Engineered T Cells for HIV/AIDS

Personalized Medicine
Engineered T cells for HIV/AIDS
Carl June, M.D.
Professor of Pathology and Lab Medicine
Abramson Cancer Center
University of Pennsylvania
April 23, 2009
Cell and Gene Based Therapies
o
o
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Personalized medicine approaches with cell
and gene therapies requires effective
academic-biotech partnerships
“Handoff point” for new therapies is later
(phase II data required) than for established
pharmaceutical approaches
Considerable infrastructure required for
GMP manufacturing, assay development,
regulatory oversight, and animal model
development
Realistic time frames: manage expectations!
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General
General Approaches
Approaches
for
for Adoptive
Adoptive T
T Cell
Cell Therapy
Therapy
HIV patients?
J Clin Invest 2007 117:1466-76
Generating
Generating an
an HIV
HIV Proof
Proof
Immune
System?
Immune System?
• Distinct roles of CD4 and CD8 T cells: implications for
various gene transfer strategies
• Mature T cell subsets: safety and feasibility of genetic
engineering now established. Long lived T cell subsets
• Stem cells have technical issues….
2
HIV Based Lentiviral Vector Design
Long antisense targeting env
(VRX496)
vpr
gag
pNL4pNL4-3
(9709bp)
env
vif
rev
pol
rev
LTR
LTR
tat vpu
pN1cptASenv
(4344bp)
J Virol 2004; 78: 7079
J Gene Med 2004; 6:963
635 458 539 937
tat
921 186 635
psi cpt ASenv RRE
non-coding marker sequence from GFP
3
Cell
Cell Culture
Culture Approaches
Approaches
for
for Adoptive
Adoptive T
T Cell
Cell Therapy
Therapy
• Functional reprogramming
• T cell selection in the host
• Treg cell depletion / augmentation
• Genetic modification
Lentiviral Vectors
o Maloney oncoretroviral
vectors
Only transduces dividing cells
Insertional mutagenesis
T cell leukemia in SCID (cγ
chain)
o Potential advantages of HIV
based lentiviral vectors
high efficiency transduction
long term expression ⇒ less
susceptible to silencing
not yet tested in humans
Practical issues
• Stable producer lines
not yet available
• Clinical grade vector is
expensive but available
- NGVL
- ViRxSys
- Lentigen
• Requirements for
release testing for
replication competent
lentivirus
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Generating
Generating Transgenic
Transgenic Human
Human Immune
Immune
Systems:
Systems: Lentiviral
Lentiviral Engineered
Engineered T
T cells
cells
99.9%
APHERESIS
1 x 10e9
T cells
CD8
Depletion
DAY 0
99.4%
1 x 10e10
1 x 10e11
Transduced
T cells
Transduced
T cells
Lentiviral
Modified
T-CELLS
INFUSED
CD38/28 beads
Cryopreserve
Harvest
+/- Fibronectin
DAY 7
DAY 14
Transduce
IL-2
Clinical Scale T Cell Culture Process
Wave
Bioreactors
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Phase I Clinical Trial of the Safety and Tolerability of a Single Dose of
Autologous T Cells Transduced with anti-sense env in AIDS
Levine et al. PNAS 2006
Ongoing Penn/ViRxSys Multi-dose STI
Study (Protocol #802456)
Pablo Tebas, MD: PI
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1st
Ex Vivo Expansion:
LV Trial vs. Multi-dose STI Trial
Assessment of Gene Modified CD4 Cell Trafficking to Mucosal IEL
Ron Collman
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Lessons from the Antisense
Lentiviral Vector Gene Transfer Trials
• HIV based vectors appear safe in 18 of 18
patients treated at Penn to date. Efficient
…
Efficient…
• VSV
-G immunogenicity: well tolerated
VSV-G
• Promising engraftment with genetically
engineered CD4 T cells
• Increased CD4 counts routinely observed
• Efficient trafficking of VRX496 to rectal
mucosa
• Multiple doses do not enhance engraftment in
PBMC
Ron Collman, Bruce Levine, Gwen Binder, Jean Boyer, Pablo Tebas
Integration Site Analysis
• Gamma
-retroviral vectors: five T cell
Gamma-retroviral
leukemias have now been reported among
the twenty patients treated for SCID.
• What is risk of insertional mutagenesis with
lentiviral vectors?
Gary Wang, Rick Bushman, Mol Ther 2009
8
Integration Site Analysis
• Integration sites were analyzed using 454
pyrosequencing, yielding a total of 7,787 unique
integration sites from the ex vivo product and 240
unique sites from cells recovered after infusion.
• Integrated vector copies were found to be strongly
enriched within active genes and near epigenetic
marks associated with active transcription units.
• Analysis of integration relative to nucleosome
structure on target DNA indicated favoring of
integration in outward facing DNA major grooves on
the nucleosomal surface.
Gary Wang, Rick Bushman, Mol Ther 2009
Using Zinc Finger Nucleases to Generate
CCR5 deficient CD4 cells: engineering resistance
Entry inhibitors
-T20: enfuvirtide
-Triple gp41 heptad repeat
-CCR5 blockers:
Maraviroc
Vicriviroc
-CCR5 knockout:
naturally occurring Δ32
genome edited CD4 cells
Hypothesis: Could zinc finger nucleases be used
to introduce a disease resistance gene by disruption of CCR5?
