Download BM Stem cell

Engineering Stem Cells to Combat
HIV Disease
Jerome A. Zack Ph.D.
David Geffen School of Medicine
At UCLA
What We Will Talk About
HIV life cycle
how the virus infects
targets of current drugs
Why gene therapy?
What hematopoietic (blood forming) stem cells are
A gene therapy strategy tested in the clinic
Several strategies under development
Human embryonic and induced pluripotent stem cells
Viral RNA
gp120
p24
RT & other
virion
proteins
Fusion & Entry
Binding
CCR5
CD4
CXCR4
Reverse
transcription
Nuclear
localization
& entry
Integration
Viral RNA
gp120
p24
Cellular Activation
Assembly
RT & other
virion prteins
Post-translational
processing
Budding
Translation
Viral Gene
Transcription
Why Gene Therapy?
Current therapies are not 100% effective
Resistance is seen, even to combined approaches
Toxicities may preclude use of certain antiretrovirals
Genetic therapies would target different aspects of
The viral life cycle
These types of therapies may be long-lasting, requiring only
A single, or limited number of treatments
Toxicities may be minimal
Stem Cell
Gene Therapy
ES/iPS Cells
Anti-Viral
Gene
Myeloid stem cell
Erythroid
progenitor
Megakaryoblast
Eosinophil
progenitor
Lymphoid stem cell
BM Stem
cell
Basophil
progenitor
Myelomonocytic
progenitor
B progenitor
T progenitor
DP Thymocyte
Monocyte
NK Cell
Megakaryocyte
HIV
CD8+
T cell
Red blood cells
Platelets
CD4+
T cell
Eosinophil Basophil Neutrophil Macrophage B cell
HIV
HIV
Phase II Ribozyme Adult Stem
Cell
Gene Transfer Protocol
An anti-viral gene therapy
approach
Hammerhead Ribozyme
Cleavage site
5'
3'
GGAGCCAGUA GAUCCUAA
TARGET RNA
RIBOZYME
CUCGGUCA CUAGGAUU5'
A CU
G
A
A
G
A U
Complementary
G
Complementary
C G
Flanking Sequence
Flanking Sequence
A U
G C Catalytic
Domain
G
C
• RNA, hybridising arms
A
G
• True enzymes - catalytic
G U
3'
domain
•
Nucleophilic attack after GUA
Haseloff & Gerlach, 1988
Protocol Design
Main Entry Criteria
• 1st or 2nd ART regimen
• Viral load < 400 c/ml for 6
Mo
• CD4+ cells > 300/mcL
• Age 18 - 45 years
G-CSF
Precursor
cells
• Randomised - active vs.
placebo
• Double blind
• 2 yr study
• 37 patients per group
Infuse
cells
HIV-Infected
individual
CD34+
cells
2 x ART
Interruptions (ATI)
• 25 - 28 weeks
• 41 - 48 weeks
Rz
1o endpoint at 48
wk
• Difference in
Viral RNA at 48
wks
Gene Therapy for HIV Disease
Published online, Feb 15, 2009
74 adult HIV+ patients enrolled
Largest cell-delivered gene therapy trial ever done
Bone marrow stem cells from half the patients were treated with an anti-viral gene
Half of the patients received control untreated stem cells
Results
Some diminishment in viral rebound when taken off of anti-retrovirals in treated group
CD4+ T cell counts somewhat higher in treated group
No adverse events due to gene therapy
The HIV co-receptor CCR5 is an
excellent HIV therapeutic target
A gene therapy approach targeting a cellular
gene critical in the initial step of HIV infection
Irvin Chen, Dong Sung An
Natural HIV resistance by CCR532/32 mutation
CCR5 32/32 homozygous mutation
1% in Caucasian population
No CCR5 expression
Naturally protected from HIV-1 infection
CCR5 32 heterozygous mutation
10% in Caucasian population
50% less CCR5 expression
Slower progression to AIDS (2-3 years)
These individuals have apparently normal health status
Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation
Hutter et.al. N Engl J Med. 2009 Feb 12;360(7):692-8.
