Transcript Stem Cell Transplantation for Sickle Cell Disease

CRISPR gene editing to cure
Sickle cell disease :
Hurdles
to overcome in
Stem
Cell Transplantation
developing an IRB approved
for Sickle Cell Disease
clinical trial
Fred Goldman MD
Director, Pediatric Blood and Marrow Transplant Program
Children’s of Alabama, University of Alabama, Birmingham
Outline
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Review sickle cell disease and therapies
Review bone marrow transplant and gene
therapy
Discuss CRISPR technology
Show our preclinical data in mice and humans
Safety concerns and FDA approval
How to best protect human subjects
Sickle cell disease (SCD)
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Most common genetic disorder
amongst African Americans
Caused by a single base pair
mutation in the betahemoglobin chain
GAG to GTG at codon 6
 Glutamic Acid to Valine
substitution produces
hydrophobic projection
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Abnormal hemoglobin
polymerizes upon
deoxygenation, RBC to take on
its characteristic sickle shape
Consequence is severely
shortened red blood cell
lifespan and severe anemia
Epidemiology and statistics
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The average life expectancy: 42 years for males and 48
years for females
Approximately 1 in 600 African American births results
in sickle cell disease
About 100,000 Americans have sickle cell disease, 2000
new births annually
1000 children with SCD in Alabama, ~100 between
18-21, ~8,000 adults
Newborn screening 50 new cases/yr in Alabama
Nigeria, 150,000 born with SCD annually
This is a WORLD WIDE problem
Long term organ damage in SCD
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Neurologic
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Pulmonary issues
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Acute chest syndrome
Pulmonary hypertension 40% by age 40
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Bone infarctions
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11% will have a stroke before age 18
1/3 will develop silent stroke by age 15
Transcranial doppler (TCD) predictive
Cognitive dysfunction
Renal insufficiency
Infection risks
Vasocclusive crises = pain crises
Iron overload from chronic transfusion also causes
end organ damage
Current treatment options in SCD
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Supportive care-manage symptoms
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Blood transfusions for acute event
Hydroxyurea (HU)
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Increases Hgb F, lowers white blood cell count
Clinical trials
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Prophylactic antibiotics
pain medications
SIT trial- transfusion better than observation
STOP trial- need transfusion for abnormal transcranial
doppler study
SWITCH- transfusion better than HU in preventing strokes
Baby HUG- fewer crises with HU vs placebo
Hematopoietic Stem Cell Transplantation
Allogeneic hematopoietic
stem cell transplantation
Bone marrow harvested
from normal donor
Patient
with
disease
Chemotherapy to
wipe out disease
and create space
Stem cell
infusion
Medication to
prevent graftversus-host
disease (GvHD)
Source of hematopoietic stem cells
for transplant
Bone
Marrow
Peripheral blood stem cells
CD34+
Cord Blood
BMT for sickle cell disease
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Only curative option
Donor identification is a problem
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15% will have matched-sibling donor
60% will have HLA-matched unrelated donor (MUD)
>25% have no suitable MUD
Due to toxicities, BMT is reserved for those
with severe disease
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Recurrent chest syndrome
CNS event
Recurrent severe pain episodes
Impaired neuropsychological functioning with abnormal
imaging
Transplant outcomes by donor type
Matched related donor
Other donor types
Expected complications of
allogeneic BMT
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Chemotherapy mediated
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Graft Failure
