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1 Cell/gene therapy Cell/Gene Therapy by:
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.

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 CCR5-Δ32 is a deletion mutation of a gene that has a specific impact on the function of T cells.[16] At least one copy of CCR5-Δ32 is found in about 4–16% of people of European descent. It has been speculated that this allele was favored by natural selection during the Black Death for Northern Europeans, but further research has revealed that the gene did not protect against the Black Death.[17] The current hypothesis is of protection vs smallpox throughout Europe,[17] especially in the major trade cities and in isolated islands and archipelagos, such as Iceland and the Azores.[18] Homozygous around 1% and then matched around 0.1% or less

4 Regenerative Medicine/Cell-Gene Therapy Maturing Gene modification of patients’ own immune cells returned to patients is saving lives. 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. These are two examples of cures achieved with Cell/Gene therapy approaches. They demonstrate that the field is achieving safe, durable results and that the underlying technology platform is becoming mature enough to investigate to treat many diseases. The presenter should also explain allogenic versus autologous approaches, that is, those that involve cells transplanted from another person and cells that are taken from a patient and then returned.

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 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

7 Cell/Gene Therapy will likely produce a functional cure, if a cure is generated
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.

8 Gene Therapy in Blood Cells Therapeutic HIV protection gene
First slide meant to give background in gene therapy of blood cells and how (broadly) it can be applied in different clinical settings. Next slide(s) will focus on a few of our success stories in the clinic and in preclinical models.

9 Some targets for gene therapy
Each X refers to a potential gene target being studied

10 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 Vectors are the means by which the anti-HIV genes are carried into cells. For instance, HIV, a lentivirus, enters immune cells and integrates itself into the DNA of cells. Using defective virus vectors loaded with anti-HIV genes is a way to get the therapeutic genes integrated into cells’ DNA.

11 Ex Vivo Gene Therapy: Putting Functional Genes Into Marrow Stem Cells or T cells Outside of the Body
Virus-Mediated Transfer of Therapeutic Gene Mobilization Leukapheresis OR Bone Marrow Harvest Isolation of Stem Cells or T cells GOAL: Gene modified cells engraft and correct or treat the disease - Cancer - Genetic disease - Infectious disease This shows the process in use in current gene-cell therapy clinical trials. The immune cells (either T cells or hematopoietic stem cells) that are to be gene modified are mobilized in a patient’s body with drugs, then either sorted from the patient’s blood through leukopheresis or harvested from the patient’s bone marrow. Then, the stem cells or T cells are isolated into a purer population. The therapeutic gene is then transferred into the DNA of the target cells using a viral vector or a non-viral technology. The gene modified cells are then re-infused back into the patient. Reinfusion Patient

12 Next Generation Technology
NH2 COOH Zinc finger Genome editing Zinc finger TAL Effector Nuclease CRISPR/Cas9 MegaTals TAL Effector Nuclease CRISPR/Cas9 These are various tools carried into cells that modify genes. For instance, the zinc finger technology, currently used in some clinical trials, acts a molecular scissors that cut out gene for CCR5, which mimics the absence of that gene that naturally occurred in the donor in Timothy Brown’s cure. megaTAL

13 Site-Specific Gene Targeting / Engineering
HIV target gene eg CCR5 The non-specific cleavage domain from the type IIs restriction endonuclease FokI is typically used as the cleavage domain in ZFNs.[1] This cleavage domain must dimerize in order to cleave DNA[2] and thus a pair of ZFNs are required to target non-palindromic DNA sites. Standard ZFNs fuse the cleavage domain to the C-terminus of each zinc finger domain. In order to allow the two cleavage domains to dimerize and cleave DNA, the two individual ZFNs must bind opposite strands of DNA with their C-termini a certain distance apart. The most commonly used linker sequences between the zinc finger domain and the cleavage domain requires the 5' edge of each binding site to be separated by 5 to 7 bp.[3]Several different protein engineering techniques have been employed to improve both the activity and specificity of the nuclease domain used in ZFNs. Directed evolution has been employed to generate a FokI variant with enhanced cleavage activity that the authors dubbed "Sharkey".[4] Structure-based design has also been employed to improve the cleavage specificity of FokI by modifying the dimerization interface so that only the intended heterodimeric species are active.[5][6][7][8] Thanks to Barry Stoddard

