1. Gene and immunomodulatory therapies to disable latent HIV Keith R. Jerome, MD PhD Fred Hutchinson Cancer Research Center University of Washington July 20, 2015

2. Why is cure of HIV so difficult? HIV integrates into the genome of infected cells Latency is established in long-lived memory T cells (Tcm, Tem, Tscm) Long-term reservoir consists of ~10 7 latently infected cells If suppressive therapy is stopped, virus quickly rebounds from the latent reservoir DE Kaufmann PLoS Med 2004

3. Take home message Cell-, gene-, and immunotherapies for HIV are highly promising; many are in clinical trials and some have already shown anti-viral activity

4. Outline Reduction of the HIV reservoir Transplantation Immunotoxins Disruption of provirus Control of viral reactivation Genetic protection of T cells CCR5 disruption CAR T cells vectored antibodies Immune modulations Concluding thoughts

5. The latent reservoir presents a substantial hurdle to cure TW Chun AIDS 2010 Participa nt SexAg e Time of initiation of ART Duration of ART at time of study (yrs) ART at time of study CD4 cell count at time of study (cells/μl) CD8 cell count at time of study (cells/μl) CD4/CD8 ratio Plasma HIV RNA at time of study (copies/ mL) Level of HIV DNA in blood (copies/ 10 6 cells) Level of infectiou s HIV in blood (/10 6 cells) Level of HIV DNA in sigmoid colon (copies/ 10 6 cells) 1Male44Chronic8.6ABC/3TC/ EFV 4105800.7<50<2.560.0575089.0 2Male46Early10.5ABC/3TC/ EFV 10608401.3<50<2.560.00064<2.56

6. Cure may not require complete elimination of the latent reservoir AL Hill, PNAS 2014

7. Types of HIV cure Sterilizing cure (the Berlin patient): no traces of replication competent virus can be found in patient Functional cure: virus can be found by sensitive testing, but controlled at extremely low levels without the need for continued therapy Remission: control of virus at extremely low or undetectable levels for an extended period, followed by subsequent viral recurrence

8. Hematopoietic Cell Transplantation HIV predisposes to leukemia and lymphoma Consequently, some HIV patients require bone or stem cell transplantation Myeloablative conditioning and chemotherapy kills cells comprising the latent HIV reservoir How effective is this? Thus, transplantation alone is not sufficient to cure HIV V Avettand-Fenoel AIDS 2007

9. Allogeneic transplantation Graft-versus-host disease (GVHD) is a common complication of allogeneic transplantation Since the HIV reservoir is recognized as foreign by the transplanted immune system, there is also a graft-versus- reservoir (GVR) effect Could allogeneic transplantation, combined with suppressive antivirals during engraftment, lead to cure?

10. Graft versus reservoir - the Boston patients Allogeneic transplants, in combination with continual antivirals throughout engraftment period GVHD contributed to a substantial reduction of the HIV reservoir Is the reservoir eliminated? Are these patients cured? TJ Henrich J Inf. Dis. 2013

11. The Boston patients Analytical treatment interruption at 4.3 and 2.6 years post-transplant Graft-versus-host can substantially reduce the size of the HIV reservoir, but does not seem adequate to eliminate it TJ Henrich Annals Int Med 2014

12. Immunotoxins PW Denton PLoS Pathogens 2014 www.pvanuden.com Latently infected cells may not be completely quiet; occasionally go through low-level reactivation Can this be exploited for elimination of such cells?

13. Targeted mutagenesis of integrated provirus Aubert PLoS ONE 2011 Targeted endonucleases allow specific disruption of specific gene targets Knockout can be directed toward essential viral genes JT Schiffer J Virol 2012

14. Targeted deletion of integrated provirus W Hu PNAS 2014 Cure requires disruption of a substantial majority of integrated virus No in vivo delivery system currently available approaches 100% in memory T cells

15. Cure may not require complete elimination of the latent reservoir AL Hill, PNAS 2014

16. Cure may not require complete elimination of the latent reservoir JM Conway, PNAS 2015

17. Protection of T cells – gene therapy Trans-dominant Rev (Rev M10) Rev M10 plus antisense TAR Antisense Env Ribozymes Fusion inhibitors Combination restriction factors

18. Genetic protection of T cells RA Morgan Hum Gene Ther 2005 Rev M10 plus antisense TAR BL Levine PNAS 2006 antisense Envelope

19. Genetic protection of T cells P Tebas Blood 2013 antisense Envelope

20. Genetic protection of T cells J van Lunzen Mol Ther 2007 entry inhibitor C46

21. Natural protection of T cells - CCR5  32 CCR5 is a critical co-receptor for HIV entry CCR5 is dispensable for normal immune cell development and function CCR5  32 is a naturally occurring variant conferring resistance to HIV infection homozygous CCR5  32 is observed in ~1% of northern Europeans; less common in other populations Tilton and Doms Antiviral Res 2010

22. Transplantation from CCR5  32 donor This is the procedure that led to the cure of the Berlin patient Cure presumably resulted from some combination of conditioning, graft-versus-reservoir, and lack of susceptible cells if reactivation occurred from any reservoir remaining Since CCR5  32 is rare, it is difficult to identify appropriately matched donors with CCR5  32 Subsequently performed in several other patients, but no additional cures documented yet

23. CCR5 disruption The gene encoding CCR5 can be targeted for disruption in a patient’s own cells Can use targeted endonucleases (ZFNs, TALENs, CRISPRs, or meganucleases) to induce mutation in CCR5 CCR5 can be disrupted in stem cells or mature T cells CXCR4, the other major co-receptor for HIV, is required for immune cell development and thus can’t be disrupted in stem cells, but disruption tolerated in T cells P Tebas NEJM 2014HP Kiem Cell Stem Cell 2012

24. Chimeric antigen receptor (CAR) T cells Latently infected cells are not completely quiet; occasionally go through low-level reactivation Can this be exploited for elimination of such cells? M Casucci J Cancer 2012 J Scholler Sci Transl Med 2012 DeeksMitsuyasuAronson Allow T cells to recognize surface antigen without need for MHC Anti-CD19 and other CAR T cells are providing dramatic responses in leukemia Clinical trials have been performed with anti-HIV CAR T cells CAR T cells persist in HIV patients Clinical benefit has been difficult to document, but published studies used early-generation CAR T cells

25. Vectored delivery of antibodies or antibody-like molecules Adeno-associated virus (AAV) transduction of muscle allows durable transgene expression MR Gardner Nature 2015 CE Deal Curr Opin HIV AIDS 2015

26. Immune modulation Therapeutic vaccination Adjuvants TLR (4/7) stimulation Cytokines (IL-7, IL-15) Reversal of immune exhaustion - PD1 pathway blockade

27. Closing thoughts on HIV cure and the role of cell-, gene-, and immuno- therapy Cell-, gene-, and immuno-therapy offer an almost unlimited number of avenues by which to attack HIV Many of these ideas are in clinical trials, and some have already shown antiviral activity Combination approaches are likely to be critical – not only combinations of gene therapies and immune modulations, but also combinations with manipulation of latency Functional cure and remission are useful concepts – even extended viral control in the absence of antivirals is likely to be beneficial

28. Acknowledgments Caladan Foundation