The goal of this study is to utilize animal models of hemophilia to develop improved, clinically relevant nonviral gene transfer methodologies. While viral gene transfer has recently shown promise for therapy of several diseases including hemophilia, many obstacles remain including: (1) difficulty of large scale virus production; (2) the host immune responses to viral vectors; (3) the potential toxicity of encapsulated viral vectors; and (4) the potential for mutagenic events initiated by random integration into the host genome. Nonviral gene transfer approaches could provide an alternative and possibly safer gene delivery strategy that may avoid some of these potential harmful effects. Our previous data clearly demonstrate the complete and sustained phenotypic correction of hemophilia B in mice following hepatic gene transfer of a high-expressing factor IX (FIX) plasmid. Furthermore, we have shown that using a combination of optimal cis-regulatory elements in FVIII gene expression cassettes, high-level expression of FVIII in the liver of hemophilia A mice was achieved. While clearly successful for plasmid delivery and expression, the rapid, high-volume tail-vein injection technique utilized in these studies, will not be suitable for direct clinical applications. Furthermore, a robust immune response against the FVIII led to complete inhibition of circulating FVIII activity despite maintenance of high level sustained FVIII expression in treated hemophilia A mice. Development of an effective method to induce tolerance will be essential for the successful application of all forms of FVIII of gene therapy including those utilizing nonviral protocols. We hypothesize that: 1.) Alternative delivery methods can be established (Transferrin-DNA conjugates, and ultrasound stimulation) that will allow efficient and clinically feasible plasmid DNA transfer into liver; 2) These combined approaches will lead to correction of disease in a hemophilia A murine model; and finally, 3) This model will also permit evaluation of the host immune response to the """"""""neo-antigen"""""""" factor VIII, including elucidation of the mechanism of immune activation, and strategies for successful immuno-modulation.
Aim I. To evaluate alternative clinically feasible methods for nonviral gene delivery of plasmid DNA into mouse livers.
Aim II. To evaluate immune response against FVIII following non-viral gene transfer, and evelop strategies to prevent and modulate alloimmunization in hemophilia A mice.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Medical Biochemistry Study Section (MEDB)
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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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