1. A N N A S L U P E C K I
C O M M U N I T Y I I A P P E
A U G U S T 2 0 1 6
N A T H W A N I A C , T U D D E N H A M E G , R A N G A R A J A N
S , R O S A L E S C , M C I N T O S H J , E T A L . ( 2 0 1 1 )
A D E N O V I R U S - A S S O C I A T E D V I R U S V E C T O R -
M E D I A T E D G E N E T R A N S F E R I N H E M O P H I L I A B .
N E N G L J M E D 3 6 5 : 2 3 5 7 – 2 3 6 5 . D O I :
1 0 . 1 0 5 6 / N E J M O A 1 1 0 8 0 4 6 . P M I D : 2 2 1 4 9 9 5 9
Gene Therapy & Hemophilia
Journal Club

2. Background Information

3. Background Information
 Currently there are 7 different pharmaceutical
companies conducting clinical trials for gene therapy
use in hemophilia A or B
 Baxter International, Uniqure, Dimensions Therapeutics,
Spark Therapeutics, BioMarin Pharmaceutical, Sangama
Biosciences/Shire, Biogen Idec
 Uniqure is using the theraputic IX gene used in the
following study by a research team at St. Jude’s/UCL
 BioMarin is using a hemophilia A program licensed
from the same research facilities
http://www.xconomy.com/national/2015/03/23/stop-the-bleeding-can-gene-therapy-finally-cure-hemophilia/?single_page=true

4. http://www.pharmacypracticenews.com/Clinical/Article/08-16/Gene-Therapy-for-Hemophilia-Gains-Ground/37554

5. Background Information
 Company: Uniqure
 Phase I/II Study of AMT-060-01 for Hemophilia
B
 30 minute IV infusion, locally prepared in pharmacy
 24 hour observation in hospital
 Adenovirus-Associated Virus (AAV5) Vector–Mediated Gene
Transfer of Human Factor VIX
 Goal: Dose-Escalation, safety, and efficacy
 Status: On going
Access date: 8/17/16 http://learningcenter.ehaweb.org/eha/2016/21st/135223/frank.leebeek.first.results.from.a.dose-
escalating.study.with.aav5.vector.html?f=p14m3s92207
Acess Date: 8/8/16 htps://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja
&uact=8&ved=0ahUKEwinp9fI5cjOAhWBcCYKHX1SBvIQFgglMAE&url=http%3A%2F%2Fwww.uniqure.com%2Fuploads%2Fpresentations%2FAMT-
060%2520Presentation%2520at%2520WFH%2C%25202016.pdf&usg=AFQjCNFmZDA3Ll0fm4-83wN56FQplevP-w

6. Background Information
 Company: BioMarin
 Phase I/II Gene Therapy Study of BMN 270 for
Hemophilia A
 BMN 270 is administered as a single IV infusion
 Adenovirus-Associated Virus (AAV8) Vector–Mediated Gene
Transfer of Human Factor VIII
 Goal: Dose-Escalation, Safety, Tolerability, and
Efficacy
 Status: Ongoing
http://www.biomarin.com/hemophilia-a Access date: 8/8/16

7. Vance et al. In Gene Therapy -Principles and Challenges (2015).

8. Approved Gene Therapies (EU only)
 Alipogene Tiparvovec (Glybera)
 Treatment of lipoprotein lipase deficiency w/ severe or
multiple attacks of pancreatitis despite low fat diet
 Derived from a virus modified to carry lipoprotein lipase gene
 October 2012: First gene therapy approved EVER in a
Western country
 Strimvelis
 Treatment of ADA-SCID for whom no suitable HLA-matched
stem cell donor is available
 Stem cell gene therapy – autologous CD34+ cells express ADA
 May 2016: First gene therapy approved for children
Access date: 8/17/16 http://www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/uman/medicines/002145/human
med_001480.jsp&mid=WC0b01ac058001d124
Access date: 8/17/16 http//www.gsk.com/en-gb/media/press-releases/2016/strimvelistm-receives-european-marketing-authorisation-to-treat-very-rare-
disease-ada-scid/

9. ADENOVIRUS-ASSOCIATED
VIRUS VECTOR-MEDIATED
GENE TRANSFER IN
HEMOPHILIA B
Study Overview

10. Relevance of Study
 Prophylactic treatment with factor is not curative, is
expensive, and is associated with inhibitor formation
 Gene therapy offers the potential cure after a single
administration assuming continuous endogenous
production of FIX
 A small rise in endogenous FIX (>1%) can decrease
bleeding