CCR5-32/ 32
BM Donor (HIV-)
HLA matched
Acute Myeloid Leukemia
Patient (HIV+)
BM transplant
Following conditioning
TBI
Nearly 100% replacement with the CCR5 negative donor cells.
HAART was discontinued after BM transplant.
HIV RNA and DNA became undetectable at 68 days post-transplant
and remained negative for 20 months. (now 3 years)
RNA interference (RNAi)
siRNA (20nt)
CCR5 mRNA
AAAAn
RNAi
Induce sequence specific
mRNA degradation
Lentiviral vector mediated stable siRNA delivery
CCR5
Vector
CCR5
mRNA
siRNA
This approach has thus far shown:
long-term engraftment in monkeys
Efficacy in mouse/human chimeric models
An analogous approach is being pursued at City of Hope/USC
This involves a reagent known as a zinc finger nuclease
The concept is to add the nuclease to stem cells ex vivo,
and this deletes the CCR5 gene. The stem cells will then be
re-introduced into the patient
Genetic Engineering of
Human Immune Responses
A stem cell genetic approach
to enhance anti-viral immunity
Scott Kitchen, PhD
Stem Cell
Gene Therapy
Class I Restricted
TCR Gene
Myeloid stem cell
Erythroid
progenitor
Megakaryoblast
Eosinophil
progenitor
Lymphoid stem cell
BM Stem
cell
Basophil
progenitor
Myelomonocytic
progenitor
B progenitor
T progenitor
DP Thymocyte
Monocyte
NK Cell
Megakaryocyte
CD8+
T cell
Red blood cells
Platelets
Eosinophil Basophil Neutrophil Macrophage B cell
CD4+
T cell
HIV Gag SL9-Specific T Cell Receptor
Restricted to HLA-A2.01
The Chimeric Model System
2. Transduce with
Anti-HIV TCR
(SL9 Peptide Specific)
Fetal Liver
Irradiate
CD34+
CD34+
CD34+
CD34+
CD34+
ESCESC
ESCESC
ESC
SCID-hu
HLA-A2.1+ 3-12 weeks
Human Thymus
3. Analyze
TCR
Expression
1. Sort CD34+
CD8
Scott Kitchen
Killing of HIV+ Target Cells by “Transgenic” T Cells
The data show us:
Stem cells can be engineered to become anti-viral T cells
These cells kill virally infected cells
The TCR must “match” the donor HLA molecules
This provides proof-of-principle that we can engineer the
human immune system.
Due to the mutation rate of the virus, for this approach to be
valid for HIV disease, multiple TCRs specific for multiple antigens,
in the context of different HLA molecules would be needed.
We are currently testing this type of approach for human melanoma
Which should not mutate as quickly as HIV, and be a better target
A Word About Totipotent Stem Cells
The previous studies all involved hematopoietic stem cells (HSC)
These are applicable for stem cell therapeutics
However, it may be difficult to obtain them, some patients will have
poor quality stem cells, and these cells are difficult to expand
Totipotent cells have some potential advantages over HSC
Human embryonic stem cells (hESC) can be expanded in vitro
These cells can be genetically manipulated easily
There are no issues with difficulty of extraction from patients
We have shown that they can be differentiated into T cells
Induced Pluripotent Stem Cells (iPS)
These cells have similar properties/advantages to hESC
However they can be obtained from any patient, and will thus be
genetically matched to the recipient, and not be rejected by the
immune system. These cells also have the potential to
differentiate along hematopoietic lineages.
