Gene therapy trial for
haemophilia B
UCL/StJude’s Trial Update at 4 years
Edward Tuddenham
Katharine Dormandy Haemophilia Centre, Royal Free Hospital
and
University College London
UK
Haemophilia B gene therapy
• Potential for a cure through continuous, 24/7,
endogenous expression of FIX protein at therapeutic
levels following a single gene transfer manoeuvre
Excellent model for gene therapy approaches
• Single gene defect (deficiency of FIX gene)
• Therapeutic goal modest; An increase in plasma FIX levels
above 1% would be sufficient ameliorate the bleeding
phenotype
• Efficacy can be assessed by validated routine laboratory
assay
• Tight regulation not required
• Wide range of FIX levels are likely to be efficacious and nontoxic
Adeno-associated viral vectors for haemophilia gene
therapy
– Non-pathogenic single stranded DNA based parvovirus
20 nm
– Replicates only in the presence of helper virus
– Easy to render “Gutless”  reduced risk of immune response to viral proteins
rep
ITR
ITR
FIX cDNA
cap
pA
LP1
ITR
ITR
Wild rAAV
type AAV
– Mediate stable long term transgenic FIX expression following a single
administration of the vector into liver
– Episomal copies; Integration rare
– Multiple serotypes with distinct tissue tropism and immunology
AAV2
AAV8
AAV5
AAV9
Key aspects of our phase I gene therapy trial for
haemophilia B
1. Vector pseudotyped with AAV8 capsid
Seroprevalence of AAV8 in humans is lower than AAV2
2. Self complementary vectors to improve potency using an
expression cassette which contained a small liver specific
promoter and codon optimised hFIX cDNA
LP1
scAAV-LP1-hFIXco
co hFIX
+
co hFIX
LP1
3. Vector administered in the peripheral circulation
A simple mode of vector delivery that takes advantage of the strong tropism of
AAV8 for the liver, the native site for FIX synthesis
Brief outline of the dose escalation arm
• Objective: Assess the safety and efficacy of a bolus
peripheral vein infusion of our novel scAAV2/8-LP1hFIXco
• In subjects with severe haemophilia B (<1% of normal, FIX:C)
• No immunosuppression at the time of vector administration
• Key entry criteria:
– No evidence of pre-existing immunity to AAV8
– No evidence of active infection with Hepatitis B, C or HIV
• Vector Production: Children’s GMP, Memphis, TN, USA
– Column chromatography purification process
• Empty: Full scAAV ratio of 10:1
• 3 Study sites: SJCRH, Stanford, UCL/Royal Free Hospital
Minimise risk
Risks largely unknown as first in
man study
•
•
•
•
Immune damage of the liver
Inhibitor formation
HCC
Sensitisation to AAV8
Elaborate 3 stage consent
process
Independent ombudsman
assessment
One subject recruited at a time
with follow-up for 6 weeks
Extensive monitoring
Characteristics of subjects in the initial
phase of the study
All patients recruited at Royal Free Hospital, UK
Patients
S1
Dose
category
S2
S3
Low
(2e11vg/kg)
S4
S5
Intermediate
(6e11vg/kg)
S6
High dose
(2e12vg/kg)
31
64
43
29
32
27
Mutation
Missense
Null
Missense
Missense
Missense
Promoter
Prophylaxis
X2/week
X2/week
X2/week
X1/week
X2/week
X3/week