11. Treatment Regimens
 Peripheral vein administration of vector particles
(220mL total, 1 hour infusion)
 No FIX administration during procedure
 Dosing regimen:
 2 × 1010 vg/kg
 6 × 1010 vg/kg
 2 × 1011 vg/kg
 A minimum of 2 subjects to start at each dose

12. Study Design
 Phase I/II Clinical Trial
 Open Label
 Dose Escalation
Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, et al. (2011) Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 365: 2357–2365. doi:
10.1056/NEJMoa1108046. pmid:22149959

13. Methods

14. Inclusion Criteria
 Males ≥ 18 years of age with established severe HB (FIX:C<1u/dl) resulting
from a missense mutation in the hFIX gene which has not been associated
with an inhibitor with detectable FIX in serum.
 Individuals with a stop codon mutation, a promoter mutation, a small
insertion or deletion causing a frame shift, a small in frame insertion or
deletion or a splice junction mutation could be enrolled if their
mutation had not been associated with an inhibitor.
 Treated/exposed to FIX concentrates for at least 10 years.
 A minimum of an average of 3 bleeding episodes per year requiring FIX
infusions or
 prophylactic FIX infusions because of frequent prior bleeding episodes.
 Able to give informed consent and comply with requirements of the trial.
 Currently free of inhibitor and have no history of inhibitors to FIX protein
and
 A negative family history for the development of an inhibitor.
 Willing to practice a reliable barrier method of contraception.
Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, et al. (2011) Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 365: 2357–2365. doi:
10.1056/NEJMoa1108046. pmid:22149959

15. Exclusion Criteria
 Evidence of active infection with Hepatitis B or C virus
 Exposure to Hepatitis B or C and on antiviral therapy
 Serological evidence of HIV or HTLV infection
 Significant liver dysfunction as defined by an abnormal ALT, bilirubin, alkaline
phosphatase or INR.
 Potential participants who have had a liver biopsy in the past 3 years were excluded if they had
significant fibrosis of 3 or 4 as rated on a scale of 0-4
 Coronary artery disease as a co-morbid condition
 Platelet count of <150 x 109/l
 Creatinine ≥ 1.5 mg/dl
 Hypertension with systolic BP consistently ≥ 130mmHg or diastolic BP consistently
≥ 90mmHg
 History of active tuberculosis, fungal disease, or other chronic infection
 History of chronic disease adversely affecting performance
 Detectable antibodies reactive with AAV8
 Subjects who were unwilling to provide the required semen samples
 Poor performance status (WHO performance status score >1) or
 Received an AAV vector previously or any other gene transfer agent in the previous
6 months
Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, et al. (2011) Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 365: 2357–2365. doi:
10.1056/NEJMoa1108046. pmid:22149959

16. Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, et al. (2011) Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 365: 2357–2365. doi:
10.1056/NEJMoa1108046. pmid:22149959

17. Endpoints
 Primary endpoint: Safety
 Emphasis on:
 Vector induced hepatitis
 Germline transmission
 Formation of neutralizing antibodies against FIX
 Efficacy was a secondary endpoint
 Persistence of biologically active FIX at ≥ 3% of normal levels
 Expression of FIX was assessed frequently during the study
period but only after a minimum of 72 hours had elapsed since
the last infusion of FIX protein concentrates
Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, et al. (2011) Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 365: 2357–2365. doi:
10.1056/NEJMoa1108046. pmid:22149959

18. Statistical Methods
 No sample size justification
 Descriptive analysis of data
 Collected in a longitudinal manner
 Analyzed using a mixed effect model
 Per protocol analysis
Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, et al. (2011) Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 365: 2357–2365. doi:
10.1056/NEJMoa1108046. pmid:22149959

19. Results

20. Primary Outcome: Safety
Germline Transmission
Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, et al. (2011) Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 365: 2357–2365. doi:
10.1056/NEJMoa1108046. pmid:22149959

21. Primary Outcome: Safety
Vector Induced Hepatitis
Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, et al. (2011) Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 365: 2357–2365. doi:
10.1056/NEJMoa1108046. pmid:22149959

22. Primary Outcome: Safety
Formation of neutralizing antibodies against FIX
 Neutralizing antibodies to FIX were not detected at
any time point in any participant
Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, et al. (2011) Adenovirus-associated virus vector-mediated gene
transfer in hemophilia B. N Engl J Med 365: 2357–2365. doi: 10.1056/NEJMoa1108046. pmid:22149959

23. Secondary Outcomes: Efficacy
4 of 6 participants were able to stop using prophylaxis with FIX concentrate
without having spontaneous hemorrhage
Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, et al. (2011) Adenovirus-associated virus vector-mediated gene
transfer in hemophilia B. N Engl J Med 365: 2357–2365. doi: 10.1056/NEJMoa1108046. pmid:22149959
Increasing Dose of AAV vector

