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Gene Therapy for Primary
Immunodeficiency
Phase l/ll studies at ICH/GOS
X-SCID (9+1 patients)
ADA SCID (1 patient)
X-CGD (2 patients)
X-linked severe combined immunodeficiency (SCIDX1)
1 in 50-100,000 live births
Major form of SCID
Severe diarrhoea,
pneumonia, septicaemia,
fungal infection, failure to
thrive, death usually within
first year of life.
HSC-multi
SCID/CID
c, IL7R, JAK3, ZAP-70
RAG1/2, artemis, ligase IV
Cernunnos
ADA, PNP
MHC I/II, CD3, CD45
T
B
NK
IL-2
 b 
JAK3
JAK1
STAT5
P
STAT5
STAT5
Gene transcription
P
Blood
Bone marrow
Stem cells
B
Bone Marrow
Transplantation: Use
donor bone marrow stem
cells to correct the defect
Thymus
NK
lymphocytes
T
Blood
Bone marrow
Stem cells
B
Gene therapy: Put the
new gene into bone
marrow stem cells to
correct the defect
Thymus
NK
lymphocytes
T
Treatment by bone marrow transplantation
 Use
of matched donors successful in 90%
of cases
 for 60% of cases, no matched donors have
to use mis-matched donors, reduces
success rate to <60%
 need to improve success for children with
no matched donor
Cumulative probability of survival after related HLAmismatched HSCT in SCID patients
3 years
survival rate
45.2%
53.8%
SCIDX1 morbidity and mortality following HLAmismatched transplantation…
20% 1 year mortality
Long term effects related to chemotherapy, usually with
alkylating agents (growth, fertility, secondary malignancy,
neuropsychological, hypodontia)
Incomplete immunological reconstitution
X-SCID: major selective growth and survival advantage
for corrected cells….
Somatic reversion events
Animal models
Y+
Gene therapy for X-SCID: phase I study
Criteria for entry:
No matched sibling donor
Molecularly confirmed diagnosis
Hin dIII (9139)
Afl III (386)
5' LTR MoLV
Bgl II (1904)
MFG-gammachain
Afl III (6694)
9142 bp
Gamma chain
Afl III (2929)
Nco I (3079)
Bam HI (3388)
Common gamma chain vector:
PG 13 producer cells (GALV envelope)
titre approximately 1x10e6 transducing units per ml
3' LTR MoLV
Retrovirus particle - pseudotyped
Transduction protocol –
SCIDX1
Harvest
CliniMacs CD34+ bone marrow
Pre-activation (40hours)
X-Vivo10 (serum free)
SCF 300ng/ml, FL 300ng/ml, TPO
100ng/ml, IL-3 20ng/ml
Transduction (3 cycles over 72 hours)
Nexell gas permeable flexible
containers
Retronectin coating
Virus pre-loading
Infusion
P2: Transduction process (days 1-5)
D1
c
CD34
D4
D5
Patient details (April 2006)
Clinical data
Age at
therapy
Maternal
graft
Mutation
(months)
Gamma
chain
expression
Total cells
infused
(x10e6)
P1
10
++
R289X
++
180
P2
10
++
S238N
-
180
P3
4
-
Y125C
+/-
P4
3y
-
R289X
++
115
P5
10
-
R222C
++
200
P6
10
-
PolyA
-
200
P7
6
-
M1i
-
84
P8
13
-
C182Y
-
207
P9
7
-
S108P
-
160
78
Lymphocyte recovery CD3/4/8 (weeks, April 2006)
P1
5000
P2
P3
1200
2000
1000
4000
1600
800
3000
1200
600
2000
800
400
1000
400
200
0
0
0
0
0
20 40 60 80 100 120 140 160 180 200 220 240
20
40
60
80
100
120
140
160
180
T cells per l
P4
1000
200
220
P5
0
20
40
60
100
120
140
160
180
200
220
P6
2500
4000
80
900
2000
800
3000
700
1500
600
500
2000
1000
400
300
1000
200
500
100
0
0
0
0
20
40
60
80 100 120 140 160 180 200 220
0
20
40
60
80 100 120 140 160 180 200 220
P8
P7
2000
1200
1000
1500
800
1000
600
400
500
200
0
0
0
20
40
60
80 100 120 140 160 180 200 220
0
20
40
60
80
100
120
140
160
180
200
220
0
20
40
60
80 100 120 140 160 180 200 220
Lymphocyte recovery CD3 (weeks, April 2006)
5000
4000
3000
P1
P5
P6
2000
P3
P7
1000
P8
P4
P2
0
0
P9
50
100
150
200
250
Flow cytometric analysis of CD45RO-CD27+ naïve T cells in P1
wk4
wk20
wk10
wk24
wk16
wk28
CD27
CD45RO
Good evidence for recent
thymic emigrants
P1 Immunoglobulin levels………
scIg
10
Ig g/l
8
6
4
2
0
0
20
40
60
weeks
IgM
IgA
IgG
80
100
Immune responses to challenge…
Somatic mutation +
10
8
6
2
0
0
20
40
60
80
100
weeks
CD19 PE
Ig g/l
4
CD27 FITC
VZV
IgG and IgM positive
Vaccine antigen positive
Development of
antigen-experienced
B cells (CD19+CD27+)
Lymphocyte proliferation responses
Patient
CD3/CD28
PHA
SEB
Con A
PWM
Candida
MLR
P1



