2. 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

3. 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 non-toxic

4. Adeno-associated viral vectors for haemophilia gene therapy
20 nm
Non-pathogenic single stranded DNA based parvovirus
Replicates only in the presence of helper virus
Easy to render “Gutless”  reduced risk of immune response to viral proteins
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
rep
cap
ITR
Wild type AAV
ITR
LP1
FIX cDNA pA
rAAV
There is currently a great deal of interest in AAV vectors which is in part due to its great safety profile.
AAV is a small non-pathogenic single stranded DNA parvovirus that is endemic in humans but does not cause disease
An additional measure of safety is afforded by the fact that this virus cannot replicate unless the target cell si also infected with a helper virus such as adenovirus.
Therefore amongst vectors of viral origin, rAAV have the best safety profile
Equally important is the fact that rAAV can transduce a variety of cell types and it is in particular able to mediate stable long term transgene expression at following a single administration of the vector into a variety of from post mitotic tissues:  Muscle, Liver, Retina, Brain
This expression in large is mediated by episomally retained copies as rAAV integrates infrequently and at a rate comparable to that of DNA. Consequently the risk of vector mediated oncogenesis with this vector is low.
Multiple serotypes of AAV derived a variety of animal species have been isolated and characterized especially by Jim Wilson’s group who will be speaking latter today. These novel serotypes have distinct biological properties that have broadened the scope for this vector system
As with most vector systems rAAV has several disadvantages. Foremost amongst these is its small packaging capacity of 4.6kb which significantly limits the potential disease targets
Additionally the majority of us have been infected with wt-AAV2, especially serotype 2 which results in long-lasting humoral and cellular immunity that has the potential to limit efficient transduction with this vector
AAV5
AAV9
AAV2
AAV8

5. 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
LP1
co hFIX + -
2. Self complementary vectors to improve potency using an expression cassette which contained a small liver specific promoter and codon optimised hFIX cDNA
scAAV-LP1-hFIXco
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

6. Brief outline of the dose escalation arm
Objective: Assess the safety and efficacy of a bolus peripheral vein infusion of our novel scAAV2/8-LP1-hFIXco
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

7. 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

8. Characteristics of subjects in the initial phase of the study All patients recruited at Royal Free Hospital, UK
Patients S1 S2 S3 S4 S5 S6
Dose category
Low
(2e11vg/kg)
Intermediate
(6e11vg/kg)
High dose
(2e12vg/kg)
Age 31 64 43 29 32 27
Mutation
Missense
Null
Promoter
Prophylaxis
X2/week
X1/week
X3/week
Spontaneous bleeds/year
2-4
4-6
1-2

9. Safety assessments Vector shedding LFTs (Subjects 1-4, 6) Vital signs
Plasma
Stools
Semen
Saliva
Urine
LFTs (Subjects 1-4, 6)

10. Low vector dose Plasma FIX levels, Subject 1: 2x1011vg/kg
FIX conc usage
down by 77%
Off prophylaxis for >48 months but no spontaneous haemorrhage

11. 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
>37
Reduced ~X1/fortnight
50%
Transgene expression of <3% is not sufficient to permit discontinuation of prophylaxis in subjects with severe pre-existing arthropathy

12. Intermediate vector dose
Plasma FIX levels, Subject 4: 6x1011vg/kg
FIX conc usage down by 88%
Off prophylaxis for >36 months. No spontaneous haemorrhage despite engaging in contact sports that had previously precipitated bleeding episode (soccer and cricket)

13. High vector dose Plasma FIX levels, Subject 5: 2x1012vg/kg
60mg
Pred
FIX conc usage down by 40%
Currently on X1/10 days prophylaxis

14. High vector dose Plasma FIX levels, Subject 6: 2x1012vg/kg
60mg
Pred
FIX conc usage
"I have not needed any of my normal treatment, either preventative or on-demand as a result of an injury. Previously, I used to infuse at home three times a week .... I play football, run and take part in triathlons - and previously I might have had to infuse both before I took part and possibly after as well. Not having to do that has been absolutely brilliant."
Off prophylaxis for >36 months.
Not required any FIX treatment

15. 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?

16. Plasma FIX level: Subject 7; 2x1012vg/kg
22Y: Null mutation; antiAAV8 IgG = 1RU;
X2/Week prophylaxis; 2 bleeds/year
60mg
Pred
Steroid course initiated at week 8 when ALT increased by 50%
Currently off prophylaxis almost 24 months post gene transfer

17. Plasma FIX level: Subject 8; 2x1012vg/kg
38Y: Missense mutation; antiAAV8 IgG = 6RU;
on demand:1Xweek; 12 bleeds/year
No spontaneous bleeding episodes following gene transfer
No transaminitis; No need for steroids
Not required FIX concentrates following gene transfer

18. Plasma FIX level: Subject 9; 2x1012vg/kg
44Y: Missense mutation; antiAAV8 IgG = 6RU;
on demand X1 week; 16 bleeds/year
No bleeding episodes over post gene transfer
No transaminitis; No need for steroids
Not required FIX concentrates following gene transfer

19. Plasma FIX level: Subject 10; 2x1012vg/kg
33Y: Null mutation; antiAAV8 IgG = 5RU;
on demand X1/Week; 24 bleeds/year
60mg
Pred
Short course of steroid started at week 9 when ALT increased by >50%
Required FIX concentrates for 3 episodes of traumatic bleeds

20. Figure 1
Factor IX:C (% of normal)
Alanine transaminase (IU/L)
Weeks after vector infusion

21. Figure 2A
Figure 2B

22. Figure 3A: Longer immunological follow-up of the dose escalation cohort
Figure 3B S7 S8 S9
S10

23. Figure 4
Weeks after vector infusion
AntiAAV IgG titre (Relative units/ml)

24. Summary:1 In total 10 subjects received scAAV8-LP1-hFIXco
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 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

25. 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

26. 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

27. Other FIX trials in the pipe line
Asklepios Bio & Baxter
PI: Samulski and Monahan
scAAV vector pseudotyped with AAV8 capsid
Codon optimised FIX cDNA containing a hyperactive FIX mutation ‘Padua’
Study is open and 2 subject recruited at 1e11vg/kg
CHOP study: PI= Kathy High
ssAAV vector pseudotyped with AAV8 capsid
Strong liver specific promoter: HCR-hAAT
Study is open and 4 subject 1e12vg/kg
Involves add-back of empties Full:empty ratio 1:5
Transaminitis occurred in both but expression could be rescued with steroid
Jichi medical school: Same approach as CHOP

28. Special acknowledgment
The patients!!!
Their bravery permitted us to perform proof-of –concept first in man study without any expectation of benefit

29. 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