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
Biswas, Moanaro; Rogers, Geoffrey L; Sherman, Alexandra et al. (2016) Combination therapy for inhibitor reversal in haemophilia A using monoclonal anti-CD20 and rapamycin. Thromb Haemost 117:
Wang, Xiaomei; Terhorst, Cox; Herzog, Roland W (2016) In vivo induction of regulatory T cells for immune tolerance in hemophilia. Cell Immunol 301:18-29
Zolotukhin, Irene; Markusic, David M; Palaschak, Brett et al. (2016) Potential for cellular stress response to hepatic factor VIII expression from AAV vector. Mol Ther Methods Clin Dev 3:16063
Vercauteren, Koen; Hoffman, Brad E; Zolotukhin, Irene et al. (2016) Superior In vivo Transduction of Human Hepatocytes Using Engineered AAV3 Capsid. Mol Ther 24:1042-9
Perrin, George Q; Zolotukhin, Irene; Sherman, Alexandra et al. (2016) Dynamics of antigen presentation to transgene product-specific CD4(+) T cells and of Treg induction upon hepatic AAV gene transfer. Mol Ther Methods Clin Dev 3:16083
Rogers, Geoffrey L; Herzog, Roland W (2015) Gene therapy for hemophilia. Front Biosci (Landmark Ed) 20:556-603
Biswas, Moanaro; Sarkar, Debalina; Kumar, Sandeep R P et al. (2015) Synergy between rapamycin and FLT3 ligand enhances plasmacytoid dendritic cell-dependent induction of CD4+CD25+FoxP3+ Treg. Blood 125:2937-47
Rogers, Geoffrey L; Suzuki, Masataka; Zolotukhin, Irene et al. (2015) Unique Roles of TLR9- and MyD88-Dependent and -Independent Pathways in Adaptive Immune Responses to AAV-Mediated Gene Transfer. J Innate Immun 7:302-14
Wang, Xiaomei; Moghimi, Babak; Zolotukhin, Irene et al. (2014) Immune tolerance induction to factor IX through B cell gene transfer: TLR9 signaling delineates between tolerogenic and immunogenic B cells. Mol Ther 22:1139-50
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:

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