The goal of this study is to develop a safe, efficient, and clinically feasible nonviral gene therapy strategy for the treatment of hemophilia A. Currently hemophilia A patients are treated with repeated infusions of protein concentrates, which is both costly and inconvenient. Furthermore, ~30% of hemophilia A patients developed inhibitory antibodies against factor VIII (FVIII) following protein replacement therapy. We have previously demonstrated that nonviral gene transfer of a liver-specific FVIII plasmid produced persistent, supra-physiological/therapeutic levels of FVIII in immunodeficient mice using a hydrodynamics-based delivery method. However, robust FVIII-specific immune responses occurred in immunocompetent HemA mice and eliminated functional FVIII two weeks following gene transfer. This murine model permitted us to develop an effective immunosuppressive regimen in combination with nonviral gene therapy to achieve a long-term therapeutic effect. Nine single or combined immunosuppressive regimens have been tested, and the best strategy of blocking the co-stimulation pathway using a combination of Ctla4-Ig and anti-murine CD40 ligand mAb (MR1) induced long- term tolerance to factor VIII in mice. Furthermore, since hydrodynamic gene delivery is unsuitable for use in humans in its current form, we have also pursued and gained preliminary success in the development of ultrasound (US)-mediated gene delivery in vivo and cell permeable peptide (CPP)-mediated gene transfer in vitro. To facilitate direct translation of these methods to human applications, we propose to further optimize the efficiency of the US-mediated gene delivery of plasmid DNA or CPP/DNA complexes, and evaluate immunomodulation therapy for the most effective and least toxic immunosuppressive regimen in the hemophilia A mouse model. The optimized delivery method will also be tested in preliminary scale-up experiments in normal dogs in preparation for further evaluation in the hemophilia A dog model. We will test the hypotheses that: 1) Safe and clinically feasible nonviral gene delivery methods, mainly US-mediated gene therapy, can be established to allow efficient plasmid DNA transfer into the mouse liver;2) Minimally toxic immunosuppressive regimens using immunosuppressive agents alone or in combination can be developed to prevent and/or modulate transgene-specific immune responses following gene therapy; 3) These combined approaches will lead to long-term correction of disease in a hemophilia A murine model;and 4) The ultrasound-mediated gene delivery method can be scaled up in normal dog experiments.

Public Health Relevance

This project will develop safe nonviral gene therapy protocols in combination with immunomodulation strategies to effectively treat hemophilia A patients. The successful protocols will significantly prolong the lifespan and improve the quality of patients with hemophilia A, and potentially be useful for developing gene therapy strategies for other genetic diseases.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Gene and Drug Delivery Systems Study Section (GDD)
Program Officer
Link, Rebecca P
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Seattle Children's Hospital
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Noble-Vranish, Misty L; Song, Shuxian; Morrison, Kyle P et al. (2018) Ultrasound-Mediated Gene Therapy in Swine Livers Using Single-Element, Multi-lensed, High-Intensity Ultrasound Transducers. Mol Ther Methods Clin Dev 10:179-188
Liu, Chao Lien; Lyle, Meghan J; Shin, Simon C et al. (2016) The dataset from administration of single or combined immunomodulation agents to modulate anti-FVIII antibody responses in FVIII plasmid or protein primed hemophilia A mice. Data Brief 7:973-80
Liu, Chao Lien; Lyle, Meghan J; Shin, Simon C et al. (2016) Strategies to target long-lived plasma cells for treating hemophilia A inhibitors. Cell Immunol 301:65-73
Liu, Chao Lien; Ye, Peiqing; Lin, Jacqueline et al. (2014) Anti-CD20 as the B-Cell Targeting Agent in a Combined Therapy to Modulate Anti-Factor VIII Immune Responses in Hemophilia a Inhibitor Mice. Front Immunol 4:502
Liu, C L; Ye, P; Lin, J et al. (2014) Long-term tolerance to factor VIII is achieved by administration of interleukin-2/interleukin-2 monoclonal antibody complexes and low dosages of factor VIII. J Thromb Haemost 12:921-31
Sun, Ryan R; Noble, Misty L; Sun, Samuel S et al. (2014) Development of therapeutic microbubbles for enhancing ultrasound-mediated gene delivery. J Control Release 182:111-20
Noble, Misty L; Kuhr, Christian S; Graves, Scott S et al. (2013) Ultrasound-targeted microbubble destruction-mediated gene delivery into canine livers. Mol Ther 21:1687-94
Song, Shuxian; Noble, Misty; Sun, Samuel et al. (2012) Efficient Microbubble- and Ultrasound-Mediated Plasmid DNA Delivery into a Specific Rat Liver Lobe via a Targeted Injection and Acoustic Exposure Using a Novel Ultrasound System. Mol Pharm :
Miao, Carol H (2011) Advances in Overcoming Immune Responses following Hemophilia Gene Therapy. J Genet Syndr Gene Ther S1:
Song, S; Shen, Z; Chen, L et al. (2011) Explorations of high-intensity therapeutic ultrasound and microbubble-mediated gene delivery in mouse liver. Gene Ther 18:1006-14

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