Spontaneous
bleeds/year
2-4
4-6
4-6
2-4
2-4
1-2
Age
Safety assessments
Vector shedding
Vital signs
Pulse
Temp
Resp
Genome copy/l
80
60
40
20
10 6
10 5
10 4
10 3
10 2
10 1
10 0
0
0
200
400
600
800
1000 1200 1400
Plasma
Vector
LFTs (Subjects 1-4, 6)
AST/ALT (IU/l)
50
40
30
20
10
0
50
10
15
20
Days post vector infusion
Minutes
0
5
100
150
200
Days post vector infusion
250
Stools
Saliva
Urine
Semen
Low vector dose
Plasma FIX levels, Subject 1: 2x1011vg/kg
FIX
7
FIX conc usage
down by 77%
5
5000
4
4000
FIX Usage
(IU/kg/year)
hFIX:C
(IU/dL)
6
3
2
3000
2000
1000
1
0
Pre
0
0
20
40
60
80 100 120 140 160 180
Weeks after vector infusion
Off prophylaxis for >48 months but no spontaneous haemorrhage
Post
Low and Intermediate vector dose
Summary of FIX expression
Subject
Vector
dose
(vg/kg)
Steady state
hFIX:C level
post gene
transfer
(% of normal)
Duration of
expression
(Months)
Prophylaxis
Reduction in
conc usage
2
2x1011
1
>39
Reduced
~X1/week
33%
3
6x1011
2
>37
Reduced
~X1/fortnight
50%
Transgene expression of <3% is not sufficient to permit discontinuation of
prophylaxis in subjects with severe pre-existing arthropathy
Intermediate vector dose
Plasma FIX levels, Subject 4: 6x1011vg/kg
FIX conc usage down
by 88%
9
8
7
6
5
4
3
2
1
0
1500
FIX Usage
(IU/kg/year)
hFIX:C
(IU/dL)
FIX
1000
500
0
0
20
40
60
80 100 120 140 160
Weeks post vector infusion
Pre
Post
Off prophylaxis for >36 months. No spontaneous haemorrhage despite
engaging in contact sports that had previously precipitated bleeding
episode (soccer and cricket)
High vector dose
Plasma FIX levels, Subject 5: 2x1012vg/kg
FIX
Pred
60mg
0
12
200
FIX conc usage
down by 40%
6
100
4
50
2
0
4000
FIX Usage
(IU/kg/year)
150
8
ALT (IU/l)
hFIX:C
(IU/dL)
10
3000
2000
1000
0
0
0
20
40
60
80
100
120
Weeks post vector infusion
hFIX
Currently on X1/10 days prophylaxis
ALT
140
Pre
Post
High vector dose
Plasma FIX levels, Subject 6: 2x1012vg/kg
Pred
FIX conc usage
60mg
0
FIX Usage
(IU/kg/year)
4000
3000
2000
1000
0
Pre
• Off prophylaxis for >36 months.
• Not required any FIX treatment
Post
Second arm: Expansion of the high dose
cohort
4 new subjects recruited over the last 12 months
Questions:
– Will perturbation of liver enzymes occur in all
subjects treated at the high dose level?
– Will early treatment with steroids reduce the
level of transaminitis and preserve stable
expression of hFIX?
– Should all high dose subjects receive prophylactic
steroids and if so when should this be started and
for how long?
Plasma FIX level: Subject 7; 2x1012vg/kg
22Y: Null mutation; antiAAV8 IgG = 1RU;
X2/Week prophylaxis; 2 bleeds/year
FIX
FIX
Pred
60mg
200
0
150
8
100
6
4
ALT (IU/l)
hFIX:C
(IU/dL)
25
20
15
10
50
2
0
0
0
10
20
30
40
50
60
70
80
Weeks post vector infusion
• Steroid course initiated at week 8 when ALT increased by 50%
• Currently off prophylaxis almost 24 months post gene transfer
Plasma FIX level: Subject 8; 2x1012vg/kg
38Y: Missense mutation; antiAAV8 IgG = 6RU;
on demand:1Xweek; 12 bleeds/year
25
20
15
10
8
200
6
100
150
4
50
2
0
0
0
5
10 15 20 25 30 35 40 45 50 55
