Zinc Finger Nuclease Gene Editing for Functional Cure in a ...
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IAS 2015 Towards an HIV Cure symposium Vancouver
Zinc Finger Nuclease Gene Editing for Functional Cure in a
Nonhuman Primate Model of HIV/AIDS
Chris Peterson Hans-Peter Kiem Lab
Fred Hutchinson Cancer Research Center Seattle, WA, USA
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Study Objectives
Establish a model of HIV infection on cART in pigtailed macaques
Edit the CCR5 gene in macaque hematopoietic stem cells (HSC’s)
Transplant autologous CCR5-deleted hematopoietic stem cells SHIV-naïve animals SHIV-infected, cART-
treated animals
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Challenge Virus and cART Regimen
• SIV-mac239 backbone • HIV-HXBc2 accessory genes tat, rev, and vpu • Env isolated from HIV-1 Clade C primary isolate • 4 NFkB binding sites in 5’ and 3’ LTR • Selected for high virulence
-Passaged through five rhesus macaques to generate a monkey adapted virus
-Isolated from monkeys with late-stage AIDS Ruprecht Lab
Shiv-1157ipd3N4
Compound Target Administration
PMPA Reverse Transcriptase (NtRTI) 1X/day Subcutaneous
FTC Reverse Transcriptase (NRTI) 1X/day Subcutaneous
Raltegravir Integrase 2X/day Oral with Food
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SHIV-C Infection and cART Suppression in Macaques
Significant SHIV-dependent depletion of gut-associated lymphoid tissue Clinically relevant patterns of tissue
viremia and cART pharmacokinetics Stable virus suppression by cART in
peripheral blood and tissues Seeding of replication competent, cART-
suppressed viral reservoirs
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Autologous Transplant of CD34+ Hematopoietic Stem Cells in the Pigtailed Macaque
Enrich CD34+ cells from primed bone
marrow
Culture ex vivo (TPO, SCF, FLT-3 Ligand)
Infuse into conditioned
animal
Animal receives myeloablative conditioning regimen consisting
of 1,020 cGy total body irradiation
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Autologous Stem Cell Transplant Alone is Not Curative in SHIV+, cART-Suppressed Animals
1.E+00
1.E+01
1.E+02
1.E+03
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-10 0 10 20 30 40 50 60 70 80 90 100
Plas
ma
Vira
l Loa
d (V
iral R
NA
Cop
ies/
mL)
Weeks Post Infection
Begin anti-retrovirals
Transplant
End anti-retrovirals
Control Transplanted Untransplanted
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Autologous Transplant of CD34+ Hematopoietic Stem Cells in the Pigtailed Macaque
Enrich CD34+ cells from primed bone
marrow
Culture ex vivo (TPO, SCF, FLT-3 Ligand)
Infuse into conditioned
animal
Animal receives myeloablative conditioning regimen consisting
of 1,020 cGy total body irradiation
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ZFN Editing of CCR5 in Pigtailed Macaque CD34+ Cells for Autologous Transplant
Peterson et al, Submitted
Enrich CD34+ cells from primed bone
marrow
Culture 24hr, 37° ex vivo (TPO, SCF, FLT-3 Ligand)
Electroporate cells with ZFN mRNA, recover overnight at 30°C
Infuse into conditioned
animal
Animal receives myeloablative conditioning regimen consisting
of 1,020 cGy total body irradiation
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Benchmarks for Efficacious ΔCCR5 Gene Therapy
Normal hematopoietic recovery following ΔCCR5 transplant
Multilineage engraftment of gene-edited cells following CD34+ infusion in vivo
Gene editing in ex vivo cultured macaque CD34+ cells
Generation of cells with biallelic editing of CCR5 Long term engraftment of edited cells, and
positive selection following SHIV challenge
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Benchmarks for Efficacious ΔCCR5 Gene Therapy
Normal hematopoietic recovery following ΔCCR5 transplant: YES
Multilineage engraftment of gene-edited cells following CD34+ infusion in vivo: YES
Gene editing in ex vivo cultured macaque CD34+ cells
Generation of cells with biallelic editing of CCR5 Long term engraftment of edited cells, and
positive selection following SHIV challenge
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Macaque CCR5 ZFNs Generate Up to 60% Bulk Gene Editing and 10% Biallelic Editing ex vivo
01020304050607080
A11217 A11210 Z12161 Z12220
Perc
ent D
isru
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5 (M
iSeq
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A11217 A11210 Z12161 Z12220
Perc
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ssay
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Col
onie
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wt/wtindel/wtindel/indel
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CCR5-Edited Stem Cells Engraft in Peripheral Blood, and Persist Following SHIV Challenge
Peterson et al, Submitted
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Modeling the Berlin Patient in M. Nemestrina
Baseline Sample Collection SHIV Infection
ΔCCR5 Transplant
SH
IV S
tatu
s?
Baseline Sample Collection
ΔCCR5 Transplant
SHIV Infection
Withdraw Antiretroviral Therapy
Antiretroviral Therapy
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Macaque CCR5 ZFN Transplants are Feasible in a Preclinical Model of Suppressed HIV Infection
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5 (M
iSeq
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SHIV Status at Transplant
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20
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Uninfected Infected, cART-suppressed
Perc
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ssay
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Col
onie
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SHIV Status at Transplant
wt/wtwt/indelindel/indel
Bulk Disruption
Mono-/Biallelic Disruption
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Conclusions and Future Directions
Efficient Targeting of CCR5 with ZFNs in Macaque HSCs; up to 10% biallelic editing Maximize ZFN delivery to true HSCs Insert a selectable marking following gene
disruption to select against CCR5wt/wt cells CCR5 Gene Editing is Equally Feasible in
SHIV+, cART-suppressed animals Define the threshold effect of ΔCCR5 therapy Combinatorial therapies
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Thank You… Kiem Lab • Hans-Peter Kiem • Jasbir Kaur • Sowmya Reddy • Krystin Norman • Kina McAllister • Willi Obenza • Andrea Repetto • Morgan Giese • Devikha Chandrasekaran
defeatHIV Collaboratory • Shiu-Lok Hu • Keith Jerome Funding • U19 AI096111 • R21 AI110154 • R01 AI080326
Sangamo Biosciences • Jianbin Wang • Michael Holmes • Philip Gregory
Kiem Animal Technicians • Kelvin Sze • Veronica Nelson • Erica Curry
• Christina Ironside • Shirley Chan • Bish Paul • Kevin Haworth • Anne-Sophie Kuhlmann • Martin Wohlfahrt • Jennifer Adair • Christopher Burtner • Olivier Humbert
• Stefan Radtke • Don Gisch • Daniel Humphrys • John Kowalski • Brian Beard • Tracey Pierce • Grace Choi • Cynthia Shen • Jacob Isenberg
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ΔCCR5 Transplant Animals Display Normal Kinetics of Hematopoietic Recovery
A11217 A11210 Z12161 Z12220 Healthy Minimum Value Peterson et al, Submitted
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Edited CCR5 Loci Are Found in All Hematopoietic Lineages
Peterson et al, Submitted
Lymphoid/Myeloid Sorts
CD4+ T-Cell Subset Sorts