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Primary rejection
Secondary rejection-occurs after 2 months
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Acute or chronic, higher with MUD
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Neurologic*** increased incidence in SCD
Hepatic
Cardiac
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Hematologic toxicity, bleeding
Increase infection risk from low white count
Mucositis, pain
Graft versus Host Disease
Other organ specific complications
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Reduced intensity regimens for SCD and
mixed donor chimerism
 Less chemo=less toxicity
 in graft rejection
 Partially engrafted in
WBC, yet fully engraft
with RBC
Hsieh et al, NEJM, 2009
Non-SCD Donor
Sickle Trait Donor
Hgb S
Donor chimerism
Hgb S
Donor Chimerism
0
67
36
25
0
74
37
60
7
11
Wu et al, BJH, 2007
Conclusion – “mixed chimerism is a suitable endpoint of stem cell-based therapies for SCD”
Gene therapy
•
Introduction of new genetic
material into the cells of an
organism for therapeutic
purposes
• Abnormal gene and normal
functioning gene identified
and cloned
• Cells responsible for
disease are identified and
accessible for manipulation
• A means of introducing and
expressing genetic material
(viral vector/gene editing)
• Over 2300 clinical trials
from 1989-2016 (per
Wikipedia)
Gene therapy for disorders of the
hematopoietic system
 Severe
immune
deficiencies
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Common g chain ~50 pts
ADA deficiency ~20 pts
CGD ~ 5 pts
WAS ~ 15 pts
 Marrow failure disorders
 Fanconi’s anemia 7 pts
 Hemoglobinopathies
 Sickle cell disease ~ 5 pts
 Thalassemia ~15 pts
Steps in ex-vivo gene therapy
Sustained Correction of X-Linked Severe Combined
Immunodeficiency by ex Vivo Gene Therapy
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12 males with X-linked
SCIDS
Bone marrow derived CD34+
HSC
Ex vivo expanded
transduced using retroviral
vector containing common g
chain
Hacein-Bey-Abina et al, NEJM 2002
Gene therapy complication
insertional mutagenesis
LMO2-Associated Clonal T Cell Proliferation in Two
Patients after Gene Therapy for SCID-X1
Hacein-Bey-Abina et al, Science 2003
BlueBird Bio
Phase 1 study HGB-206 for SCD:
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Gene therapy using
autologous CD34+ cells
transduced ex vivo with
lentiglobin BB305
lentiviral vector
Eligibility
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18 or older
Severe SCD
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Primary endpoint: safety
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Success and kinetics of
HSC engraftment
Incidence of mortality
Detection of vector
mediated insertional
mutagenesis
Clinical adverse events
Data from Bluebird study reported
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Bone marrow harvesting
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Transduction efficiency of HgbAT87Q
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Transfused to Hgb 10-12 with <30% HgbS
Harvest volume 15-20 ml/kg, repeated to obtain minimum
.5-1.3 vector copy number/CD34+ cell pre infusion
.1 vector copy/peripheral blood leukocyte 2-3 months post
Full dose myeloablation
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Busulfex 3.2 mg/kg IV x 16 dose, AUC=1100
Hematologic toxicity expected
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Neutrophil engraftment d16-18
Platelet engraftment d 23-29
Outcomes
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Patients transfusion dependent with minimal expression of
HgbAT87Q
Safety summary
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marrow harvest
2 non serious grade 3 SAE- pain
1 serious SAE with prolonged hospitalization due to pain
N=4, 2,2,2 and 4 harvests needed to achieve
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volume 1.9-3.2L
TNC 7-12 x 10>8/kg
 Safety
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of infused subjects
No AE from BB305 infusion
1 serious AE d+42 (bacteremia)
2 serious AE >d+42 pain crisis