Weeks post vector infusion
• No spontaneous bleeding episodes following gene transfer
•No transaminitis; No need for steroids
•Not required FIX concentrates following gene transfer
ALT (IU/l)
hFIX:C
(IU/dL)
FIX
Plasma FIX level: Subject 9; 2x1012vg/kg
44Y: Missense mutation; antiAAV8 IgG = 6RU;
on demand X1 week; 16 bleeds/year
hFIX
ALT
200
8
150
hFIX:C
(IU/dL)
4
100
2
50
0
0
0
10
20
30
40
50
Weeks post vector infusion
• No bleeding episodes over post gene transfer
•No transaminitis; No need for steroids
•Not required FIX concentrates following gene transfer
ALT (IU/l)
6
Plasma FIX level: Subject 10; 2x1012vg/kg
33Y: Null mutation; antiAAV8 IgG = 5RU;
on demand X1/Week; 24 bleeds/year
Pred
200
60mg
0
150
6
100
4
ALT (IU/l)
hFIX:C
(IU/dL)
8
50
2
0
0
0
10
20
30
40
Weeks post vector infusion
ALT
hFIX
• Short course of steroid started at week 9 when ALT increased by >50%
• Required FIX concentrates for 3 episodes of traumatic bleeds
Figure 1
2 0 0
3 0
2 0 0
3 0
S 2
S 1
1 5 0
6
1 5 0
6
4
1 0 0
4
1 0 0
2
5 0
2
5 0
0
0
0
0
2 5
5 0
7 5
1 0 0
1 2 5
1 5 0
0
0
1 7 5
2 0 0
3 0
2 5
5 0
7 5
1 0 0
1 2 5
1 5 0
2 0 0
3 0
S 4
S 3
6
1 5 0
4
1 0 0
4
1 0 0
2
5 0
2
5 0
Factor IX:C (% of normal)
0
0
0
2 5
5 0
7 5
1 0 0
1 2 5
0
0
0
1 5 0
2 5
5 0
7 5
1 0 0
1 2 5
1 5 0
P
P
3 0
2 0 0
3 0
2 0 0
S 6
S 5
1 2
1 2
1 5 0
1 0
1 5 0
1 0
8
8
1 0 0
1 0 0
6
6
4
4
5 0
5 0
2
2
0
0
0
2 5
5 0
7 5
1 0 0
0
0
0
1 2 5
2 5
5 0
7 5
1 0 0
1 2 5
1 5 0
P
2 0 0
3 0
S 8
2 0 0
3 0
1 2
S 7
1 5 0
1 2
1 5 0
1 0
1 0
8
8
1 0 0
6
1 0 0
6
4
4
5 0
2
5 0
2
0
0
0
0
0
0
2 5
5 0
7 5
2 5
5 0
7 5
1 0 0
P
3 0
2 0 0
S 9
1 2
3 0
2 0 0
S 1 0
1 5 0
1 2
1 0
1 5 0
1 0
8
1 0 0
8
6
4
5 0
1 0 0
6
4
2
0
0
5 0
2
0
0
2 5
5 0
0
7 5
0
Weeks after vector infusion
2 5
5 0
Alanine transaminase (IU/L)
1 5 0
6
H
D
-P
o
-P
o
-P
-P
D
ll
ll
t
e
t
e
s
r
s
r
4 0
H
A
A
A n n u a lis e d b le e d r a t e ( N o /Y e a r )
H
D
-P
o
-P
o
-P
-P
ll
D
ll
H
A
A
t
e
t
e
s
r
s
r
( IU /k g /y e a r )
F IX U s a g e
8 0 0 0
Figure 2A
6 0 0 0
4 0 0 0
2 0 0 0
0
Figure 2B
3 0
2 0
1 0
0
0
baseline
100
0
nd
1
*
*
*
2
3
4
*
5
6
weeks
* *
7
*
*
8
9
0
0
1
2
3
4
5
4
w
weeks
5
6
6
7
7
8
8
9
*
9
12
8
w
7
w
6
w
5
w
4
w
200
3
w
300
2
3
# no sample
1
w
weeks
0
2
STJ 003
0
w
9
1
8
w
100
in
e
w
ee
k
1
w
ee
k
2
w
ee
k
3
w
ee
k
4
w
ee
k
5
w
ee
k
6
w
ee
k
7
w
ee
k
w 8
ee
k
11
400
lin
e
200
ba
se
l
300
SFU per 106 PBMC
Figure 3B
S7
ba
se
SFU per 106 PBMC
STJ 002
300
200
SFU per 106 PBMC
7
week 9
6
week 8
5
week 7
4
week 6
400
3
week 5
S9
2
week 4
1
week 3
0
week 2
in
e
w
ee
k
1
w
ee
k
2
w
ee
k
3
w
ee
k
4
w
ee
k
5
w
ee
k
6
w
ee
k
7
w
ee
k
8
w
ee
k
9
ba
se
l
0
week 1
SFU per 106 PBMC
Figure 3A: Longer immunological follow-up of the dose escalation cohort
RFH 201
S8
400
300
200
100
11
weeks
STJ 004
S10
400
100
*
12
Figure 4
5 1 2
5 1 2
2 5 6
2 5 6
1 2 8
1 2 8
6 4
6 4
3 2
3 2
1 6
1 6
8
8
4
4
2
2
S 2
S 1
1
1
0 .5
0 .5
AntiAAV IgG titre (Relative units/ml)
0
1 0
2 0
3 0
4 0
5 0
0
5 1 2
5 1 2
2 5 6
2 5 6
1 2 8
1 2 8
6 4
6 4
3 2
3 2
1 6
1 6
8
8
4
1 0
2 0
3 0
4 0
5 0
4
2
2
S 3
1
1
0 .5
0 .5
0
1 0
2 0
3 0
4 0
5 0
S 4
0
5 1 2
5 1 2
2 5 6
2 5 6
1 2 8