24. Clinical Significance
 Therapeutic expression of a transferred factor IX
gene can be achieved in humans
Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, et al. (2011) Adenovirus-associated virus vector-mediated gene
transfer in hemophilia B. N Engl J Med 365: 2357–2365. doi: 10.1056/NEJMoa1108046. pmid:22149959

25. Author’s Primary Conclusion
 FIX level increases are dose dependent
 Individual variations in immunity & exposure to AAV
influence the type and magnitude of immune responses
to this gene therapy
 To determine the frequency & clinical significance of ALT
elevations a larger number of participants needs to be
treated at the high dose level
 Routine use of corticosteroids is not indicated since the incidence
and timing of immune mediated ALT elevations is uncertain
 This gene therapy approach has the potential to convert
severe bleeding hemophilia to a mild form or reverse it
entirely
Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, et al. (2011) Adenovirus-associated virus vector-mediated gene
transfer in hemophilia B. N Engl J Med 365: 2357–2365. doi: 10.1056/NEJMoa1108046. pmid:22149959

26. Study Analysis & Critique

27. Internal Strengths Internal Weaknesses
 2 times per week
observations
 Restart prophylactic
FIX if levels fall below
1%
 Small cohort (N=6)
 Open label
Study Validity

28. External Strengths External Weaknesses
 Only male participants
 Only severe hemophilia
patients
 (FIX:C < 1U/dL)
 Exclusion of:
 Patients with inhibitor
 Patients with active or
treatment for Hep B/C
 Patients with HIV
 Age range: 27-64 y.o.
 Treatment with FIX for
at least 10 years
Study Validity

29. Conclusion & Role in Clinical Practice
 Hemophilia gene therapy has the potential to be an
effective way to reduce the need for factor replacement
 Larger trials are necessary to determine liver safety
 Clinical trials are set to finish Phase I/II as early as 2020
 The cost of gene therapy may be prohibitive to how many
people it actually treats
 Average factor replacement cost = $250,000 per year
 Current gene therapy cost in the EU: $650,000 - $1 million once
 Durability of effect ~ 5 years (mean follow up of 3.2)
 No data available if a dose of vector may need to be repeated
Nathwani AC, Reiss UM, Tuddenham EG, Rosales C, Chowdary P, et al. (2014) Long-term safety and efficacy of factor IX gene
therapy in hemophilia B. N Engl J Med 371: 1994–2004. doi: 10.1056/NEJMoa1407309. pmid:25409372

30. References
 Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, et al. (2011) Adenovirus-associated virus
vector-mediated gene transfer in hemophilia B. N Engl J Med 365: 2357–2365. doi:
10.1056/NEJMoa1108046. pmid:22149959
 Nathwani AC, Reiss UM, Tuddenham EG, Rosales C, Chowdary P, et al. (2014) Long-term safety and efficacy
of factor IX gene therapy in hemophilia B. N Engl J Med 371: 1994–2004. doi: 10.1056/NEJMoa1407309.
pmid:25409372
 http://www.pharmacypracticenews.com/Clinical/Article/08-16/Gene-Therapy-for-Hemophilia-Gains-
Ground/37554
 http://www.xconomy.com/national/2015/03/23/stop-the-bleeding-can-gene-therapy-finally-cure-
hemophilia/?single_page=true
 Access date: 8/17/16
http://learningcenter.ehaweb.org/eha/2016/21st/135223/frank.leebeek.first.results.from.a.dose-
escalating.study.with.aav5.vector.html?f=p14m3s92207
 Acess Date: 8/8/16
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=0ahUKEwi
np9fI5cjOAhWBcCYKHX1SBvIQFgglMAE&url=http%3A%2F%2Fwww.uniqure.com%2Fuploads%2Fpresenta
tions%2FAMT-
060%2520Presentation%2520at%2520WFH%2C%25202016.pdf&usg=AFQjCNFmZDA3Ll0fm4-
83wN56FQplevP-w
 http://www.biomarin.com/hemophilia-a Access date: 8/8/16
 Vance et al. In Gene Therapy -Principles and Challenges (2015).
 8/17/16
http://www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/human/medicines/002145/human_med_
001480.jsp&mid=WC0b01ac058001d124
 8/17/16 http://www.gsk.com/en-gb/media/press-releases/2016/strimvelistm-receives-european-marketing-
authorisation-to-treat-very-rare-disease-ada-scid/
 http://www.pharmacypracticenews.com/Clinical/Article/08-16/Gene-Therapy-for-Hemophilia-Gains-
Ground/37554