ND
ND
~
ND
P2


ND
ND
ND

ND
P3


ND




P4


ND


ND

Gene expression in CD3/CD28 stimulated CD4 cells of
ATLp1 vs control
100000
ATLp1 (AU)
10000
1000
100
10
1
1
10
100
1000
Control (AU)
10000
100000
Integration analysis
– efficiency and quality of transduction




RT-PCR, linker adaptor mediated (LAM)-PCR and
related techniques
Copy number
Numbers of integration sites
Integration destinations
Unknown host
Genomic DNA
Proviral gene
Unknown host
Genomic DNA
5’LTR
Host/Integrant/Junction (HIJ)
3’LTR
HIJ
CD3
CD19
CD33
<3 copies
0.01-0.2 copies
0.001-0.1 copies
CD34
+ve (P1, P2, P3)
CD19
CD13
CD3
0.00025
0.0025
0.025
0.125
0.25
1.25
2.5
Efficiency of gene transfer…..
100bp
LAM-PCR analysis...
#1
#2
3 19 M G NK 3 G M 19 NK
Summary of results:
 Immunological
reconstitution 
 Immune cell function 
 Immunisation 
 Children at home, off therapy 
Adult patient details
Clinical data
P5
Bronchiectasis
Liver disease
Age at
therapy
20y
Details
at treatment
Previously undergone
mismatched transplant,
judged to be failing,
previous donor no
longer available
Similar case with another adult patient in Paris
Follow up
(months)
17m
Current
status
No
change
Paris study - update
11 children treated (1 atypical)
Good response in 10 patients
However:
Patients 4, 5 and 10 – Serious adverse event
Full immune reconstitution but developed monoclonal T cell
lymphoproliferation
- CD3+ leukemia
All presented at between 30-36 months post-treatment
All treated by chemotherapy & BMT
Conclusion:
P5 AW
P10 AW
P4 relapsed and died
Mechanism of leukaemogenesis?
Insertional mutagenesis
P4 & P5 - Single insertions into LMO-2 gene
(P4 in intron 1, P5 5’ upstream, promoter region)
MFG c
P5
1
MFG c
2
3
4
5
P4
LMO-2
Oncogene, known to be expressed in T cell leukemia,
LMO-2 is highly expressed in leukaemic clones
Other factors?
P10 - 3 different integration sites (Lyl-1, Bmi-1, ?)
Synergistic effect of c + LMO-2?
6
Patient follow up (April 2006)…
Follow up
months
P1
AW*
56
P2
AW
52
P3
AW*
47
P4
AW*
39
P5
AW*
27
P6
AW*
24
P7
AW
17
P8
AW
4
P9
AW
0
*off prophylactic therapy
5%
4%
3%
Insertions in %
Insertions around Transcription Start Sites
TSS
8%
7%
6%
2%
1%
9 to 10
8 to 9
7 to 8
6 to 7
5 to 6
4 to 5
3 to 4
2 to 3
1 to 2
0 to 1
Distance from Transcription Start Site TSS (kb)
-10 to -9
-9 to -8
-8 to -7
-7 to -6
-6 to -5
-5 to -4
-4 to -3
-3 to -2
-2 to -1
-1 to 0
0%
Analysis of Retroviral Integration Sites (IS)
Post-Transplantation
Absolute
Percentage
Total number of integration sites
(IS)
344
Exactly mappable IS
252
100%
IS in RefSeq genes
99
39%
IS in RefSeq genes including the
region 10 kb upstream and
downstream of the gene
148
59%
IS more than 10 kb away from
RefSeq genes
104
41%
IS within +/- 5 kb around
transcription start site
58
23%
Lymphocyte recovery CD3 (Paris, March 2005)…..
9000
8000
P
4
T Lymphocytes/µl
7000
*
6000
P
8
5000
P10*
4000
3000
P2
P7
2000
P5
P
6
1000
P9
*
P1
0
0
10
20
30
40
Months after Gene Therapy
50
60
70
Lymphocyte recovery CD3 (Paris and London, March 2005)…..
9000
8000
P
4
T Lymphocytes/µl
7000
*
6000
5000
*
*
*
4000
3000
P1
0
*
P
8
P2
P7
2000
P5
P
6
1000
P9
*
P1
0
0
10
20
30
40
Months after Gene Therapy
50
60
70
UK SCIDX1 protocol….
(MFG, GALV-pseudotype, no B2
mutation)
Harvest
CliniMacs CD34+ bone marrow
Pre-activation (40hours)
X-Vivo10 (serum free)
SCF 300ng/ml, FL 300ng/ml, TPO
100ng/ml, IL-3 20ng/ml
Transduction (3 cycles over 72 hours)
Nexell gas permeable flexible
containers
Retronectin coating
Virus pre-loading
No protamine sulphate
Infusion
Will it happen to children treated in
London?
We don’t know, but:
1.
At least 4 of our patients are out of the
“time-frame”
2. We used a slightly different virus
3. We used a different protocol
Development of safer vectors
New Improved Retroviral
Vectors