Conclusions
Stem cell based therapies have been tested in the clinic, and
have relevance to HIV disease
Stem cell based therapeutics could offer life-long benefit, as stem
cells themselves survive for the life of the individual
These approaches are continually evolving
Approaches attacking viral gene products, cellular gene products
and that manipulate the immune system are in development
It is likely that ablation of existing stem cell components (I.e. bone
marrow) will be needed to increase the efficiency of reconstitution
of newly introduced cells
Development of ES and iPS technology may facilitate genetic
therapeutic approaches to a variety of diseases
CCR5 down-regulation in CCR5 shRNA transduced primary human T cells
Control
Mock
% CCR5 +
in Vector +population
30%
CCR5
30.3
No-shRNA
N/A
0.019
lacZ-shRNA CCR5-shRNA
29%
16.4
17.6
33%
30.8
4.31
3%
29.1
1.38
Reduced
CCR5
Expression
69.7
0.019
23
43.1
56
EGFP
(indicates vector)
8.9
30.1
39.4
Reduction of virus production in CCR5 tropic HIV-1
infected CCR5-shRNA transduced T cells in vitro
p24 (ng/ml)
200
150
100
50
0
mock
No-shRNA
lacZshRNA
CCR5shRNA
CCR5 tropic HIV inhibition in human splenocytes
ex vivo
CCR5 tropic HIV
CXCR4 tropic HIV
18
shRNA
no shRNA
50
the amount of p24
(ng/mL)
the amount of
p24 (ng/ml)
60
40
30
20
10
shRNA
no shRNA
16
14
12
10
8
6
4
2
0
0
0
2
4
6
8
days
10
12
14
0
2
4
6
8
days
10
12
14
T Cell Selection
Lineage
Commitment
MHC I
MHC II
CD8+
CD4+
SCID-hu mouse as a model for human thymopoiesis
Human fetal
thymus
Human fetal
liver
Thy/Liv implant
3-4 months
SCID-hu mouse
CD8
hESC-derived Progenitors were injected into irradiated SCID-hu mice
CD8+ TCR expressing cells are made and
exported to the periphery
Thymus
10 3
50.5
0.25
10 2
Untransferred
Control
Mouse #17
6.7
10 3
79.5
10
1
10
1
10
0
10
0
0.12
10 0
10 1
10 2
10 3
13.1
2
10
1
10
0
11.7
10.6
1.89
73.3
10
0
10
1
SL9 Tetramer
10
2
0.003
10
3
2
10
2
10
1
10
1
10
0
10
0
3
10
0.48
10 1
10 2
93.8
10
CD8
0.06
0.01
5.6
3
10 0
10 3
SL9 Tetramer
10
10
0.003
99.95
CD8
3
10
4.03
9.79
SL9 Tetramer
10
0.04
10 2
0.2
49.1
HIV-TCR
Transduced
CD34+
Recipient
Mouse #24
Spleen
0
10
1
10
2
10
0.01
99.72
3
0.21
10
0
10
1
SL9 Tetramer
10
2
10
3
The HLA*A2.01 Molecule is Required for Development of Transgenic T Cells
HLA-A*2.01+
Recipient
Mouse # 47-29
3
10
3
10 2
10
2
10 1
10
1
10
0
10
10
0
80.2
10 3
HLA-A*2.01Recipient
Mouse # 47-15
15.4
15.4
4.28
0.061
10 0
10 1
10 2
10 3
2
10 2
10
1
10
1
0
10
0
10
10
10
SL9 Tetramer
1
10
2
10
0
10
1
10
69.3
2
3
10 3
30.6
0
0.15
0
42.2
10
10
95.4
15.8
12
10 3
3.41
3.35
1.02
30
0.1
10
CD8
0
10
1
10
2
10
3
No
CD8+
Cells
Analysis of HIV-Specific TCR on Transgenic T Cells
SCID-hu
Biopsy
Thymocytes
T1 cells
(A2.1+)
T1 cells
(A*0201+)
“load”
with
SL-9
peptides
T1 cells
(A2.1+)
T1 cells
(A2.1+)
Mix,
1 Week
Culture
w/ IL-2
IFN- ELISPOT
(3 additional days),
Killing of targets