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

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

16 Current Clinical Approaches

17 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.” Clinical trials are ongoing! Sangamo Trials- CCR5 Targeting T cell approach targeting the gene for CCR5 in clinical trials Hematopoietic Stem Cell approach targeting CCR5 about to begin a clinical trial Both enroll patients whose virus has been tested to ensure that CCX4 tropic virus is not present. Calimmune Trials – CCR5 and C46 T cell and Hematopoietic stem cell approach In both cell types, the gene for CCR5 is targeted. Another gene therapy approach is added, C46, in both cell types. This approach mimics an class of drugs, fusion inhibitors and blocks CCX4 tropic virus.

18 SB-728-T + cyclophosphamide
Thank you Richard Jeffrey and TAG. SANGAMO AUTOLOGOUST CELL TRIALS WITH CONDITIONING AGENT SB-728mR-T (autologous CD4T cells genetically Modified at the CCR5 gene) + cyclophosphamid NCT Phase I/II June 2018 SB-728-T + cyclophosphamide NCT (closed to enrollment) Phase I/II Dec. 2013

19 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. failed chemotherapy and are indicated for the transplant of hematopoietic stem cells (blood forming stem cells-HSCs) following drug or radiation destruction of their immune system to kill their cancer

20 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 Immunologic non-responders are HIV patients who achieve viral suppression with anti-retroviral therapy but whose T cells never fully recover to normal levels. They are increased risk for opportunistic infections and have more challenges managing their HIV.

21 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. The CXCR4 complication was seen in patients who underwent the same procedure as Tim Brown, the Berlin Patient.

22 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. The yield of modified cells is low at this time, and the question remains, what percentage of the cell population needs to be modified to create a clinical benefit? One hypothesis is that the protected cells will be able to expand in vivo due to HIV targeting of the non-modified cells. These drugs have side effects

23 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.

24 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

25 Basic Science Approaches- Improving the Technology and Engineering Possible Solutions

26 Hematopoietic Stem Cell Gene Therapy / Editing for HIV
Patient HSC Collection Vector mediated gene therapy Nuclease-mediated protection from HIV Nuclease-mediated disruption of integrated HIV Generation of genetically modified HIV protected blood and immune system inside the patient in vivo selection Expansion of gene-edited and HIV protected HSCs Collaboration Dr. Sauvageau (new UM171 molecule Fares et al Science 2014) Patient The Kiem lab has recently developed a novel and highly efficient strategy that exclusively uses the purine analog 6-thioguanine (6TG) for both pretransplantation conditioning and post-transplantation chemoselection of hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient bone marrow (BM). In a mouse BM transplantation model, combined 6TG preconditioning and in vivo chemoselection consistently achieved >95% engraftment of HPRT-deficient donor BM and long-term reconstitution of histologically and immunophenotypically normal hematopoiesis in both primary and secondary recipients, without significant toxicity and in the absence of any other cytotoxic conditioning regimen. To translate this strategy for combined 6TG conditioning and chemoselection into a clinically feasible approach, it is necessary to develop methods for genetic modification of normal hematopoietic stem cells (HSC) to render them HPRT-deficient and thus 6TG-resistant. Here we investigated a strategy to reduce HPRT expression and thereby confer protection against 6TG myelotoxicity to primary murine BM cells by RNA interference (RNAi). Accordingly, we constructed and validated a lentiviral gene transfer vector expressing short-hairpin RNA (shRNA) that targets the murine HPRT gene. Our results showed that lentiviral vector-mediated delivery of HPRT-targeted shRNA could achieve effective and long-term reduction of HPRT expression. Furthermore, in both an established murine cell line as well as in primary murine BM cells, lentiviral transduction with HPRT-targeted shRNA was associated with enhanced resistance to 6TG cytotoxicity in vitro. Hence this represents a translationally feasible method to genetically engineer HSC for implementation of 6TG-mediated preconditioning and in vivo chemoselection. Development of novel conditioning regimens, treosulfan, Astatine-211-based RIT, CAR-T cells Kiem et al. Cell Stem Cell 2012 (modified)

