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Prof. Jamal Tazi, PhD University of Montpellier, France Head of Splicos Therapeutics

The HIV-1 splicing inhibitor , SPL-464, compromises viral replication in vitro and induces a long lasting anti-viral effect in humanized mice infected with HIV-1. Prof. Jamal Tazi, PhD University of Montpellier, France Head of Splicos Therapeutics.

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Prof. Jamal Tazi, PhD University of Montpellier, France Head of Splicos Therapeutics

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  1. The HIV-1 splicinginhibitor, SPL-464, compromises viral replication in vitro and induces a long lasting anti-viraleffect in humanizedmiceinfectedwith HIV-1 Prof. Jamal Tazi, PhD University of Montpellier, France Head of SplicosTherapeutics

  2. Alternative splicing and HIV life cycle

  3. Alternative splicinginitiates HIV replication Viral RNA Viral entry Reverse transcription Integration Viral DNA Non splicing Splicing ARN viral Protease Régulatory proteins (Tat and Rev) Viral structural proteins

  4. Alternative splicingis a keyprocess for HIV replication

  5. HAT TFIID Tat ARN polII Rev TFIIH CycT1 Rev CDK9 Tat Late transcripts Early transcripts classe 9 kb classe 4 kb classe 2 kb RRE RRE Viral proteins (Env/Vpu, Vif, Tat, Vpr) Precursors Nef NUCLEUS LTR LTR gag pol env Tat TRANSCRIPTION RRE gag pol env Splicing export CYTOPLASM Translation

  6. HIV-1 RNA has weak 3’ splice sites snRNP U2 U2AF65 U2AF35 YNYURY (Y)n AG A Regulatory sequence BP PPT C Y(n)AG G Consensus U AAUUUUCGGGUUUAUUACAG G A1 UAUUACUUUGACUGUUUUUCAG A A2 ACAACUGCUGUUUAUCCAUUUCAG A A3 AGUUUGUUUCACAACAAAAG C A4a AGUUUGUUUCACAACAAAAGCCUUAG G A4b AAGUGUUGCUUUCAUUGCCAAG U A4c AGUUUGUUUCACAACAAAAGCCUUAG G A5 GGAUAUUCACCAUUAUCGUUUCAG A A7

  7. U2AF65 U2AF35 YNYUR Y A HIV-1 RNA splicingdepends on splicingregulatorsthattarget HIV-1 RNA sequences Splicingactivators SnRNP U2 (Py) AG n 3’ss Splicingrepressors

  8. Splicosinhibitors Splicos has a propriatorylibrary of smallchemical compounds targeting the splicingmachinery Soret et al PNAS 2005 Tazi et al Mol Phar 2005 Tazi et al TIBS 2005 Soret al Prog Mol SubcellBiol 2006 Bakkour et al PloSPath 2007 Keriel et al PloS One, 2009 Tazi et al BBA Mol Bio 2009 Tazi et al FEBS J 2010 2 manuscripts Patents Lejeune et al 2007 Tazi et al 2005 Tazi et al 2008 Tazi et al 2009 Tazi et al 2009 Tazi et al 2010

  9. Lead SPL-464 characterization

  10. SPL-464 induces a dose-dependent inhibition of HIV-1 replication in primary macrophages fromdifferentdonnors • SPL-464 inhibits viral replication of different HIV-clades including B and C types • SPL-464 inhibits viral replication of ART escape mutants • SPL-464 inhibits viral replication of HIV-2 • Aftersix months of in vitrotreatmentwith SPL-464 no resistantviruses have emmerged, whereasdrug-resistantviruses are selectedfollowingthreeweeks of treatmentwitheither 3TC or EFV

  11. Deepsequencing of YU-2 virus after SPL-464 treatmentdid not revealanyselected mutation

  12. LONG SHORT ‘Percent Spliced In’, psi or  fwd rev [LONG] x 100%  = [LONG + SHORT] Profiling cellular alternative splicing events shifted by Splicos drugs Robotic liquid handling High throughput capillary electrophoresis

  13. Darunavir SPL- 464 Control Cells SPL-464 did not induce global changes of alternative splicing in PBMCs

  14. Efficacy of SPL-464 in mouse models

  15. SPL- 464 HIV-1 inhibition on hu-PBL-SCID mouse after 40 mg/kg/daytreatment by twice-daily per os administration startedsimultaneously to HIV-1 infection.

  16. Treatmentwith SPL- 464 rescues CD8/CD4 ratio in infectedmice

  17. SPL-464 (40mg/kg daily by gavage) reduces viral loads in engraftedhumanized NSG miceinfected by YU2 HIV-1 strain. Comparisonwith ART

  18. Long lasting HIV-1 inhibitoryeffect of SPL-464 in infectedhumanizedmice.

  19. Summary • SPL-464 is a novel anti-HIV agent active againstdifferent HIV-1 clades and mutants as well as HIV-2 • SPL-464 did not induceemergence of HIV-1 mutants • SPL-464 has a new mode of action inhibiting HIV-1 splicing but not splicing of cellular genes • SPL-464 induces a long lasting effect in humanizedmice • SPL-464 rescues CD8/CD4 ratio in infectedhumanizedmice

  20. Anti – HIV therapeuticstrategies Entry Inhibiteur Viral RNA Reverse transcriptase inhibitors Viral entry Reverse transcription IntegraseInhibitors Integration Viral DNA Non splicing Splicing ARN viral ProteaseInhibitors Protease Régulatory proteins (Tat and Rev) Viral structural proteins SPL-464 Confidentiel

  21. Acknowledgements Splicos, Montpellier, France Didier Scherrer Aude Garcel Noëlie Campos Audrey Vautrin Julian Venables Mc Gill University, Canada Mark Wainberg UniversityHospital of Zürich, Switzerland Roberto Speck Renier Myburgh Erika Schlaepfer IRD, Montpellier, France Eric Delaporte Curie Institute, Paris, France Florence Manhuteau Romain Najman

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