9 non-hematologic grade 3-4 AE in 2 subjects
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Fever, mouth pain, mucositis, febrile neutropenia, anorexia,
fatigue, dyspnea (gr3), bacteremia (gr 4)
 Safety
profile consistent with autologous HSCT
Problems with current gene addition
therapy trials
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Failure to control integration sites
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Random gene insertion in or near proto
oncogenes
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Limited cell numbers
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Common gamma chain SCIDs 5/20
WAS trial 5/10
CGD 5/5
due to primary disease state
Age of patient and challenge of harvest
Inefficiency/inconsistency of transduction
Not under control of endogenous
regulators
CRISPR-Cas System
(Clustered Regularly Interspersed Palindromic Repeats)
CRISPR-Cas System
Provides bacteria
with innate
immunity to
defend against
invading viruses
Gene correction via CRISPR/Cas
• The CRISPR/Cas system
has been used for gene
editing (adding, disrupting
or changing the sequence
of specific genes)
• By delivering the Cas9
protein/enzyme and
appropriate guide RNAs
into a cell, the genome can
be cut/repaired at any
desired location
Phase I Trial of PD-1 Knockout Engineered T
Cells for the Treatment of Metastatic Nonsmall Cell Lung Cancer
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Evaluate the safety of
CRISPR-mediated PD-1
knockout engineered T
cells
Primary outcomes
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Number of participants
with adverse events
and/or dose limiting
toxicities as a measure of
safety
Sponsor
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Sichuan University
UAB IRB approved protocol to collect
bone marrow from SCD patients
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Sickle cell adult patients
consent to undergo a
bone marrow aspirate
Patients financially
compensated
Collect 30 ml of marrow
from hip
Isolate CD34 cells by
selection column
Test in vitro and in NSG
mice
Differentiation in vitro of CRISPR/Cas corrected
sickle CD34+ stem cells
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CRISPR/Cas modified
to allow optimal
correction and
preserve viability
CD34+ cells undergo
nucleoporation with
RNP/ssODN complex
Cells are plated onto
methocult where they
differentiate
Red blood cell
precursor colonies are
picked and DNA is
sequenced
CD34+
BFU-E
GEMM
S/S
Selected Sanger
sequencing
results of six
genotypes of
GEMM/BFU-E
colonies
S/Indel
Indel/Indel
A/S
A/Indel
A/A
mr5Cas9 complex nucleofected human sickle CD34+
GEMM/BFU-E sanger sequencing results
1
2
3
4
5
6
7
8
9
10
11
12
A
SS
A/IND
A/S
SS
SS
A/IND
SS
SS
A/S
IND/IND
A/S
S/IND
B
SS
A/S
SS
S/IND
S/IND
SS
SS
AA
S/IND
SS
AA
SS
C
SS
A/IND
SS
SS
IND/IND
A/S
S/IND
SS
S/IND
SS
A/S
SS
D
S/IND
SS
S/IND
AA
SS
SS
IND/IND
SS
SS
A/S
SS
A/IND
E
SS
SS
S/IND
SS
A/S
SS
A/S
S/IND
S/IND
SS
SS
IND/IND
F IND/IND
SS
SS
A/IND
SS
SS
A/IND
IND/IND
SS
SS
A/S
SS
G IND/IND
SS
SS
A/S
IND/IND
A/IND
A/S
A/IND
SS
A/S
S/IND
A/IND
H
SS
SS
S/IND
SS
SS
S/IND
SS
S/IND
A/S
SS
SS
A/IND
Complex for nucleofection
mr1Cas9 + T2gRNA + ssODN
GEMM /BFU-E colonies
Total colonies
A/A
A/S
S/S
A/indel
S/indel
Indel/indel
Colonies with at least 1 allele corrected
Colonies with indels
Colonies with genome modification
Total number of alleles
16/80
96
3.1%
14.6%
47.9%
10.4%
15.6%
8.3%
28.1%
42.7%
52.1%
3
14
46
10
15
8
27
41
50
192
Total “A” alleles (corrected)
30
Total “S” alleles (uncorrected)
Total “indel” alleles
121
41
15.6%
63.7%
21.6%
GEMM
correction:
4/16
Total
Colonies:
2.5X
CAS9WT
Deep sequencing data for 5 Off-Target (OT) sites in
Sickle BM CD34+ GEMM/ BFU-E
0.1200%
0.1000%
0.0800%
Neg ctrl
wtCas9
0.0600%
mr5Cas9
mr4Cas9
0.0400%
0.0200%
0.0000%
OT1
OT2
OT3
OT4
OT5
CRISPR corrected human sickle CD34+ bone
marrow produce RBCs with normal hemoglobin A
Qualitative mass spec analysis of betaglobin in sample marked with red
rectangle above.
Quantitative data obtained from
comparison to a standard curve of
known mixtures of betaS and betaA
peptides demonstrate 35% betaA.
