Gene therapy and therapeutic gene editing:
what are the predictors of success?
Bobby Gaspar
Professor of Paediatrics and Immunology
Centre for Immunodeficiency
UCL Institute of Child Health/Great Ormond Street NHS Trust
Gene therapy and paediatric conditions
Retinal
abnormalities
Skin conditions
EB
Cystic fibrosis
Inborn errors
OCT, Hurlers
X-ALD
Haemophilia
Duchenne’s MD
Severe
immunodeficiencies
Fanconi’s anaemia
Haemoglobinopathies
Acute leukaemias
Primary Immunodeficiency
stem cells
red blood cell
platelets
white blood cells
neutrophils /monocytes
white blood cells / Immune cells
Severe combined immunodeficiency (SCID)
Bacteria
Fungi
Virus
Pneumonia, diarrhoea, repeated infections
Molecular defects in SCID
SCID/CID
HSC-multi
c, IL7Ra, JAK3, ZAP-70
RAG1/2, artemis, ligase IV,
Cernunnos
ADA, PNP
MHC I/II, CD3//, CD45,
ORAI1
T
B
NK
How do we get genes into cells?
Disabled viruses
Adenovirus
Adeno-associated virus
Retrovirus
Non-viral vectors
Pathology of SCID-X1
Blood
Bone marrow
Stem cells
B
X
Thymus
In X-SCID growth of
lymphocytes is
blocked
NK
lymphocytes
T
ab
X
SCID-X1 gene therapy protocol
LTR-driven gammaretroviral vector: MFG C
MoLV
R
U5
Y
Q
SD
SA
IL2RG
Criteria for entry:
No matched sibling donor
Molecularly confirmed diagnosis
Common gamma chain vector:
PG 13 producer cells (GALV envelope)
titre approximately 1x10e6 transducing
units per ml
MoLV
R
U5
Lymphocyte recovery CD3
5000
P10
P9
T Lymphocytes/µl
4000
P8
3000
P5
P7
P1
P6
2000
P3
P4
1000
P2
0
0
50
100
150
Weeks
200
250
300
Patient details
Age at
therapy
Maternal
graft
Mutation
Gamma
chain
expression
Total cells
infused
(x10e6)
Current
status
(months)
P1
10
++
R289X
++
180
A+W
P2
10
++
S238N
-
180
A+W
P3
4
-
Y125C
+/-
78
A+W
P4
3y
-
R289X
++
115
A+W
P5
10
-
R222C
++
200
A+W
P6
10
-
PolyA
-
200
A+W
P7
6
-
M1i
-
84
A+W
P8
13
-
C182Y
+
207
A+W
P9
7
-
S108P
+
160
A+W
P10
12
-
del
-
60
A+W
Biochemical defect in ADA deficiency
DNA
d-ATP
increase is toxic to
lymphocyte function
dCydK
d-adenosine
d-adenosine
d-inosine
ADA
d-adenosine
T cell recovery post ADA gene therapy
Metabolic correction post gene therapy
1600
1400
1000
800
80
60
40
20
0
Pre-diagnosis
Pre-GT
Post-GT
50
ADA Activ ity (nmol/mgHb/h)
dATP (mmol/L)
1200
40
30
20
10
0
Pre-diagnosis
Pre-GT
Post-GT
Summary of ADA-Deficient SCID Patients
Retroviral Vectors, Myeloreductive Conditioning
–Milan/London/CHLA-NHGRI, NIH-UCLA
Center
# Pts
Milan
18
0.8 – 11.5
London
8
CHLA-NHGRI
UCLA-NHGRI
TOTAL
1 As
F/U (yrs)1 Off Enzyme
Survival
DFS2
15/18
100%
83.3%
0.5 – 7.5
4/8
100%
50%
6
8
2– 5
0.1-2
3/6
7/8
100%
100%
50%
87.5%
40
0.1 – 11.5
29/40
100%
72.5%
of April 2012
2DFS ≡ Alive without BMT or PEG-ADA reData: Courtesy HB Gaspar (London) and Alessandro Aiuti (Milan)
start
Patient details
Age at
Maternal
therapy
graft
(months)
Mutation Gamma
chain
expression
Total cells
infused
(x10e6)
Current
status
P1
10
++
R289X
++
180
A+W
P2
10
++
S238N
-
180
A+W
P3
4
-
Y125C
+/-
78
A+W
P4
3y
-
R289X
++
115
A+W
P5
10
-
R222C
++
200
A+W
P6
10
-
PolyA
-
200
A+W
P7
6
-
M1i
-
84
A+W
P8
13
-
C182Y
+
207
A+W
P9
7
-
S108P
+
160
A+W
P10
12
-
del
-
60
A+W
Activation of LMO2
Chromosome 11p13
Forward Strand
33.65Mb
33.85Mb
34.05Mb
Fold
difference
gene
expression
Fold difference
in geneinexpression
Fold
difference
in gene
expression
Fold
difference in gene expression
Folddifference
difference
gene
expression
Fold difference
in gene
expression
Fold
inin
gene
expression
Fold difference
in gene expression
100
100
100
100
C11orf41
100
CD59 FBXO3
100100
100
LMO2
CAPRIN1 NAT10 ABTB2
O2
C11orf41
0.1
11
1
CD59
C11orf41
FBXO3
C11orf41
C11orf41
CD59
LMO2
CD59
CD59
FBXO3
Relative to:
Leukaemia panel
DP1 T cells
DP2 T cells
Nat10
ABTB2
Gene
Leukemia
DP1 T cells
DP2 T cells
Difference
Difference
DP1DP1
T cells
T cells
DP1 T cells
DP2 T cellsDP2 T cellsDP2
DP2
T cells
T cells
DP2 T cells
DP2DP2
T cells
T cells
DP2 T cells
X
0.1
Gene
Gene
1 1
Nat10 CAPRIN1
ABTB2Nat10
Nat10
ABTB2
ABTB2
C11orf41
CD59 FBXO3
FBXO3
LMO2 CAPRIN1
CAPRIN1
Nat10 ABTB2
ABTB2
C11orf41C11orf41
CD59 CD59
FBXO3
LMO2 LMO2
CAPRIN1
Nat10 Nat10
ABTB2
LMO2
ABTB2
0.1
Gene
CAPRIN1
DP1 T cells
1
CAPRIN1
Nat10
ABTB2
FBXO3
LMO2
CAPRIN1
FBXO3
LMO2
LMO2
Nat10
C11orf41CAPRIN1
CD59 CAPRIN1
FBXO3
Leukemia
LeukemiaLeukemia
DP1 T cellsDP1 T cellsDP1
DP1
T cells
T cells
1
0.1
0.1
0.1
Difference
Difference
Difference
Difference
1
10 10
VIRUS
Leukemia Leukemia Leukemia
Leukemia
Leukemia
INTEGRATION
10
10
1010
10
Difference
10
Difference
Fold Difference
in gene expression
Gene
0.1 0.1
Gene
Gene Gene
Development of a new vector for SCID-X1
LTR-driven gammaretroviral vector: MFG gC
MoLV
R U5
Q
Y
SA
SD
IL2RG
MoLV
R U5
New gammaretroviral SIN vectors: Sin11 / SRS11
MP
RSV
R U5
Q
SD
Y
Prom.
