1. CELL/GENE THERAPY HIV Cure Research Training Curriculum Cell/Gene Therapy by: Jeff Sheehy, the California Institute for Regenerative Medicine (CIRM) Jerome Zack, UCLA Hans-Peter Kiem, The Fred Hutchinson Cancer Research Center Jessica Handibode, AVAC July, 2015 The HIV CURE training curriculum is a collaborative project aimed at making HIV cure research science accessible to the community and the HIV research field.

2. Session Goals/Objectives  Learn about how therapies that insert genes and use cells is on the brink of transforming medicine and curing disease.  Learn how Gene/Cell therapies fit into HIV cure efforts  Learn the targets, techniques, and cell types used in HIV Gene/Cell Therapy  Understand the risks associated with Gene/Cell therapy clinical trials

3. Timothy Brown Road to a Cure for HIV HIV+ Acute Myeloid Leukemia Patient Identification of HLA-identical, CCR5Δ32 homozygous bone marrow donor Chemo- and Radiotherapy Conditioning Allogeneic stem cell transplant 6 years later: remains cured

4. GOOD MORNING AMERICA UCLA Researchers Announce Gene Therapy Cure for 18 ‘Bubble Baby’ Patients Nov 18, 2014  18 patients with Severe Combined Immunodeficiency Disease (SCID) ranging in age from 3 months to 4 years at the time of treatment.  Their blood stem cells (hematopoietic stem cells) were removed from their bone marrow and genetically modified to correct the gene defect that had left the children without a working immune system.  The children were cured without any side effects. New York Times In Girl’s Last Hope, Altered Immune Cells Beat Leukemia December 9, 2012  Juno Therapeutics, the company developing the therapy, in a study found an 89 percent remission rate among 27 adults with acute lymphoblastic leukemia no longer responding to other treatments.  Doctors remove millions of the patient’s T- cells and insert new genes that enable the T- cells to kill cancer cells.  The new genes program the T-cells to attack B-cells, a normal part of the immune system that turn malignant in this leukemia.  The altered T-cells — called chimeric antigen receptor cells — are then dripped back into the patient’s veins, and if all goes well they multiply and start destroying the cancer. Regenerative Medicine/Cell-Gene Therapy Maturing Gene modification of patients’ own immune cells returned to patients is saving lives.

5. What is Cell/Gene Therapy  A branch of Regenerative Medicine, an emerging field that involves the "process of replacing, engineering or regenerating human cells, tissues or organs to restore or establish normal function”.  Gene therapy is the the delivery of therapeutic gene into a patient's cells to treat disease.  Cell therapy is the delivery of intact, living cells into a patient to treat disease.  Combination Cell/Gene Therapy approaches that seek to insert genes into a patients’ own cells to control or kill HIV are in clinical trials now.

6. Ex vivo gene therapy - Usually with blood cells (lymphocytes or blood stem cells) for diseases affecting the hematopoietic system In vivo gene therapy - Oncolytic adenoviruses for the treatment of cancer Adeno-associated vectors for the treatment of Duchenne muscular dystrophy or hemophilia Non-viral for cancer Different Routes of Gene Therapy

7.  Sterilizing Cure  complete eradication of all replication competent forms of HIV. The reservoir is gone.  Timothy Brown received a sterilizing cure.  Functional Cure  Life-long control of virus in the absence of antiretroviral therapy, but without achieving complete eradication of HIV.  Virus remains in reservoirs in the body. Sterilizing vs functional cure

8. Gene Therapy in Blood Cells

9. Therapeutic HIV protection gene Gene Therapy in Blood Cells

12. Therapeutic HIV protection gene Gene Therapy in Blood Cells

13. Some targets for gene therapy

18. Gene Therapy- Vectors to deliver anti- HIV genes  LV- Lentivirus vectors  RV- gammaretroviral vectors,  AAV – adeno- associated vectors  Adenovirus vectors  Vectors are replication defective – so they cannot replicate and spread once they are inside the cells and after delivering the anti-HIV genes

19. Patient Ex Vivo Gene Therapy: Putting Functional Genes Into Marrow Stem Cells or T cells Outside of the Body

20. Patient Ex Vivo Gene Therapy: Putting Functional Genes Into Marrow Stem Cells or T cells Outside of the Body Mobilization Leukapheresis OR Bone Marrow Harvest

21. Patient Ex Vivo Gene Therapy: Putting Functional Genes Into Marrow Stem Cells or T cells Outside of the Body Virus-Mediated Transfer of Therapeutic Gene GOAL: Gene modified cells engraft and correct or treat the disease - Cancer - Genetic disease - Infectious disease

22. Patient Ex Vivo Gene Therapy: Putting Functional Genes Into Marrow Stem Cells or T cells Outside of the Body Reinfusion