1 2 8
6 4
6 4
3 2
3 2
1 6
1 6
8
8
1 0
2 0
3 0
4 0
5 0
4
4
2
2
S 5
1
1
0 .5
0 .5
0
1 0
2 0
3 0
4 0
5 0
0
5 1 2
5 1 2
2 5 6
2 5 6
1 2 8
1 2 8
6 4
6 4
3 2
3 2
1 6
1 6
8
8
4
S 6
1 0
2 0
3 0
4 0
5 0
4
2
2
S 7
1
1
0 .5
0 .5
0
1 0
2 0
3 0
4 0
5 0
0
5 1 2
5 1 2
2 5 6
2 5 6
1 2 8
1 2 8
6 4
6 4
3 2
3 2
1 6
1 6
8
8
4
S 8
1 0
2 0
3 0
4 0
5 0
4
2
2
S 1 0
S 9
1
1
0 .5
0 .5
0
1 0
2 0
3 0
4 0
5 0
0
Weeks after vector infusion
1 0
2 0
3 0
4 0
5 0
Summary:1
• In total 10 subjects received scAAV8-LP1hFIXco
• Evidence of vector mediated human FIX
expression in all 10 subjects treated to date
– Level of transgenic FIX achieved is dose dependent with an
average of 5% of normal at the high vector dose
– Expression has remained remarkable stable over time with
follow-up between 14-49 months
– 5/7 able to stop prophylaxis, with significant improvement in
quality of life and reduction in frequency of bleeding
episodes
• Net saving to the UK government from decrease in FIX usage is
>£1.5M
Summary:2
• Perturbation of liver enzymes has been
observed in 4/6 subjects treated at the high
dose level
• Not related to pre-treatment anti-AAV IgG levels
• Not related to HLA
• Not related to previous infection or treatment for
HBC
– ?use steroids prophylactically between 7-9
weeks after gene transfer
• These results are encouraging and support
further evaluation of the AAV approach
Our next haemophilia B gene
therapy study
• High purity (“fewer empties”) scAAV-LP1-hFIXco
• Commenced in Feb 2014
– Questions:
– Will reducing the capsid protein load reduce the risk of
transaminitis?
– Will the reduction in empty particles improve hFIX
expression?
• 1st subject treated with new prep stable at
11 weeks after gene transfer with no
evidence of transaminitis
Other FIX trials in the pipe line
Asklepios Bio & Baxter
PI: Samulski and Monahan
1. scAAV vector pseudotyped with AAV8 capsid
2. Codon optimised FIX cDNA containing a hyperactive FIX
mutation ‘Padua’
3. Study is open and 2 subject recruited at 1e11vg/kg
CHOP study: PI= Kathy High
1. ssAAV vector pseudotyped with AAV8 capsid
2. Strong liver specific promoter: HCR-hAAT
3. Study is open and 4 subject recruited @ 1e12vg/kg
4. Involves add-back of empties Full:empty ratio 1:5
5. Transaminitis occurred in both but expression could be
rescued with steroid
Jichi medical school: Same approach as CHOP
Special acknowledgment
• The patients!!!
– Their bravery permitted us to perform proof-of –
concept first in man study without any expectation
of benefit
Acknowledgments
UCL/Royal Free Hospital: Amit Nathwani, Jenny Mcintosh, David Linch,
Cecilia Rosales, Pratima Chowdary, Anne Riddell, Jun Pie, Chris Harrington, James
O'Beirne
St Jude Children’s Research Hospital: Andrew Davidoff, Ulrike Reiss,
Cathy Ng, Junfang Zhou, Yunyu Spence, Chris Morton,, John Gray, Arthur
Nienhuis
Children’s GMP: John Coleman, Jim Allay, Susan Sleep
Basingstoke Haemophilia Centre: Savita Rangarajan
CBC (Bristol): Keith Smith, David Anstee and Paul Lloyd-Evans
CHOP: Kathy High, Federico Mingozzi, Etiena Basner-Tschakarjan
ALSAC