CELL/GENE THERAPY

HIV Cure Research Training CurriculumCell/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

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

GOOD MORNING AMERICAUCLA 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 TimesIn Girl’s Last Hope, Altered Immune Cells Beat LeukemiaDecember 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 MaturingGene modification of patients’ own immune cells returned to patients is saving lives.

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.

• 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

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.

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

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 – 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

Patient

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

MobilizationLeukapheresis

OR Bone Marrow Harvest

Reinfusion

Isolation of Stem Cells or T cells

Virus-Mediated Transfer of Therapeutic Gene

GOAL: Gene modified cells engraft and correct or treat the disease

- Cancer - Genetic disease - Infectious disease

Next GenerationTechnologyGenome editing Zinc finger

TAL Effector Nuclease

CRISPR/Cas9

MegaTals

NH2

COOH

Zinc finger

TAL Effector Nuclease

CRISPR/Cas9

megaTAL

HIV target gene eg CCR5

Thanks to Barry Stoddard

Site-Specific Gene Targeting / Engineering

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

Expanding gene-edited and corrected

HSCs

Development of novel conditioning regimens

for efficient engraftment

Generation of HIV protected blood and immune system

inside the patientIIn 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

Patient

Hematopoietic Stem Cell Modification and Transplantation to Cure HIV/AIDS

Current Clinical Approaches

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

SANGAMO AUTOLOGOUST CELL TRIALS WITH CONDITIONING AGENT

SB-728mR-T (autologous CD4T cells genetically Modified at the CCR5 gene) + cyclophosphamid

NCT02225665 Phase I/II June 2018

SB-728-T + cyclophosphamide NCT01543152 (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 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.

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

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

In vivo Selection to increase the Percent HIV-protected cells

O6BG/BCNU

% G

ene

Mar

king

Days After Transplantation

Gene Marking

Therapeutic Threshold

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

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 componentExpressed on outside of cell;This can be part of an antibody, or other molecule Usually binds HIV envelope on infected cellsHLA independent;

Signaling ComponentSends signal into the cellDirects the cell to kill HIV infected target

CD3ζ

BindsViral protein

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.

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

New avenues: Induced pluripotent stem cells (iPSC)

Transfer

Skin biopsy

Fibroblast reprogramming

iPSCs generation

HIV-resistant CD4+ T cells or NK cells

HIV-resistant HSCs Gene-modified

• 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

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