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Combinatorial Strategy with ZFNs To
Achieve Genome Specific Targeting
FokI nuclease
domain
FokI nuclease
domain
Porteus, Nat Biotech 2005
ZFN-Mediated Genome Editing
Endogenous gene targeted for
disruption (CCR5)
1.
2.
x
Break repaired by nonhomologous end-joining
(NHEJ) – resulting in loss of
genetic information
3.
4.
ZFNs dimerize and introduce
a double stranded DNA
break in the gene
x
Gene disrupted
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Rationale for Selection of CCR5 ZFN
Target for Gene Modification Therapy
• Primary HIV-1 coreceptor
• People carrying mutations of both CCR5 alleles
(homozygotes for CCR5 delta 32 deletion) are resistant for
HIV infection (1% of Caucasian population
• No adverse effects of the mutation in humans for mice
• “Hit-and-Run Delivery” – Transient delivery of CCR%-ZFNs
can permanently disrupt the CCR5 coding sequence,
generating modified cells resistant to R5-tropic HIV
• T cells can be isolated, modified, and expanded >100-fold
ex vivo. These cells can be reinfused over a period of time
to maintain a resistant population of CD4+ T cells.
NEJM 2009; 360: 692
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Can ZFNs Phenocopy the CCR5Δ32 mutation?
Specific Disruption of the CCR5 Gene
Cleavage products
(ZFN-modified DNA)
Perez, Nature Biotech, 2008
High Efficiency
Disruption of
CCR5 in Primary
Human CD4 T
cells Via Ad5/35
Transduction of
ZFNs
Genome Editing to Delete CCR5
Pre-Clinical Approach
CD4 cells
Donor
( HIV +/-)
Activate CD4 Cells
PHA/IL2
CD3/28
Introduce ZFN plasmids
Electroporation
Ad5/35
Infect HIV-1
Culture 10 - 70 days
Cell lines
PM-1
Ghost
Transfer to
NOD/SCID/γc-/-
Perez, Nature Biotech
Assess for genotoxicity
Assess CCR5 disruption (cel1)
Assess function
HIV resistance
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In Vivo Selection of CCR5-ZFN Modified
Primary CD4 T-cells in HIV Infected
NOD/SCID IL-2Rγnull (NSG) Mice
PBMCs
CCR5 or GFP modification
In vitro expansion
PBMCs
CD4 cells
Inject
Inject
NOG (NOD/scid/IL-2Rgc-)
HIV-1
(2 - 12 weeks)
Analysis
In Vivo Reduction of Viral Load and
Selection of Genome Edited CD4 Cells
by HIV-1
NSG mice xenografted with human CD4 cells
13
Summary – Preclinical Studies
• CCR5 has been validated as a major target for
inhibition of HIV infection: genetic accidents of nature
and small molecule compounds
• Genome editing has been accomplished at therapeutic
levels of efficiency at the CCR5 locus when introduced
into primary human T cells, confering robust resistance
to HIV infection in vitro and in vivo
• Targeted CCR5 gene disruption in T-cells could provide
an effective therapy for HIV infection/AIDS
• Genome editing of T cells (and stem cells?) may be a
promising strategy for a variety of acquired and
congenital diseases
Pilot Test of Adoptive Transfer of CCR5
Deleted CD4 T Cells using ZFNs to
Introduce HIV resistance
pUC ori
CMV promoter
EcoRI (740)
KpnI (791)
pVAX-SB-728
5016 bp
BamHI (1145)
8267-FokEL (ZFN1)
Clinicaltrials.gov NCT00842634
KanR
BglII (1739)
2A peptide
BGH polyA
XhoI (2804)
Ad5/35
Transduction
AvrII (1805)
KpnI (1853)
BamHI (2204)
8196z-FokKK (ZFN2)
Human Gene Transfer Protocol #0704-843
NIH/OBA/RAC approval June 20, 2007; FDA Approval Feb 20009
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Take Home Messages
o VRX496 anti-sense env engineered
CD4 T cells are safe (n=~60
infusions) and can traffic to IEL
o Zinc finger nucleases permit
generation of genetically edited T
cells that are resistant to HIV.
Pilot test ongoing.
Disclaimer
o
o
Under a licensing agreement between the University of
Pennsylvania and Invitrogen, Inc., the University is
entitled to a share of royalty received by the University
on sales of some of the technology described in this
presentation.
This arrangement is being managed by the University
in accordance and compliance with its conflict of
interest policies.
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Collaborators and Acknowledgements
Path
Path &
& Lab
Lab Medicine
Medicine
Richard
Richard Carroll
Carroll
Tatiana
Tatiana Golovina
Golovina
Bruce
Bruce Levine
Levine
James
James Riley
Riley
CVPF
Bruce Levine
Don Siegel
Gwen Binder
Anne Chew
Zoe Zheng
Julio Cotte
Penn CFAR
Faten Aberra
Jean Boyer
Rick Bushman
Gary Wu
Farida Shaheen
Ronald Collman
Penn ACTU
Pablo Tebas
Larisa Zifchak
Joe Quinn
Ian Frank
Children’s
Children’s Hospital
Hospital
Jordan
Jordan Orange
Orange
Elena
Elena Perez
Perez
Stephan
Stephan Grupp
Grupp
ViRxSys
Laurent Humeau
Cathy Afable
Tessio Rebello
Vladimir Slepushkin
Gary McGarrity
Sangamo
Sangamo Biosciences
Biosciences
Mike
Mike Holmes
Holmes
Philip
Philip Gregory
Gregory
Dale
Dale Ando
Ando
Support
NIH: NIAID
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