NEW TREATMENTS AND TARGETS P174 Universal Tre (uTre) recombinase specifically targets the majority of HIV-1 isolates Karpinski, J*; Chemnitz, J; Hauber, I; Abi-Ghanem, J; Paszkowski-Rogacz, M; Surendranath, V; Chakrabort, D; Hackmann, K; Schröck, E; Pisabarro, M; Hauber, J; Buchholz, F (Dresden, Germany) P175 Targeted destruction of HIV positive cells Sharma, J*; Dodgen, C; Skepu, A; Meyer, M (Cape Town, South Africa) *Indicates presenting author. Universal Tre (uTre) recombinase specifically targets the majority of HIV-1 isolates Janet Karpinski1, Jan Chemnitz2, Ilona Hauber2, Helga Hofmann-Sieber2, Carola Schäfer2, Niklas Beschorner2, Josephine Abi-Ghanem3, Maciej Paszkowski- Rogacz1, Vineeth Surendranath4, Debojyoti Chakraborty1, Karl Hackmann5, Evelin Schröck5, María Teresa Pisabarro3, Joachim Hauber2, Frank Buchholz1,4 1Department of Medical Systems Biology, Medical Faculty Carl Gustav Carus, TU Dresden, Germany, 2Heinrich Pette Institute - Leibniz Institute for Experimental Virology, Hamburg, Germany, 3Department of Structural Bioinformatics, TU Dresden, Germany, 4Max-Planck-Institute for Molecular Cell Biology and Genetics, Dresden, Germany, 5Institute for Clinical Genetics, Medical Faculty Carl Gustav Carus, TU Dresden, Germany ABSTRACT Background: Current drugs against HIV can suppress the progression to AIDS but Methods: We employed the search tool SeLOX [3] in Results: Highly specific enzymatic activity on loxLTRu is demonstrated for cannot clear the patient from the virus. Because of potential side effects of these drugs order to find a well-conserved HIV-1 proviral sequence uTre in both Escherichia coli and human cells. Naturally occurring viral variants and the possible development of drug resistance, finding a cure for HIV infection that could serve as target site for a universal Tre from with single mutations within the loxLTRu sequence are also shown to be remains a high priority of HIV/AIDS research. We recently generated a recombinase sequences compiled in the Los Alamos HIV Sequence efficiently targeted by uTre, further increasing the range of applicability of the (termed Tre) tailored to efficiently eradicate the provirus from the host genome of HIV-1 Database. We selected a candidate (termed loxLTRu) recombinase. Antiviral uTre activity is also exhibited in human Jurkat T cell infected cells by specifically targeting a sequence that is present in the long terminal with a mean conservation rate of 94% throughout the culture and in HIV patient derived primary T cells. Potential off-target sites in repeats (LTRs) of the viral DNA [1]. In vivo analyses in HIV-infected humanized mice major HIV-1 subtype groups A, B, and C. We applied the human genome are not recombined by uTre. Furthermore, uTre expression demonstrated highly significant antiviral effects of Tre recombinase [2]. However, the loxLTRu as substrate in our established substrate-linked in Jurkat or primary T cells shows no obvious Tre-related cytopathic or fact that Tre recognises a particular HIV-1 subtype A strain may limit its broad protein evolution (SLiPE) process [4] and evolved the genotoxic effects. Finally, uTre expressing mice show no undesired therapeutic application. To advance our Tre-based strategy toward a universally uTre recombinase in 146 evolution cycles. phenotypes during their normal lifespan. efficient cure, we have engineered a new, universal recombinase (uTre) applicable to the majority of HIV-1 infections by the various virus strains and subtypes. loxLTRu is a highly conserved target sequence in the LTR of HIV-1 Generation of of uTre recombinase by directed evolution A uTre uTre host DNA 5LTR HIV-1 proviral genome 3LTR A B C LTR U3 R U5 loxLTRu B C (A) Strategy to generate uTre recombinase. A summary of the 145 substrate-linked directed-evolution cycles and the subsequent steps to select for well-tolerated recombinases in human (A) Location of uTre recombinase target site loxLTRu in HIV-1 LTR (long cells are shown. Recombination was assayed by restriction enzyme digest, resulting in a smaller fragment for recombined (one triangle) and a larger fragment for non-recombined terminal repeat). (B) loxLTRu sequence aligned to Tre target site (loxLTR) substrate (two triangles). (B) Sequence diversity of evolved Tre recombinase libraries. (C) Molecular modelling of evolutionary conserved mutations. and Cre target site (loxP). (C) Representations (in %) of the in (A) shown target sequences in HIV-1 subtypes A, B, and C combined, and of loxLTRu in the three individual main subtypes uTre recombines loxLTRu efficiently and specifically in human cells A uTre efficiently and specifically targets loxLTRu and similar viral variant sites B A B D C (A) Recombination efficiency of uTre on loxLTRu in comparison to that of Tre on loxLTR. (B) Activity of C uTre on related recombinase target sites. (C) LacZ- based recombination reporter assay. (D) Activity of uTre on loxLTRu-like sites VS1, VS2, VS3 and VS4 present in 28 different HIV-1 isolates. uTre expression does not result in cytotoxicity A (A) Recombination efficiency and specificity of uTre in transiently transfected HeLa cells and (B) in the genomic context of Hela cells with stably integrated target sites. (C) Antiviral uTre activity in Jurkat T cell culture. uTre eradicates HIV infection in patient derived primary T cells B C ] 6 A control uTre Cell count [x10 !"