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.
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
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



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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


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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 Tcells and insert new genes that enable the Tcells 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 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
Sterilizing vs functional cure

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.
Gene Therapy in Blood Cells
Gene Therapy in Blood Cells
Therapeutic HIV
protection gene
Gene Therapy in Blood Cells
Gene Therapy in Blood Cells
Gene Therapy in Blood Cells
Therapeutic HIV
protection gene
Some targets for gene therapy
Some targets for gene therapy
Some targets for gene therapy
Some targets for gene therapy
Some targets for gene therapy
Gene Therapy- Vectors to deliver antiHIV 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
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
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
Patient
Ex Vivo Gene Therapy: Putting Functional Genes Into Marrow Stem Cells or
T cells Outside of the Body
Reinfusion
Patient
Next Generation
Technology
Genome editing
 Zinc finger

TAL Effector
Nuclease

CRISPR/Cas9

MegaTals
COOH
Zinc finger
NH2
TAL Effector
Nuclease
CRISPR/Cas9
megaTAL
Site-Specific Gene Targeting / Engineering
HIV target gene eg CCR5
Thanks to Barry Stoddard
Scarless Repair Of Genetic Defect or
Targeted Insertion Of New Genetic Material
Scarless Repair Of Genetic Defect or
Targeted Insertion Of New Genetic Material
Scarless Repair Of Genetic Defect or
Targeted Insertion Of New Genetic Material
Scarless Repair Of Genetic Defect or
Targeted Insertion Of New Genetic Material
Hematopoietic Stem Cell Modification and
Transplantation to Cure HIV/AIDS
Patient
Kiem et al. Cell Stem Cell 2012 (modified)
Hematopoietic Stem Cell Modification and
Transplantation to Cure HIV/AIDS
Collection HSCs
Patient
Kiem et al. Cell Stem Cell 2012 (modified)
Hematopoietic Stem Cell Modification and
Transplantation to Cure HIV/AIDS
Collection HSCs
Patient
1) Vector mediated
gene transfer of
HIV resistance
genes
2) Nucleases for
CCR5 disruption
3) Nucleases to
eliminate
integrated Virus
Kiem et al. Cell Stem Cell 2012 (modified)
Hematopoietic Stem Cell Modification and
Transplantation to Cure HIV/AIDS
Collection HSCs
Patient
1) Vector mediated
gene transfer of
HIV resistance
genes
2) Nucleases for
CCR5 disruption
3) Nucleases to
eliminate
integrated Virus
Expanding geneedited and
corrected
HSCs
Kiem et al. Cell Stem Cell 2012 (modified)
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 CCR5
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.”
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
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


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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 UM171
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
O6BG/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 CD4+ T-cells
Cytolytic Activity
X4- tropic
R5- tropic
Dampening
of IR
Resistance to
Direct Infection
Dual-tropic
B-Cell
function
Peripheral
Tolerance
Decreased Viremia
Long-term
protection
Maintenance
of SHIVSpecific CD4+
T-Cells
Macrophage
Activation
CD8+ T-Cell
function
Maintenance of
Lymphoid Tissue
Younan…Kiem Blood 2013
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)
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
CD3ζ
CD4-zeta CAR Vector For Introduction
into Stem Cells
D
1
D
2
CD4 extracellular Domain
D
3
D
4
CD4Tm
Zeta
5’ LTR
H1
CCR5
sh1005
Signaling Domain
7SK
sh516
UbC
Anti-HIV protective genes
EGFP
2A
CD4-zeta
ΔLTR
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+ 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
Conclusions
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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

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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