IL2RG
EF1a(S)
SF
PRE
Δ R U5
Reduced mutagenesis with SIN configuration
P < 0.001
Control Cohort Survival (n=14)
Percent survival
100
80
60
40
detection limit
20
0
0
100
200
300
400
Days
23/24 mice long term survival
SF
EFS
SF.HS
Gene transfer for SCID-X1 using a
self-inactivating (SIN) gammaretroviral
vector
A multi-institutional phase I/II trial evaluating the
treatment of SCID-X1 patients with retrovirusmediated gene transfer
Sites:
Great Ormond Street Hospital, UK (1)
Hôpital Necker Enfants Malades, France (4)
Children’s Hospital Boston, US (2)
Cincinnati Children’s Hospital Medical Center, US
Mattel Children’s Hospital, Los Angeles, US (1)
Lentiviral vector gene therapy for ADA-SCID
Phase I/II, non-controlled, open-label,
nonrandomised, trial to assess the
safety and efficacy of EF1αS-ADA lentiviral
vector mediated gene modification of
autologous
CD34+ cells from ADA-deficient individuals
Sites:
Great Ormond Street Hospital, UK (2)
Mattel Children’s Hospital, Los Angeles, US
Vector production:
Indiana University Vector Production Facility
Lentiviral vector mediated Gene Therapy for ADA SCID
MRC DPFS
MRC Research Grant
Development of vector
January 2007
July 2009
Collaborations:
UCLA
Indiana University Vector Production Facility
MRC DCS
Testing of efficacy and
safety
December 2012
Phase I/II study of
10 patients
January 2018
1st patient treated Feb 2012
3 patients treated in total
LV Phase I/II trial for Wiskott-Aldrich syndrome
Lentiviral Vector for CGD
Ch imeric p ro mo ter
HIV1 RRE
HIV1 p si
W PRE* (PRE4)
HIV1 PBS
d eltaU3
HIV U5
HIV1 R
HIV1 R
d eltaU3
HIV1-p o lyA
cPPT cts
G P91
Provirus pCCL chimGP91 WPRE4
5 487 bp
HIV U5
Therapeutic gene editing
Gene therapy and paediatric conditions
Retinal
abnormalities
Skin conditions
EB
Cystic fibrosis
Inborn errors
OCT, Hurlers
X-ALD
Haemophilia
Duchenne’s MD
Severe
immunodeficiencies
Fanconi’s anaemia
Haemoglobinopathies
Acute leukaemias
Gene therapy in PID
Cures now available for specific SCID conditions
First ever cures with Gene Therapy
Morbidity and mortality still low compared to other
treatments and prev innovations
Need new safer vectors
Each disease should be considered individually (gene
regulation, expression, tissue specificity)
Many thanks to
Institute of Child Health
Emma Bjorkeren
Kate Parsley
Kimberly Gilmour
Sam Cooray
Elena de Falco
Jo Sinclair
Doug King
Steve Howe
Stuart Adams
Suzy Thornhill
Michelle Quaye
Daleen Lopez-Begg
Sue Swift
Fang Zhang
Lin Zhang
Claudia Montiel Equiha
Maria Alonso-Ferrero
Marlene Carmo
Christine Rivat
Claire Booth
Karen Buckland
Sue Swift
Nourredine Himoudi
Anne-Marie McNicol
Great Ormond Street Hospital
Collaborators
Paul Veys
Persis Amrolia
Kanchan Rao
Graham Davies
Alison Jones
Cathy Cale
Lesley Henderson
Jane Gaspar
Jin Hua Xu-Bayford
Lucie Brown
Nursing and support staff
Chris Baum
Axel Schambach
Christof von Kalle
Manfred Schmidt
Mik Antoniou
Marina Cavazzana-Calvo
Alain Fischer
David Williams
Manuel Grez
Vicky Bordon
Theoni Petropolou
Jude Cope
Jodi New
Purine Res Lab, Guys
Hospital
Lynette Fairbanks
Manchester
Brian Bigger
Fiona Wilkinson
Adrian Thrasher
Bobby Gaspar