Curing Bleeding Disorders:

1
Curing Bleeding Disorders Gene Therapy- A Means
to the End?
5th WFH Global Forum on Safety and Supply of
Treatment Products for Bleeding Disorders 25
September 07 Glenn Pierce MD PhD San Francisco,
California
2
The First "Gene Transfer" Experiments Early
Years- Proof of Principle Established
In parallel

• Brinkhous allotransplantation of spleen into
  FVIII deficient dogs (1967)
• short term cures, not reproduced in humans
  (Hathaway, 1969)
• Thomas orthotopic liver transplants in dogs
  successful (1969)
• Hemophilia A cured in humans (Lewis, Starzl,
  1985)
• FIX deficiency also cured
• Brinkhous liver transplants in porcine vWD
  successful (1976)
• not effective in humans, species differences
  (1991)

• GenesDNA, 1953
• Watson and Crick
• Biotech industry born
• 1976 (Genentech)
• 1st recombinant DNA produced drugs early 1980s
  (insulin)

3
Gene Therapy 1980s to Early 1990s

• Genes for FVIII and FIX cloned early 1980s
• Concept of gene therapy established hemophilia
  recognized as low hanging fruit
• Cause of hemophilia known, 1-2 would be useful,
  precise regulation not required, protein needs to
  circulate in blood
• First immunodeficiency disease trial initiated
  1989
• First NHF-NIH collaborative workshop organized
  1992
• Education, advocacy, cross-fertilization between
  HTCs-Gn Therapists
• Hemophilia gene therapy unit established at NIH
• 11 million funding for 5 years obtained from
  Congress 1994
• 8 grants distributed- most seeded clinical trials
  in late 1990s
• Biotechnology companies have high level interest
• Genetic Therapy Inc obtained rights to Factor IX
  (Brit. Biotech) and VIII (Genetics
  Institute/Wyeth) genes for Gene Rx
• Viagene (Chiron) developed collaboration for
  Factor VIII gene (Bayer)
• Somatix starts FVIII gene therapy program
  patents to use specific cell types for ex vivo
  therapy collaboration to use devices (Baxter)
• Academic centers established early research
  programs
• UNC, Chapel Hill Salk U Wash Baylor

4
Will the prediction come true?
Pierce, Hemalog, Summer 1990
This goal was a catalyst
5
Advocacy, Education and Fundraising
6

• Gene Therapy Workshops Established
• Initiated 1996, 8 held to date with scientists
  from academia, industry, government
• Review status, identify scientific priorities for
  further research and funding
• Chair Pierce
• Co-chairs, Verma, High, VandenDriessche (2008)
• Novel technologies sessions included

7
Animal Models Essential in Hemophilia Research

• Dog models
• Naturally occurring FVIII and FIX
• Mimics human condition
• Basis for all current products
• Mouse models
• FVIII and FIX knockout mice created
• Lifelong cures in animals beginning in late 1990s

Hem B
8
Liver and Muscle AAV2 Injection in Hem B Dogs
Multi-year Cures Factor IX Expression More
Efficient in Liver
12
9
5
0.3
Nakai et al, Blood 1998 Grimm et al Blood 2003
9
NHF MASAC recommendations
Ethics, responsibilities
Research, Clinical Trials
10
US Gene Therapy Clinical Trials 41 Subjects
Started Dec 1998 Jun 1999 Jun 1999 Jun
2001 Jun 2001
HemophiliaA (12) B (7) A (13) B (8) A (1)
Trial TranskaryoticTherapies High/Kay/ Avigen C
hiron High/Kay/ Avigen GenstarTherapeutics
Therapy typeEx vivo plasmid i.p. injection In
vivo AAVi.m. injection In vivo retroviral i.v.
infusion In vivo AAVliver-targeted infusion In
vivo adenoviral i.v. infusion
StatusCompletedPhase I CompletedPhase
I CompletedPhase I CompletedPhase I Trial
Terminated
One Factor IX trial in China 2 subjects, early
90s, ex vivo, likely insufficient numbers of
cells delivered, little reported
11
1 Step Forward, 2 Steps Back?

• NHF workshop topics have covered the entire field
• Sept. 1999 - Patient death attributed to
  adenoviral therapy
• Subsequent safety reviews, Congressional
  Inquiries
• 2002 Two X-SCIDS subjects cured by gene therapy
  develop leukemia FDA places all Retroviral
  Clinical Trials on Hold
• Today, over 30 patients treated and cured
• Multiple diseases cured in animals, variable
  toxicities infrequently observed
• Hemophilia gene therapy trials 2 unexpected
  adverse events, little efficacy detected in
  humans- in contrast to animals
• 2007- Rheumatoid arthritis patient receiving AAV
  gene therapy dies, investigation in progress