23. Next Generation Technology Genome editing  Zinc finger  TAL Effector Nuclease  CRISPR/Cas9  MegaTals NH2 COOH Zinc finger TAL Effector Nuclease CRISPR/Cas9 megaTAL

24. HIV target gene eg CCR5 Thanks to Barry Stoddard Site-Specific Gene Targeting / Engineering

25. Scarless Repair Of Genetic Defect or Targeted Insertion Of New Genetic Material

26. Scarless Repair Of Genetic Defect or Targeted Insertion Of New Genetic Material

27. Scarless Repair Of Genetic Defect or Targeted Insertion Of New Genetic Material

28. Scarless Repair Of Genetic Defect or Targeted Insertion Of New Genetic Material

29. Kiem et al. Cell Stem Cell 2012 (modified) Patient Hematopoietic Stem Cell Modification and Transplantation to Cure HIV/AIDS

30. Collection HSCs Kiem et al. Cell Stem Cell 2012 (modified) Patient Hematopoietic Stem Cell Modification and Transplantation to Cure HIV/AIDS

31. Collection HSCs Kiem et al. Cell Stem Cell 2012 (modified) 1)Vector mediated gene transfer of HIV resistance genes 2)Nucleases for CCR5 disruption 3)Nucleases to eliminate integrated Virus Patient Hematopoietic Stem Cell Modification and Transplantation to Cure HIV/AIDS

32. Expanding gene- edited and corrected HSCs Collection HSCs Kiem et al. Cell Stem Cell 2012 (modified) 1)Vector mediated gene transfer of HIV resistance genes 2)Nucleases for CCR5 disruption 3)Nucleases to eliminate integrated Virus Patient Hematopoietic Stem Cell Modification and Transplantation to Cure HIV/AIDS

33. Expanding gene- edited and corrected HSCs Development of novel conditioning regimens for efficient engraftment Generation of HIV protected blood and immune system inside the patient IIn vivo selection Collection HSCs Kiem et al. Cell Stem Cell 2012 (modified) 1)Vector mediated gene transfer of HIV resistance genes 2)Nucleases for CCR5 disruption 3)Nucleases to eliminate integrated Virus Patient Hematopoietic Stem Cell Modification and Transplantation to Cure HIV/AIDS

34. Current Clinical Approaches

35. Timothy Brown--cured of HIV through a transplant of hematopoietic stem cells with a natural mutation that largely prevents HIV infection. This mutation can be replicated via gene therapy. Timothy received the stem cells from a donor and the resulting graft vs host disease was likely a factor in his cure. Attempts to replicate have failed in 6 patients due to the severity of their cancer. Matt Sharp took part in a clinical trial in which his own T-cells were removed from whole blood via apheresis and then gene modified and returned into his body. The Phase I trial recruited immunologic non-responders and Matt experienced a rise in his T-cell count. Sangamo, the sponsor, reported Phase II trial results in late 2014, that a “single infusion” of modified T cells “resulted in sustained reduction and control of viral load in the absence of antiretroviral drugs in several subjects..” and “a decrease in the size of the HIV reservoir.” Cell/Gene Therapy—Why? One cure, human trials underway

36. Current Clinical Trials MazF-T Redirected MazF- CD4 autologous T-cells Phase 1 Study Completion: 2016 SB728-mR-T + cyclophosphamid Phase 1/II Study Completion: 2018 Genetically modified periphal blood stem cell transplant in treating patients with HIV-associated non-Hodgkin’s lymphoma or Hodgkin lymphoma Study Completion: 2019 VRX- 496 University of Pennsylvania Pase I/II Study Completion: 2020 www.treatmentactiongroup.org/cure/trials

37. Clinical trials—blood cancer patients  Many trials recruit lymphoma or leukemia patients who need a transplant  Undergo conditioning to eliminate current immune system to create space for a new system  The HSCs used in these trials are autologous, meaning that they are taken from the patients not from a donor.  Their HSCs are gene modified to resist HIV, and are then transplanted back into the participant in a mix of modified and unmodified cells.

38. Clinical trials-other patient populations  Other trials propose going into healthier patients—currently, either immunologic non-responders or patients who have quit taking ART (treatment fatigue) as participants.  Some of these trials include conditioning regimens which present toxicity issues

39. Clinical Trial Issue  CCR5 deletion is unlikely to be sufficient by itself in many patients.  Mutated HIV that uses the CXCR4 receptor to infect cells is a potential complication  Gene therapy that blocks HIV in multiple ways will be needed.

40. Clinical Trial Issue  During cell modification, the percentage of cells modified varies, and a low yield of modified cells is a barrier.  Enough cells must be modified to achieve a therapeutic effect.  Hematopoietic cells are stimulated in a patient using drugs prior to apheresis to increase their number and percentage in the blood and enable more cells to be modified and returned.

