Slide1: 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


The First "Gene Transfer" Experiments: Early Years- Proof of Principle Established: The First "Gene Transfer" Experiments: Early Years- Proof of Principle Established 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) Genes=DNA, 1953 Watson and Crick Biotech industry born 1976 (Genentech) 1st recombinant DNA produced drugs: early 1980s (insulin) In parallel:


Gene Therapy: 1980s to Early 1990s: 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


Will the prediction come true?: Will the prediction come true? Pierce, Hemalog, Summer 1990 This goal was a catalyst


Advocacy, Education and Fundraising: Advocacy, Education and Fundraising


Slide6: 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


Animal Models Essential in Hemophilia Research: 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


Liver and Muscle AAV2 Injection in Hem B Dogs: Multi-year Cures; Factor IX Expression More Efficient in Liver: 12% 9% 5% 0.3% Liver and Muscle AAV2 Injection in Hem B Dogs: Multi-year Cures; Factor IX Expression More Efficient in Liver Nakai et al, Blood 1998; Grimm et al Blood 2003


NHF MASAC recommendations: NHF MASAC recommendations Ethics, responsibilities Research, Clinical Trials


US Gene Therapy Clinical Trials: 41 Subjects: US Gene Therapy Clinical Trials: 41 Subjects Trial Transkaryotic Therapies High/Kay/ Avigen Chiron High/Kay/ Avigen Genstar Therapeutics Started Dec 1998 Jun 1999 Jun 1999 Jun 2001 Jun 2001 Hemophilia A (12) B (7) A (13) B (8) A (1) Therapy type Ex vivo plasmid i.p. injection In vivo AAV i.m. injection In vivo retroviral i.v. infusion In vivo AAV liver-targeted infusion In vivo adenoviral i.v. infusion Status Completed Phase I Completed Phase I Completed Phase I Completed Phase I Trial Terminated One Factor IX trial in China: 2 subjects, early 90s, ex vivo, likely insufficient numbers of cells delivered, little reported


1 Step Forward, 2 Steps Back?: 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?


Delivery of New Genes: Delivery of New Genes Naked DNA Target Cell Therapeutic Protein AAV Retrovirus/Lentivirus Adenovirus Nucleus


Slide13: circular concatemer + ss rAAV mRNA ? nucleus - ss rAAV ss rAAV pool annealed ds rAAV circular monomer integrated concatemer mRNA circular dimer mRNA mRNA cytoplasm episomal linear concatemer integrated monomer ? now investigating Amazing Protein Viral Vector Interactions with Cells are Poorly Understood If this looks complicated…Interactions between host defenses and virus even more poorly understood


Slide14: Kay et al, Nat Genet 2000; 24:257 Manno et al, Blood 2003; 101:2963 Arruda et al, Mol Therapy 2006; 14:452 Manno et al, Nat Med, 2006 12:342-7 Mingozzi et al, Nat Med, 2007 13: 419-22 Muscle delivery: Long term local expression (3.7 years) but insufficient circulating FIX Liver Delivery: Therapeutic circulating FIX levels but not sustained


Activation of AAV-specific Memory CD8+ T cells Requires Cross Presentation of AAV Capsid: 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? Manno, Pierce, Arruda et al, Nat Med, 2006 12:342-7 Mingozzi et al, Nat Med, 2007 13: 419-22 Vendenburghe et al, Nat Med 2006 12:967-71 Gene therapy needs to solve the 600 million year old mystery of the battle between viruses and hosts: get along or die….


Slide16: Progress in AAV-mediated Gene Transfer for Hemophilia B Couto, Pierce, Curr Opin Mol Ther 2003; 5:517


Workshops 2001/02: Unmet needs: 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


Slide18: Ways of Improving rFVIII for Protein and Gene Therapy Secretion Potency Resistance to inactivation Resistance to inhibitors Prolonged lifetime A1 mutation F309S B domain variants 226aa/N6 Improved activation FVIII/HCII hybrid  Specific Activity E113A Porcine-human hybrids LRP/HSPG binding site mutations PEGylated liposomes PEG-FVIII IR8 Disulfide Bond-FVIII Porcine-human hybrids D318G/M337R Factors IX and VIIa:  potency, life, secretion


Why Pursue Gene Therapy? Global Reality: 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


Slide20: Relationship of Economic Capacity to Number of Adults with Hemophilia* *Data from 17 Randomly Selected Countries Evatt and Robillard, 2000; 6:131-4 Haemophilia Adults Adults


Can Gene Transfer Cure Hemophilia in the Developing World?: 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, 8:261-267) Academic-Industry, Government-Nonprofit collaborations Status >$500 million USD invested 2006 Workshop: J Thromb Haemost. 2007;55:901-6. 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


Slide22: Reduced infusion frequency How does one prevent the pathology of hemophilia (eg, arthropathy, death)? Prevent bleeding… Pathology CURE Symptomatic treatment: on-demand therapy Regular, preventative treatment Curative therapy Hemophilia: The March Towards a Cure Paradigm Shift 1950s Proteins Genes 20xx If not gene therapy, then what?