engineering stem cells to combat hiv disease jerome a. zack ph.d. david geffen school of medicine at...
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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 cyclehow the virus infectstargets 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
CD4
CXCR4
Binding
Fusion & Entry
Nuclear localization & entry
Reverse transcription
Integration
Infection
gp120
p24
Viral RNA
RT & othervirion proteins
CCR5
Expression
gp120
p24
Viral RNA
RT & othervirion prteins
Budding
Assembly
Viral Gene Transcription
Translation
Post-translationalprocessing
Cellular Activation
Why Gene Therapy?
Current therapies are not 100% effectiveResistance is seen, even to combined approachesToxicities may preclude use of certain antiretrovirals
Genetic therapies would target different aspects ofThe viral life cycle
These types of therapies may be long-lasting, requiring onlyA single, or limited number of treatments
Toxicities may be minimal
Neutrophil
BM Stemcell
Myeloid stem cell
T progenitor
DP Thymocyte
CD4+
T cellB cell
MegakaryoblastErythroid progenitor
Eosinophilprogenitor
PlateletsRed blood cells
Myelomonocytic progenitor
Basophilprogenitor
BasophilEosinophil
Lymphoid stem cell
Megakaryocyte
B progenitor
Macrophage
Monocyte
CD8+
T cell
Stem Cell
NK Cell
Gene Therapy
HIV HIV
HIV
Anti-ViralGene
ES/iPS Cells
Phase II Ribozyme Adult Stem Cell
Gene Transfer Protocol
An anti-viral gene therapy approach
Hammerhead Ribozyme
AA
CU
GA
UGAG
CUCGGUCA CUAGGAUU
C GA UG CG C
AG U
G
GGAGCCAGUA GAUCCUAA
Cleavage site
Complementary
Flanking Sequence
Complementary
Flanking Sequence
CatalyticDomain
5'3'
5' 3' TARGET RNA
RIBOZYME
• RNA, hybridising arms
• True enzymes - catalytic
domain
• Nucleophilic attack after GUAHaseloff & Gerlach, 1988
2 x ART2 x ARTInterruptions (ATI)Interruptions (ATI)• 25 - 28 weeks25 - 28 weeks• 41 - 48 weeks41 - 48 weeks
11oo endpoint at 48 endpoint at 48 wkwk
• Difference in Difference in Viral RNA at 48 Viral RNA at 48 wkswks
CD34CD34++ cellscells
HIV-Infected HIV-Infected individualindividual
G-CSF G-CSF
RzRz
Precursor Precursor cellscells
Infuse Infuse cellscells
Main Entry CriteriaMain Entry Criteria• 1st or 2nd ART regimen1st or 2nd ART regimen• Viral load < 400 c/ml for 6 Viral load < 400 c/ml for 6
MoMo• CD4CD4++ cells > 300/mcL cells > 300/mcL• Age 18 - 45 yearsAge 18 - 45 years
DesignDesign• Randomised - active vs. Randomised - active vs.
placeboplacebo• Double blindDouble blind• 2 yr study2 yr study• 37 patients per group37 patients per group
Protocol
74 adult HIV+ patients enrolled
Bone marrow stem cells from half the patients were treated with an anti-viral gene
Half of the patients received control untreated stem cells
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
Published online, Feb 15, 2009
Largest cell-delivered gene therapy trial ever done
Results
Gene Therapy for HIV Disease
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 CCR532/32 mutation
CCR5 32/32 homozygous mutation 1% in Caucasian populationNo CCR5 expressionNaturally protected from HIV-1 infection
CCR5 32 heterozygous mutation 10% in Caucasian population50% less CCR5 expressionSlower progression to AIDS (2-3 years)
These individuals have apparently normal health status
Hutter et.al. N Engl J Med. 2009 Feb 12;360(7):692-8.
Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation
CCR5-32/ 32BM Donor (HIV-)HLA matched
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-transplantand remained negative for 20 months. (now 3 years)
BM transplant
Acute Myeloid LeukemiaPatient (HIV+)
Following conditioning TBI
RNA interference (RNAi)
siRNA (20nt)
RNAi
CCR5 mRNAAAAAn
Induce sequence specificmRNA degradation
Lentiviral vector mediated stable siRNA delivery
Vector
siRNA
CCR5mRNA
CCR5
This approach has thus far shown:
long-term engraftment in monkeysEfficacy 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
Scott Kitchen, PhD
A stem cell genetic approach
to enhance anti-viral immunity
Neutrophil
BM Stemcell
Myeloid stem cell
T progenitor
DP Thymocyte
CD4+
T cellB cell
MegakaryoblastErythroid progenitor
Eosinophilprogenitor
PlateletsRed blood cells
Myelomonocytic progenitor
Basophilprogenitor
BasophilEosinophil
Lymphoid stem cell
Megakaryocyte
B progenitor
Macrophage
Monocyte
CD8+
T cell
Stem Cell
NK Cell
Gene TherapyClass I RestrictedTCR Gene
HIV Gag SL9-Specific T Cell Receptor
Restricted to HLA-A2.01
ESCESC
ESCESCESC
CD34+CD34+
CD34+CD34+
CD34+
1. Sort CD34+
3. Analyze TCR
ExpressionSCID-hu
Irradiate
3-12 weeks
Fetal Liver
2. Transduce withAnti-HIV TCR
(SL9 Peptide Specific)
Scott Kitchen
HLA-A2.1+Human Thymus
CD8
The Chimeric Model System
Killing of HIV+ Target Cells by “Transgenic” T Cells
The data show us:
Stem cells can be engineered to become anti-viral T cellsThese cells kill virally infected cellsThe 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 melanomaWhich 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 vitroThese cells can be genetically manipulated easilyThere are no issues with difficulty of extraction from patientsWe 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.
ConclusionsStem cell based therapies have been tested in the clinic, andhave relevance to HIV disease
Stem cell based therapeutics could offer life-long benefit, as stemcells themselves survive for the life of the individual
These approaches are continually evolving
Approaches attacking viral gene products, cellular gene productsand that manipulate the immune system are in development
It is likely that ablation of existing stem cell components (I.e. bonemarrow) will be needed to increase the efficiency of reconstitutionof 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
Mock No-shRNA lacZ-shRNA CCR5-shRNACC
R5
30.1 39.4
29.1 1.38
23 43.1
16.4 17.6
56 8.9
30.8 4.31
69.7 0.019
30.3 0.019
EGFP
% CCR5 + in Vector +population N/A 3%33%29%
ReducedCCR5Expression
(indicates vector)
Control
30%
Reduction of virus production in CCR5 tropic HIV-1 infected CCR5-shRNA transduced T cells in vitro
0
50
100
150
200
mock No-shRNA lacZ-shRNA
CCR5-shRNA
p24
(ng/
ml)
CCR5 tropic HIV
0
2
4
6
8
10
12
14
16
18
0 2 4 6 8 10 12 14
days
the amount of p24
(ng/mL)
shRNAno shRNA
CXCR4 tropic HIV
0
10
20
30
40
50
60
0 2 4 6 8 10 12 14
days
the amount of p24 (ng/ml)
shRNAno shRNA
CCR5 tropic HIV inhibition in human splenocytes ex vivo
MHC I
CD8+
MHC II
CD4+
Lineage Commitment
T Cell Selection
SCID-hu mouse
3-4 months
SCID-hu mouse as a model for human thymopoiesis
Thy/Liv implant
CD8
Human fetal liver
Human fetal thymus
hESC-derived Progenitors were injected into irradiated SCID-hu mice
100 101 102 103
100
101
102
103
0.48 0.01
93.85.6100 101 102 103
100
101
102
103
13.1 11.7
1.8973.3
10.6
SL9 Tetramer CD8
SL9 Tetramer
SL9 Tetramer
UntransferredControl
Mouse #17
Thymus Spleen
HIV-TCRTransduced
CD34+Recipient
Mouse #24
SL9 Tetramer
100 101 102 103
100
101
102
103
50.5 0.25
0.1249.1
0.2
6.7 79.5
4.039.79100 101 102 103
100
101
102
103
0.04
0.00399.95
0.003
CD8100 101 102 103
100
101
102
103
0.06 0.21
0.0199.72
CD8+ TCR expressing cells are made and exported to the periphery
SL9 Tetramer
HLA-A*2.01+Recipient
Mouse # 47-29
100 101 102 103
100
101
102
103
69.3 30.6
0.10100 101 102 103
100
101
102
103
1.02 3.41
0.1595.4
3.35
CD8
HLA-A*2.01-Recipient
Mouse # 47-15
100 101 102 103
100
101
102
103
4.28 15.4
0.06180.2
15.4
100 101 102 103
100
101
102
103
30 15.8
42.212
The HLA*A2.01 Molecule is Required for Development of Transgenic T Cells
NoCD8+ Cells
Analysis of HIV-Specific TCR on Transgenic T Cells
Biopsy
T1 cells(A*0201+)
“load”with SL-9
peptides
T1 cells(A2.1+)
T1 cells(A2.1+)
T1 cells(A2.1+)
Mix,1 Week Culturew/ IL-2
IFN- ELISPOT(3 additional days), Killing of targets
Thymocytes
SCID-hu