Self-inactivating (SIN) vectors
- promoter and enhancer element deletion in 3’LTR
- should have no LTR-directed transcription
- should reduce the risk of insertional mutagenesis

Tissue specific promoters
- viral promoters, SFFV LTR is active in stem
and progenitor cells
- constitutive eukaryotic promoters, EF1α
- haematopoietic cell specific promoters, WASp
A.
B.
C.
D.
MLV LTR
IL2RG
MLV LTR
MLV LTR
SFFV
IL2RG
wpre
MLV LTR delU3
MLV LTR
EF1α
IL2RG
wpre
MLV LTR delU3
MLV LTR
WASP
IL2RG
wpre
MLV LTR delU3
Schematic representation of SIN vectors
(A) Vector used in our current clinical trial
(B-D) SIN vectors in which the IL2RGγc transgene is regulated by
internal regulatory sequences:
(B) spleen focus forming virus promoter (SFFV)
(C) elongation factor 1α promoter (EF1α)
(D) Wiskott Aldrich syndrome protein promoter
Mock
wt
SF
EF
CD4
wt
B220
CD8
SIN gammaretroviral reconstitution of murine T and B
cell compartments....
EF
IgM
5 months after gene transfer
GFP expression from modified lentiviral vectors
100%
B cells
80%
Myeloid cells
Progenitor cells
60%
40%
20%
0%
pHR'SIN.CEW
pHR'SIN.SEW pHR'SIN.cPPT-SEWpHR'SIN.cPPT-SEW
MOI <30
MOI 70
Future gene therapy:
Other primary immunodeficiency
disorders
-ADA-SCID (1 patient, 3 further planned)
-X-CGD (2 patients)
-WAS (hope to start 2007?)
Primary haematological disorders,
including leukemia
Stem cell protection strategies
The Molecular Immunology Unit at ICH
ICH
Kate Parsley
Kimberly Gilmour
Jo Sinclair
Steve Howe
Doug King
Suzy Bailey
Fang Zhang
Aris Giannakopoulos
Meera Ulaganathan
Mohammed Osman
Mike Blundell
Graham Davies
Christine Kinnon
Bobby Gaspar
Adrian Thrasher
Funded by:
The Wellcome Trust
MRC, BBSRC,
Department of
Health,
CGD Trust, Primary
Immunodeficiency
Association, CF Trus
EU, LRF, ARC,
SPARKS
and others…
GOS
Nursing Staff
Pharmacy Staff
EUFETS
Klaus Khulke
Cincinatti/ Freiberg
Manfred Schmidt
Christof Von Kalle
Frankfurt
Manuel Grez
Stefan Stein
Hannover
Christopher Baum