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

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

29 Potential purification of modified cells, reaching almost 95% purity.
A genetic “handle” attached to modified cells, enabling better screening of unmodified cells Potential purification of modified cells, reaching almost 95% purity. Once cells are taken from a patient either through leukapheresis or bone marrow harvest, they are sorted into the cell type needed, then those cells are gene modified. However, the methods currently used leave a large percentage of unmodified cells in the mix. The new method described here allows better screening of modified cells leading to a much purer population of sorted cells with the therapeutic modification that will then be returned to patients.

30 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, (26 July 2012)

31 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 CD4ζ binding to HIV envelope protein was expressed using a gamma-retroviral vector in peripheral T-cells and evaluated in three clinical trials10, 31, 50 more than 20 years ago. Treatment was well-tolerated and safe, but these studies were all confounded by the concurrent administration of combination antiretroviral therapy, although it appeared in one trial that tissue viral replication was reduced. Furthermore, a significant problem was that the re-infused gene modified T-cells were pre-morbid and dysfunctional due to massive ex vivo expansion and persisted only at low levels. By contrast, stem cells, HSPC and TSCM, would provide for long-term maintenance of functional gene-modified effector T-cells of both CD4+ and CD8+ lineages. Recently, better expression and safety using lentiviral vectors as well as new designs for CARs to enhance T-cell signaling provide opportunities for anti-HIV CARs to cure. However CD3ζ

32 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 Zack (UCLA) is engineering immune system stem cells (hematopoietic and T stem cells) with molecules that will directly mature immune cells developed from stem cells to attack and kill cells infected with the AIDS virus. This approach provides a self-renewing population of both CD8+ and CD4+ HIV-targeted T-cells that are resistant to direct THE IDEA OF DIRECT VS INDIRECT INFECTION NEEDS TO BE EXPLAINED IN THE NOTES AND HIGHLIGHTED AS A PRESENTER’S NOTE TO EXPLAIN HIV infection. These cells bypass the mechanisms THIS NEEDS TO BE EXPLAINED IN MY MORE DETAIL. A LAY PERSON WOULDN’T KNOW WHAT IT MEANS by which HIV usually evades the immune response. This is the same technology that is leading to remission in some cancer patients and may revolutionize oncology. 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

33 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 Paula Cannon conducts most of this work Paula Cannon (USC) is developing a new class of genetic engineering tools called targeted nucleases to make genetic engineering of stem cells much more precise and therefore safer. She seeks to deliver these reagents directly to the stem cells in the body, without the current necessary steps of first removing the cells and performing the genetic engineering in a lab. Uses a viral vector that specifically targets hematopoietic cells in vivo. HSC

34 New avenues: Induced pluripotent stem cells (iPSC)
Skin cells are converted back into embryo-like state (pluripotency) The pluripotent cells are modified to have a deletion of CCR5Δ32 mutation Modified cells differentiated and returned Transfer Skin biopsy Fibroblast reprogramming iPSCs generation HIV-resistant CD4+ T cells or NK cells HIV-resistant HSCs Gene-modified Y.W. Kan (UCSF) published in 2014 an iPSC approach. Fibroblasts were removed from a skin biopsy taken from a patient. Genes were delivered into the cells that reprogrammed them to back to an embryo-like state of pluripotency. These wild-type induced pluripotent stem cells were gene modified to have the natural CCR5Δ32 mutation (“exactly mimicking the natural mutation”) like the cells of Timothy Brown’s donor. The pluripotent cells were differentiated into monocytes and macrophages The modified cells were resistant to HIV infection in vitro. Could be a limitless source of gene modified immune cells matched to the patient who is the source of cells.

35 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.

36 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.

37 Acknowledgements

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