) Depletion of latent HIV-1 infection in vivo: a proof-of-concept study Lancet 366, 549-555. Lewandoski, M. (2001) Conditional control of gene expression in the mouse. Nat. Rev. Genet. 2, 743-755. Lin Q, Jo D, Gebre-Amlak K D, Ruley H E (2004) Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells. BMC Biotechnol. 4, 25. Little S J, Holte S, Routy J P, Daar E S, Markowitz M, Collier A C, Koup R A, Mellors J W, Connick E, Conway B, Kilby M, Wang L, Whitcomb J M, Hellmann N S, Richman D D (2002) Antiretroviral-drug resistance among patients recently infected with HIV. N. Engl. J. Med. 347, 385-394. Loonstra A, Vooijs M, Beverloo H B, Allak B A, van Drunen E, Kanaar R, Berns A, Jonkers J (2001) Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. PNAS 98, 9209-9214. Macara I G (2001) Transport into and out of the nucleus. Microbiology and molecular biology reviews 65, 570-594. Malim M H, Hauber J, Fenrick R, Cullen B R (1988) Immunodeficiency virus rev trans-activator modulates the expression of the viral regulatory genes. Nature 335, 181-183. Marcello A (2006) Latency: the hidden HIV-1 challenge. Retrovirology 3, 7. Matsumura I, Ellington A D (2001) In vitro evolution of beta-glucuronidase into a beta-galactosidase proceeds through non-specific intermediates. J. Mol. Biol. 305, 331-339. Minshull J, Stemmer W P. (1999) Protein evolution by molecular breeding. Curr. Opin. Chem. Biol. 3, 284-290. Nagy A (2000) Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99-109. Needleman S B, Wunsch C D (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol. 48, 443-453. Nolden L, Edenhofer F, Haupt S, Koch P, Wunderlich F T, Siemen H, Brustle O. (2006) Site-specific recombination in human embryonic stem cells induced by cell-permeant Cre recombinase. Nat. Methods 3, 461-467. Oess S, Hildt E., Novel cell permeable motif derived from the PreS2-domain of hepatitis-B virus surface antigens, Gene Ther. 2000 May; 7(9):750-8 O'Doherty U, Swiggard W J, Malim M H (2000) Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding. J Virol. 74, 10074-10080. Pearson W R, Lipman D J (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85, 2444-2448. Peitz M, Pfannkuche K, Rajewsky K, Edenhofer F. (2002) Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: A tool for efficient genetic engineering of mammalian genomes. Proc. Natl. Acad. Sci. USA 99, 4489-4494. Ratner L, Starcich B, Josephs S F, Hahn B H, Reddy E P, Livak K J, Petteway S R, Jr., Pearson M L, Haseltine W A, Arya S K, (1985) Polymorphism of the 3 open reading frame of the virus associated with the acquired immune deficiency syndrome, human T-lymphotropic virus type III. Nucl. Acids Res. 13, 8219-8229. Richard J P, Melikov K, Brooks H, Prevot P, Lebleu B, Chemomordik L V (2005) Cellular uptake of the unconjugated TAT peptide involves clathrin-dependent endocytosis and heparin sulfate receptors. J. Biol. Chem. 280, 15300-15306. Rufer A W, Sauer B (2002) Non-contact positions impose site selectivity on Cre recombinase. Nucl. Acids Res. 30, 2764-2771. Ruhl M, Himmelspach M, Bahr G M, Hammerschmid F, Jaksche H, Wolff B, Aschauer H, Farrington G K, Probst H, Bevec D, Hauber J (1993) Eukaryotic initiation factor 5 A is a cellular target of the human immunodeficiency virus type 1 Rev activation domain mediating trans-activation J Cell Biol. 123, 1309-1320. Sanger F, Nickler S, Coulson A R (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. Santoro S W, Schultz P G (2002) Directed evolution of the site specificity of Cre recombinase. Proc. Natl. Acad. Sci. USA 99, 4185-4190. Saraf-Levy T, Santoro S W, Volpin H, Kushnirsky T, Eyal Y, Schultz P G, Gidoni D, Carmi N (2006) Site-specific recombination of asymmetric lox sites mediated by a heterotetrameric Cre recombinase complex. Bioorg. Med. Chem. 14, 3081-3089. Sauer B, McDermott J (2004) DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-cl regions of PI-related phages. Nucl. Acids. Res. 32, 6086-6095. Schambach A, Bohne J, Chandra S, Will E, Margison G P, Williams D A, Baum C (2006) Equal potency of gammaretro viral and lenti viral SIN vectors for expression of O.sub.6-methylguanine-DNA methyltransferase in hematoietic cells. Molecular Therapy 13, 391-400. Scherr M, Eder M (2002) Gene Transfer into Hematopoietic Stem Cells Using Lentiviral Vectors. Current Gene Therapy 2, 45-55. Shehu-Xhilaga M, Tachedjian G, Crowe S M, Kedzierska K. (2005) Antiretro viral compounds: mechanisms underlying failure of HAART to eradicate HIV-1. Curr. Med. Chem. 12, 1705-1719. Shimshek D R, Kim J, Hubner M R, Spergel D J, Buchholz F, Casanova E, Stewart A F, See-burg P H, Sprengel R (2002) Codon-improved Cre recombinase (iCre) expression in the mouse. Genesis 32(1), 19-26. Smith T f, Waterman M S (1981) Overlapping genes and information theory. J Theor. Biol. 91, 379-380. Stark W M, Boocock M R, Sherratt D J (1992) Catalysis by site-specific recombinases. Trends Genet. 8, 432-439. Stemmer WPC (1994) Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389-391. Sternberg N, Hamilton D (1981) Bacteriophage P I site-specific recombination. I. Recombination between loxP sites. J. Mol. Biol. 150, 467-486. Van Duyne G D (2001) A structural view of cre-loxp site-specific recombination. Annu. Rev. Biophys. Biomol. Struct. 30, 87-104. Vives E, Brodin P, Lebleu B (1997) A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 272, 16010-16017. Vives E (2003) Cellular uptake of the TAT peptide: an endocytosis mechanism following<> ionic interactions. J. Mol. Recognit. 16, 265-271. Volkert F C, Broach J R (1986) Site-specific recombination promotes plasmid amplification in yeast. Cell 46, 541-550. Voziyanov Y, Konieczka J H, Stewart A F, Jayaram M (2003) Stepwise manipulation of DNA specificity in Flp recombinase: progressively adapting Flp to individual and combinatorial mutations in its target site. J. Mol. Biol. 326, 65-76. Yuan L, Kurek I, English J, Keenan R (2005) Laboratory-directed protein evolution Microbiol. Mol. Biol. Rev. 69, 373-92. WO 2002/44409 WO 2008/083931 WO 2011/147590.