Or 2 Steps Forward, 1 Step Back?
12
Delivery of New Genes
Target Cell
AAV
Nucleus
Adenovirus
Retrovirus/Lentivirus
Therapeutic Protein
Naked DNA
13
Viral Vector Interactions with Cells are Poorly
Understood
ss rAAV
- ss rAAV
mRNA
cytoplasm
mRNA

annealed ds rAAV
mRNA
circular dimer
circular monomer
ss rAAV pool
nucleus
?
episomal linear concatemer
If this looks complicatedInteractions between
host defenses and virus even more poorly
understood
now investigating
circular concatemer
?
integrated monomer
Amazing Protein
integrated concatemer
mRNA
14
Kay et al, Nat Genet 2000 24257 Manno et al,
Blood 2003 1012963 Arruda et al, Mol Therapy
2006 14452
Muscle delivery Long term local expression (3.7
years) but insufficient circulating FIX
Liver Delivery Therapeutic circulating FIX
levels but not sustained
Manno et al, Nat Med, 2006 12342-7 Mingozzi et
al, Nat Med, 2007 13 419-22
15
Activation of AAV-specific Memory CD8 T cells
Requires Cross Presentation of AAV Capsid

• 60 of human population have prior exposure to
  natural AAV infection, hence AAV neutralizing
  antibodies detected in blood
• As expected, AAV-specific memory CD8 T cells are
  also detected in healthy humans
• The activation of AAV-specific memory CD8 T
  cells by AAV-FIX vector requires cross
  presentation of AAV capsid peptides by MHC class
  I
• Intracellular trafficking of AAV from cytoplasm
  to nucleus involves proteasome, cross over to MHC
  I presentation pathway
• Solutions to this problem?

Gene therapy needs to solve the 600 million year
old mystery of the battle between viruses and
hosts get along or die.
Manno, Pierce, Arruda et al, Nat Med, 2006
12342-7
Mingozzi et al, Nat Med, 2007 13 419-22
Vendenburghe et al, Nat Med 2006 12967-71
16
Progress in AAV-mediated Gene Transfer for
Hemophilia B
Couto, Pierce, Curr Opin Mol Ther 2003 5517
17
Workshops 2001/02 Unmet needs

• Safer vectors
• Control immune responses
• Regulate gene expression, protein secretion
• Effect of secondary diseases
• Hepatitis, inhibitors
• Recommendations for conduct of trials
• Alternatives to gene therapy
• Longer-lived molecules enabling for gene
  therapy?
• Transgenic animal produced clotting factors?
• Progress with FIX/FVIII in dogs, humans
• Science has changed, but Priorities, Issues have
  changed little through 2006 Workshops

18
Secretion
Potency

• A1 mutation
• F309S
• B domain variants
• 226aa/N6

• Improved activation
• FVIII/HCII hybrid
• ? Specific Activity
• E113A

Ways of Improving rFVIII for Protein and Gene
Therapy
Resistance to inhibitors
Resistance to inactivation
Prolongedlifetime

• IR8
• Disulfide Bond-FVIII
• Porcine-human hybrids
• D318G/M337R

• Porcine-human hybrids

• LRP/HSPG binding site
• mutations
• PEGylated liposomes
• PEG-FVIII

Factors IX and VIIa ? potency, life, secretion
19
Why Pursue Gene Therapy? Global Reality

• Emerging discussions at Workshops
• 400,000 worldwide 100,000 receive some Rx
• 75 not diagnosed
• Many die in childhood
• Hemophilia not a priority with governments
• Lack of infrastructure, training, education
• Cost of treatment prohibitive
• Therapy will not become affordable to most
• Prevention of bleeding even more difficult

20
Relationship of Economic Capacity to Number of
Adults with Hemophilia
Adults
Adults
Evatt and Robillard, 2000 6131-4 Haemophilia
Data from 17 Randomly Selected Countries
21
Can Gene Transfer Cure Hemophilia in the
Developing World?

• Barriers
• Cost precedents established with recombinant
  factor
• Specialized expertise and equipment
• Highly experimental technology
• Safety not established Risk/Benefit
• Ethical issues
• Solutions
• Vaccine model (Kelley, Verma, Pierce Haemophilia
  2002, 8261-267)
• Academic-Industry, Government-Nonprofit
  collaborations
• Status
• gt500 million USD invested
• 2006 Workshop J Thromb Haemost. 200755901-6.
  Gene therapy, bioengineered clotting factors and
  novel technologies for hemophilia treatment.
  Pierce GF, Lillicrap D, Pipe SW, Vandendriessche
  T
• Next steps New research hypotheses, new DNA
  delivery vectors required, evading host immunity,
  designer molecules
• Next workshop February 2008

22
Hemophilia The March Towards a Cure
How does one prevent the pathology of hemophilia
(eg, arthropathy, death)? Prevent bleeding
Pathology
CURE
Paradigm Shift
Curative therapy
Reduced infusion frequency
Symptomatic treatment on-demand therapy
Regular, preventative treatment
1950s Proteins
Genes 20xx
If not gene therapy, then what?