#$% D B E !"#%&'"()"*+,%+-%.+/0,*"(%)120320("/04%+53/"260/%0507/$8%%!$#%90((%62+:/;%",4%70((%7#7(0%.2+620$$<+,% ","(#$<$%",4%".+./+$<$%"$$"#%+,%=)2>"/%1%70(($8%!%#%?,"(#$<$%+-%;)@",%.2<@"2#%9!AB%1%70(($%"C02%DE?%F% <+,+@#7<,%$*@)("*+,8%!&#'9!GAB%HI9%4<5020,*"*+,%"$$"#8%!(#%IJK%L$.07/2"(%>"2#+/#.<,6M%","(#$<$%+-% metaphase%$.20"4$%<$+("/04%-2+@%.2<@"2#%;)@",%9!AB%1%70(($%+'020N.20$$<,6%)1208 uTre expressing transgenic mice A B !"#%?,*'<2"(%)120%"7*'</#%<,%."*0,/%402<'04%9!AB%1%70((%7)(/)208%!$#%!00.%$0O)0,7<,6%","(#$<$%+-% )120%/20"/04%'$8%),/20"/04%."*0,/%70(($8 !"#%P0,02"*+,%+-%/2",$60,<7%)120%@<708%% C !$#% P0,+/#.<,6% +-% -+),402% ",4% /2",$60,<7% +5$.2<,68% !%#% D9Q3R"$04% ","(#$<$%+-%)120%0N.20$$<+,%<,%/2",$60,<7% +5$.2<,68 CONCLUSIONS References: We have developed a broad-range HIV-1 LTR specific recombinase that has the potential to be [1] Sarkar I, Hauber I, Hauber J, Buchholz F. HIV-1 proviral DNA excision using an evolved recombinase. Science. 2007;316(5833):1912-5. Epub 2007/06/30. effective against the vast majority of HIV-1 strains and to cure HIV-1 infected cells from the infection. [2] Hauber I, Hofmann-Sieber H, Chemnitz J, Dubrau D, Chusainow J, Stucka R, et al. Highly Significant Antiviral Activity of HIV-1 LTR-Specific Tre-Recombinase in Humanized Mice. PLoS pathogens. 2013;9(9):e1003587. Epub 2013/10/03. These results strongly encouraged us in our confidence that a Tre recombinase-mediated HIV [3] Surendranath V, Chusainow J, Hauber J, Buchholz F, Habermann BH. SeLOX--a locus of recombination site search tool for the detection and directed evolution of site-specific recombination systems. Nucleic acids research. 2010;38(Web Server issue):W293-8. Epub 2010/06/10. eradication strategy may become a valuable component of a future therapy for HIV-infected patients. [4] Buchholz F, Stewart AF. Alteration of Cre recombinase site specificity by substrate-linked protein evolution. Nature biotechnology. 2001;19(11):1047-52. Epub 2001/11/02. Targeted destruction of HIV positive cells Sharma J1, Dodgen CG1, Skepu A2 Rees DJG1 and Meyer M1 1DST/Mintek Nanotechnology Innovation Centre, Department of Biotechnology, University of the Western Cape, Private Bag X17, Bellville 7535, Republic of South Africa 2Advanced Materials Division, Mintek, 200 Malibongwe Drive, Randburg, 2125 INTRODUCTION Human immunodeficiency virus (HIV), the virus that causes acquired immunodeficiency syndrome (AIDS), (UNAIDS, 2012) has become one of the world’s most serious health and development challenges. A continuing expansion of human HIV/AIDS pandemic poses an exceptional threat to global health, international development and world security. Numerous prevention interventions exist to combat HIV but nothing has been proven very effective for the eradication of HIV load. Therefore in the proposed study, a strategy demonstrated by Vocero-Akbani et al in 1999 will be used and an effort will also be made to develop a gold nanoparticle for the targeted destruction of HIV infected cells which will be a well-planned strategy to stem the relentless global growth of AIDS deaths and new HIV infections. OBJECTIVES Since HIV primarily infects CD4+ cells; we aim to use CD4 as a selectable target to deliver a pro-apoptotic protein to HIV infected cells using nanoparticles as carriers. The aim of study was to develop a nanotechnology-based death inducing delivery system for the destruction of CD4+HIV infected cells through the activation of caspase-3. METHODS AND RESULTS Experiment 1. Construction of an “HIV-infected cell” model system Experiment 2. Synthesis and Purification of Mutant caspase-3 HIV-1 protease cleavage site D Cloning of CD4 into a green fluorescent protein (GFP) Expressing Vector 1 2 1 2 3 4 MGSSHHHHHHSSGCTERQANFLGKIWPGISLDNSYKMDYPEMGLCIIINN WT Caspase 1500bp KNFHKSTGMTSRSGTDVDAANLRETFRNLKYEVRNKNDLTREEIVELMR p17 1500bp 850bp 1000bp DVSKEDHSKRSSFVCVLLSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLT HIV-1 protease cleavage site A GKPKLFIIQACMRGTELDCGIETGGSQVSQNYPIVQNLQGGVDDDMACH PCR amplification of CD4 pEGFP-N3 mammalian Colony PCR screening for expression vector CD4 in pEGFP-N3 KIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCAMLKQYADKLEFMH p12 Mutant Caspase ILTRVNRKVATEFESFSFDATFHAKKQIPCIVSMLTKELYFYH