41. Gene therapy clinical trial concerns  Gene therapy trials involve different gene editing/modifying techniques.  Precision is key, a serious concern is “off target” editing.  If the genes other than those targeted are modified (off target editing), the potential for serious adverse events exist, including cancer.

42. Treatment Interruptions  Seen as essential to allow modified cells to engraft and increase as a proportion of the cell population and to allow HIV to kill unprotected cells, and thus select for modified cells.  This process carries potential risks like treatment regimen resistance

43. Basic Science Approaches- Improving the Technology and Engineering Possible Solutions

44. Patient Expansion of gene-edited and HIV protected HSCs Collaboration Dr. Sauvageau (new UM171 molecule Fares et al Science 2014) Development of novel conditioning regimens, treosulfan, Astatine-211-based RIT, CAR-T cells Generation of genetically modified HIV protected blood and immune system inside the patient in vivo selection HSC Collection Kiem et al. Cell Stem Cell 2012 (modified) 1)Vector mediated gene therapy 2)Nuclease-mediated protection from HIV 3)Nuclease-mediated disruption of integrated HIV Patient Hematopoietic Stem Cell Gene Therapy / Editing for HIV

45. O6BG/BCNU % Gene Marking Days After Transplantation Gene Marking Therapeutic Threshold In vivo Selection to increase the Percent HIV- protected cells

46. Macrophage Activation B-Cell function CD8 + T-Cell function Cytolytic Activity Long-term protection Dampening of IR Peripheral Tolerance Maintenance of Lymphoid Tissue Maintenance of SHIV- Specific CD4 + T-Cells Resistance to Direct Infection R5- tropic X4- tropic Dual-tropic Development of Gene Modified, Infection Resistant CD4 + T-cells Decreased Viremia HSC Modification Results in the Development of Infection Resistant Immune Cell Populations and an Enhanced Immune Response HSC Modification Results in the Development of Infection Resistant Immune Cell Populations and an Enhanced Immune Response Younan…Kiem Blood 2013

47. Other Gene/Cell therapy approaches  T cells are taken from the peripheral blood of patients suppressed on antiretroviral therapy and presented with multiple HIV antigens before expansion  Cells are functional and have broadly specific and potent HIV infected cell killing ability and the ability to suppress HIV replication  Can be used with latency reversing agents as a “kill” strategy HIV: Shock and Kill. Steven G Deeks. Nature 487, 439-440 (26 July 2012)

48. Chimeric antigen receptor (CAR) Antigen binding component Expressed on outside of cell; This can be part of an antibody, or other molecule Usually binds HIV envelope on infected cells HLA independent; Signaling Component Sends signal into the cell Directs the cell to kill HIV infected target CD3 ζ Binds Viral protein

49. CD4-zeta CAR Vector For Introduction into Stem Cells UbC H1 CCR5 sh1005 CCR5 sh1005 ΔLTR 5’ LTR EGFP CD4-zeta 7SK sh516 2A D1D1 D1D1 D2D2 D2D2 D3D3 D3D3 D4D4 D4D4 CD4- Tm Zeta CD4 extracellular Domain Anti-HIV protective genes Signaling Domain

50. Other approaches: Chimeric antigen receptor T cells (CAR T cells)  Engineering hematopoietic and T stem cells that attack and kill cells infected with HIV.  Provides a self-renewing population of both CD8+ and CD4+ HIV- targeted T-cells resistant to direct HIV infection  Also used in cancer Jacobson, Caron A., and Jerome Ritz. "Clinical Trials Time to Put the CAR-T before the Horse." Blood Journal. American Society of Hematology, 3 Nov. 2011.

51. New avenues: In vivo gene modification  A new class of genetic engineering tools called targeted nucleases make genetic engineering of stem cells much more precise and therefore safer  Deliver these reagents directly to the stem cells in the body,  Uses a viral vector that specifically targets hematopoietic cells in vivo. HSC T cells

52. Conclusions  Regenerative Medicine/Cell-Gene Therapy is a rapidly maturing field offering potential for cures and therapies in several diseases and conditions  Clinical trials in HIV are underway or planned  A functional cure may result, but clinical benefit such as increased T cells for immunological non-responders would also help some patients greatly. And cell/gene therapy could provide the “kill” in “kick and kill”. It doesn’t have to lead to a cure by itself.

53. Conclusions  Current approaches in trial are very complex, but as the technologies develop, easier to administer and cheaper therapies will be available.  Risks, such as off-target effects and the need for treatment interruptions, are high in early trials and participants should carefully consider all risks before entering a trial.

54. Acknowledgements