CELLGENE THERAPY HIV Cure Research Training Curriculum CellGene

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 January, 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.

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

Timothy Brown Road to a Cure for HIV+ Acute Myeloid Leukemia Patient Identification of HLA-identical, CCR 5Δ 32 homozygous bone marrow donor Chemo- and Radiotherapy Conditioning Allogeneic stem cell transplant 6 years later: remains cured

Regenerative Medicine/Cell-Gene Therapy Maturing Gene modification of patients’ own immune cells returned to patients is saving lives. GOOD MORNING AMERICA New York Times UCLA Researchers Announce Gene Therapy Cure for 18 ‘Bubble Baby’ Patients In Girl’s Last Hope, Altered Immune Cells Beat Leukemia 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. December 9, 2012 § § Juno Therapeutics, the company developing 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.

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 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.

Different Routes of Gene Therapy • 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

Cell/Gene Therapy will likely produce a functional cure, if a cure is generated § Sterilizing cure § Functional Cure § complete eradication of all replication competent forms of HIV. The reservoir is gone. § Life-long control of virus in the absence of antiretroviral therapy, but without achieving complete eradication of HIV. § Timothy Brown received a sterilizing cure. § Virus remains in reservoirs in the body.

Gene Therapy in Blood Cells Therapeutic HIV protection gene

Some targets for gene therapy

Gene Therapy- Vectors to deliver anti -HIV genes LV- Lentivirus vectors RV- gammaretroviral vectors, § AAV – adenoassociated 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

Ex Vivo Gene Therapy: Putting Functional Genes Into Marrow Stem Cells or T cells Outside of the Body Virus-Mediated Transfer of Mobilization Therapeutic Gene Leukapheresis OR Bone Marrow Harvest Isolation of Stem Cells or T cells GOAL: Gene modified cells Reinfusion engraft and correct or treat the disease - Cancer - Genetic disease - Infectious disease Patient

Next Generation Technology Genome editing § Zinc finger § TAL Effector Nuclease § CRISPR/Cas 9 § Mega. Tals COOH Zinc finger NH 2 TAL Effector Nuclease CRISPR/Cas 9 mega. TAL

Site-Specific Gene Targeting / Engineering HIV target gene eg CCR 5 Thanks to Barry Stoddard

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

Hematopoietic Stem Cell Modification and Transplantation to Cure HIV/AIDS Collection HSCs Patient Generation of HIV protected blood and immune system inside the patient IIn vivo selection Patient Development of novel conditioning regimens for efficient engraftment 1) Vector mediated gene transfer of HIV resistance genes 2) Nucleases for CCR 5 disruption 3) Nucleases to eliminate integrated Virus Expanding geneedited and corrected HSCs Kiem et al. Cell Stem Cell 2012 (modified)

Current Clinical Approaches

Cell/Gene Therapy—Why? One cure, human trials underway 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. ”

SANGAMO AUTOLOGOUST CELL TRIALS WITH CONDITIONING AGENT SB-728 m. R-T (autologous CD 4 T cells genetically Modified at the CCR 5 gene) + cyclophosphamid B-728 -T + cyclophosphamide NCT 02225665 Phase I/II June 2018 NCT 01543152 (closed to enrollment) Phase I/II Dec. 2013

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. §

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 §

Clinical Trial Issue § CCR 5 deletion is unlikely to be sufficient by itself in many patients. § Mutated HIV that uses the CXCR 4 receptor to infect cells is a potential complication § Gene therapy that blocks HIV in multiple ways will be needed.

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. §

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. §

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 §

Basic Science Approaches- Improving the Technology and Engineering Possible Solutions

Hematopoietic Stem Cell Gene Therapy / Editing for HIV HSC Collection 1) Vector mediated gene therapy 2) Nuclease-mediated protection from HIV Patient Generation of genetically modified HIV protected blood and immune system inside the patient in vivo selection 3) Nuclease-mediated disruption of integrated HIV Expansion of gene-edited and HIV protected HSCs Collaboration Dr. Sauvageau (new UM 171 molecule Fares et al Science 2014) Patient Development of novel conditioning regimens, treosulfan, Astatine-211 -based RIT, CAR-T cells Kiem et al. Cell Stem Cell 2012 (modified)

In vivo Selection to increase the Percent HIVprotected cells % Gene Marking O 6 BG/BCNU Gene Marking Therapeutic Threshold Days After Transplantation

HSC Modification Results in the Development of Infection Resistant Immune Cell Populations and an Enhanced Immune Response Development of Gene Modified, Infection Resistant CD 4+ T-cells Cytolytic Activity X 4 - tropic R 5 - tropic Dampening of IR Resistance to Direct Infection Dual-tropic B-Cell function Peripheral Tolerance Decreased Viremia Long-term protection Maintenance of SHIVSpecific CD 4+ T-Cells Macrophage Activation CD 8+ T-Cell function Maintenance of Lymphoid Tissue Younan…Kiem Blood 2013

§ A genetic “handle” attached to modified cells, enabling better screening of unmodified cells § Potential purification of modified cells, reaching almost 95% purity.

Other Gene/Cell therapy approaches The “kill” in “Kick and Kill”, (Lam, Baylor) T cells are taken from the peripheral blood of patients suppressed on antiretroviral therapy. § The cells are presented with multiple HIV antigens and then expanded. § 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 to kill the “kicked” HIV. § § HIV: Shock and Kill. Steven G Deeks. Nature 487, 439 -440 (26 July 2012)

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 Binds Viral protein CD 3ζ

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 CD 8+ and CD 4+ HIVtargeted 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.

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. § T cells HSC

New avenues: Induced pluripotent stem cells (i. PSC) • Skin cells are converted back into embryo-like state (pluripotency) • The pluripotent cells are modified to have a deletion of CCR 5Δ 32 mutation • Modified cells differentiated and returned Skin biopsy Fibroblast reprogramming i. PSCs generation HIV-resistant HSCs Transfer Genemodified HIV-resistant CD 4+ T cells or NK cells

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. §

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. §

Acknowledgements