After more than a decade of clinical studies, gene therapy for the X-linked bleeding disorder hemophilia B (factor IX, F.IX) deficiency) based on in vivo adeno-associated viral (AAV) gene transfer has enjoyed a major success in 2011. Several patients showed sustained therapeutic expression after hepatic gene transfer. Despite these encouraging results, immune responses against vector and transgene product continue to be of concern for safety and success of gene therapy for hemophilia. At the same time, our prior animal studies demonstrated that hepatocyte-restricted transgene expression from an AAV vector induced immune tolerance to the F.IX transgene product. Tolerance induction critically depended on a CD4+CD25+FoxP3+ regulatory T cell (Treg) response. Optimal hepatic gene transfer increased Treg induction and thus the success rate of tolerance induction. Conversely, we found that innate TLR9-dependent immune responses to the vector may increase adaptive immune responses. This proposal addresses several immunological aspects that are critical to future development of the approach. First, we will further define the mechanism of innate immune responses to AAV vectors and determine links to adaptive responses against vector and F.IX transgene product. Second, we will uncover a mechanism by which optimal hepatic gene transfer can not only prevent but also reverse antibody (inhibitor) formation against F.IX. Our preliminary data support the notion that hepatic AAV gene transfer represents an alternative to current clinical immune tolerance induction (ITI) protocols for hemophilia (which are suboptimal and expensive). Finally, we will extent investigations on the immunology of hepatic AAV gene transfer to treatment of hemophilia A (factor VIII, F.VIII, deficiency), the more common form of hemophilia. F.VIII is typically regarded as more immunogenic and is also more difficult to express at high level. In conventional protein therapy, inhibitor formation occurs far more frequently in hemophilia A than in hemophilia B. For all these reasons, development of a gene therapy for hemophilia A has not been straightforward. We propose that use of a codon-optimized F.VIII cDNA and transient depletion of B cells with anti-CD20 can be exploited to accomplish tolerance induction to F.VIII by hepatic AAV gene transfer. In summary, the following hypotheses will be tested: i) toll-like receptors, inflammatory cytokine production and type I interferon responses are critical components of the innate response to AAV vector;ii) innate immune responses and Treg shape adaptive responses to vector and F.IX transgene product, iii) blockage of innate immunity reduces adaptive responses against vector and transgene product;iv) optimal hepatic gene transfer (under conditions of limited innate immunity and high levels of expression) can effectively reverse inhibitor and anaphylactic responses to F.IX in hemophilia B mice;and v) use of a codon-optimized F.VIII cDNA and transient B cell depletion facilitate tolerance induction to F.VIII by liver gene transfer in hemophilia A mice.

Public Health Relevance

This project will develop novel to prevent or eliminate undesired immune responses in treatment of the inherited bleeding disorder hemophilia. Immune responses to coagulation factors represent a severe complication of treatment of patients with hemophilia, increase morbidity and mortality, and substantially increase cost of treatment. A gene therapy that also promotes immune tolerance would substantially improve the lives of hemophilic patients and lower health care costs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI051390-13
Application #
8586838
Study Section
Gene and Drug Delivery Systems Study Section (GDD)
Program Officer
Johnson, David R
Project Start
2002-04-01
Project End
2017-11-30
Budget Start
2013-12-01
Budget End
2014-11-30
Support Year
13
Fiscal Year
2014
Total Cost
$302,569
Indirect Cost
$100,069
Name
University of Florida
Department
Pediatrics
Type
Schools of Medicine
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Rogers, Geoffrey L; Martino, Ashley T; Zolotukhin, Irene et al. (2014) Role of the vector genome and underlying factor IX mutation in immune responses to AAV gene therapy for hemophilia B. J Transl Med 12:25
Sarkar, Debalina; Biswas, Moanaro; Liao, Gongxian et al. (2014) Ex Vivo Expanded Autologous Polyclonal Regulatory T Cells Suppress Inhibitor Formation in Hemophilia. Mol Ther Methods Clin Dev 1:
Sherman, Alexandra; Schlachterman, Alexander; Cooper, Mario et al. (2014) Portal vein delivery of viral vectors for gene therapy for hemophilia. Methods Mol Biol 1114:413-26
Markusic, David M; Hoffman, Brad E; Perrin, George Q et al. (2013) Effective gene therapy for haemophilic mice with pathogenic factor IX antibodies. EMBO Mol Med 5:1698-709
Hoffman, Brad E; Herzog, Roland W (2013) Covert warfare against the immune system: decoy capsids, stealth genomes, and suppressors. Mol Ther 21:1648-50
Martino, Ashley T; Suzuki, Masataka; Markusic, David M et al. (2011) The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9-dependent innate immune responses in the liver. Blood 117:6459-68
Hoffman, Brad E; Martino, Ashley T; Sack, Brandon K et al. (2011) Nonredundant roles of IL-10 and TGF-? in suppression of immune responses to hepatic AAV-factor IX gene transfer. Mol Ther 19:1263-72
Jayandharan, Giridhara R; Aslanidi, George; Martino, Ashley T et al. (2011) Activation of the NF-kappaB pathway by adeno-associated virus (AAV) vectors and its implications in immune response and gene therapy. Proc Natl Acad Sci U S A 108:3743-8
Moghimi, B; Sack, B K; Nayak, S et al. (2011) Induction of tolerance to factor VIII by transient co-administration with rapamycin. J Thromb Haemost 9:1524-33
Nayak, S; Herzog, R W (2010) Progress and prospects: immune responses to viral vectors. Gene Ther 17:295-304

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