Mouse model of sickle cell disease
Science 245: 971-973 (1989)
Science 247: 566-568 (1990)
Science 278: 873-876 (1997)
Science 318:1920-1923 (2007)
Sickle gene correction in long-term
reconstituting HSC
mouse control
mouse AS control
mouse AA control
mouse SS control
mr5Cas9-BM#30
mr5Cas9-BM#10
mr5Cas9-BM#3
mr5Cas9-BM#1
IEF gel analysis of hemoglobin at
12 weeks after secondary transplant (24 weeks total)
Blood counts 12 weeks after
secondary transplant
RBC (x10^6 cells/µL)
HGB (g/dL)
HCT (%)
% Retic
AS
SS
Corrected
11.26
10.4
46.1
17.47
6.38
6.5
34.4
56.8
10.53
9.4
40.5
6.38
Spleens at 12 weeks after secondary transplant
AA Control
SS Corrected SS Uncorrected control
Human A/S (sickle trait) cord blood CD34+
mr5Cas9/ssODN nucleoporation
Intra-femoral injection into NSG mice
FACS Purification of BM Cells at 13 Weeks Post-Transplantation
Multi-lineage long-term reconstitution of
corrected human CD34+ cells in NSG Mice
FACS Sorted Cells
A*/A*+ S
B cells
(CD19)
T cells
(CD7)
monocyte RBC pre Stem cell
(CD11b) (CD235a) (CD34)
Exp 1
Exp 2
2.00E+05
2.60E+05
1.10E+05
1.80E+05
3.30E+05
3.20E+05
1.80E+04
4.10E+04
2.60E+05
7.80E+05
Exp 1
Exp 2
6.00%
60.70%
6.80%
78.10%
7.70%
82.20%
5.40%
66.70%
2.80%
72.20%
Summary of pre-clinical data
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CRISPR/Cas complex can correct the sickle
mutation in both human and murine HSC
Efficiency of correction (25-50%) should be
sufficient to correct the disease
CRISPR can correct true HSC as
demonstrated by secondary transplants
Minimal off target sites and no evidence of
mutagenesis thus far
Challenges for a phase 1 study
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Collection of HSC from sickle cell patients
Preparative therapy
Must complete preclinical safety studies in
mice transplanted with “therapeutic dose”
Patient enrollment
UAB GMP facility
Safety issues of marrow
collection
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Requires sufficient volume to get desired
CD34 cell count
 General
anesthesia and transfusions
 Need 2-3 liters of marrow
 Recovery and pain post procedure, repeat?
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Alternatives
 GCSF
mobilization of peripheral blood stem cells
not feasible in SCD
 Expansion techniques ex vivo, successful for cord
blood (SR-1)
Safety issue of chemo-ablation
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Full dose Busulfan and associated toxicity
Partial dose Busulfan (25%)
 Works
for SCIDs
 In sickle cell disease, may be inadequate for
engraftment
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Alternatives
 Non
myeloablative conditioning and use of marrow
niche clearing agents (ACK2)
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Preclinical testing ongoing in sickle mice
Successful in other murine disease models
CRISPR issues
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Must produce in bulk in a GMP facility
Consistent nucleoporation and correction
efficiency
Malignant transformation potential low
 Monitor
sites
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and sequence off target and oncogene
Antibody formation
Patient issues
Who would be the best candidate
 What side effects will be expected
 What if there is no engraftment
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What the FDA will say?
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Medical standards are disease specific
CRISPR editing of sickle CD34+ cells is more
than “minimal manipulation” and thus will be
considered a “drug” therapy needing an IND
GMP facility for stem cell processing and
CRISPR production
Work with local IRB in tandem in developing
plan
Comparison of transplant options
Allogeneic
MUD HSCT
Allogeneic
Haplo HSCT
Allogeneic
MRD HSCT
Lentivi
HSCT
CRISPR
HSCT
donor
60%
100%
15%
100%
100%
GVHD
+++
+/++
+
-
-
Infection
++
++
++
+
+
reject
+
+
+
++
?
# done
~100
~100
~500
5
0
Cost
~ 600k
~ 600k
~ 400k
?
?
Acknowledgements
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Tim Townes and the lab
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Lei Ding
Chao Li
Chai Wei Chang
Li-Shin Lai
Kevin Pawlik
Joe Sun
Jane Wu
Dewang Zhou
Erik Westin
Divya Devadasan
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IRB
UAB
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Hematology service
OB/GYN
Stem Cell Institute
All of our sickle cell
patients and their
families