of Project 1 Hemophilia B (HB) is an X-linked disease resulting from the deficiency of clotting factor IX (FIX). Skeletal muscle is an attractive target tissue for gene therapy using adeno-associated viral vectors (AAV). Early-phase clinical studies, using recombinant adeno-associated virus (rAAV) vectors encoding the human FIX gene for intramuscular (IM) injection of HB subjects, were associated with an excellent safety profile, but low efficacy. One strategy for increasing efficiency is to transduce larger areas of muscle. We accomplished this by developing a non-invasive, peripheral transvenular delivery of AAV vectors to skeletal muscle of HB dogs via afferent transvenular retrograde extravasations (ATVRX). Vector doses tested were within the range of those safely delivered to human subjects. In this application we propose to further improve the therapeutic potential of ATVRX for HB gene therapy by using a naturally occurring FIX variant, FIX-Padua or FIX-R338L. This FIX variant is associated with an 8-fold higher specific activity than the wild-type FIX. Early data in HB dogs using AAV-6-encoding canine (c) FIX-Padua to skeletal muscle via ATVRX resulted in expression of a cFIX protein at 10-30% of normal FIX activity in plasma, but with only 1-3% antigen levels. The HB dogs used were from University of North Carolina-Chapel Hill (UNC-CH) colony and harbor a missense mutation in the FIX gene. We now seek to carry out a comprehensive study in two distinct dog models of HB to assess the efficacy and safety of cFIX-Padua. We hypothesize that the use of a FIX protein with high specific activity will allow us to (1) lower the effective rAAV vector doses required for hemostatic efficacy and at the same time not increase local antigen production of FIX and (2) overcome the limited capacity of skeletal muscle in performing all the post- translational FIX modifications for a fully functional protein. There are 3 specific aims to this proposal.
Aim 1 : To determine the safe and therapeutic dose range of AAV-6-cFIX-Padua following delivery to skeletal muscle via ATVRX. We will perform a dose-escalation study to define the minimal and maximal vector doses that would result in sustainable and safe therapeutic levels of FIX.
Aim 2 :To determine the immunological profile of expression of cFIX-Padua in two canine HB models: (a) High risk model for inhibitor formation using inhibitor- prone HB dogs from the University of Alabama at Birmingham (UAB) dog colony (b) HB dogs from the UNC- CH dog colony expressing subtherapeutic levels of wild-type (WT) FIX from previous injections of AAV-2-cFIX.
Aim 3. To determine the biological function and immunogenicity of recombinant FIX-Padua protein. We will characterize FIX-Padua by a series of biochemical and functional kinetic assays and carry out immunologic challenges in AAV-FIX treated dogs with these FIX proteins. Thus, the overall goal of this proposal is to establish the efficacy and safety of a novel therapeutic protocol that combines an optimized peripheral intravascular delivery of rAAV to skeletal muscle with the use of an effective AAV serotype (AAV-6), and the use of a novel highly active FIX as the transgene product that should be translatable to patient care in the future.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
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Heart, Lung, and Blood Initial Review Group (HLBP)
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Children's Hospital of Philadelphia
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Sauna, Zuben E; Lozier, Jay N; Kasper, Carol K et al. (2015) The intron-22-inverted F8 locus permits factor VIII synthesis: explanation for low inhibitor risk and a role for pharmacogenomics. Blood 125:223-8
Sullivan, Spencer K; Mills, Jason A; Koukouritaki, Sevasti B et al. (2014) High-level transgene expression in induced pluripotent stem cell-derived megakaryocytes: correction of Glanzmann thrombasthenia. Blood 123:753-7
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
Yazicioglu, Mustafa N; Monaldini, Luca; Chu, Kirk et al. (2013) Cellular localization and characterization of cytosolic binding partners for Gla domain-containing proteins PRRG4 and PRRG2. J Biol Chem 288:25908-14
Callejas, David; Mann, Christopher J; Ayuso, Eduard et al. (2013) Treatment of diabetes and long-term survival after insulin and glucokinase gene therapy. Diabetes 62:1718-29
Siner, Joshua I; Iacobelli, Nicholas P; Sabatino, Denise E et al. (2013) Minimal modification in the factor VIII B-domain sequence ameliorates the murine hemophilia A phenotype. Blood 121:4396-403
Mingozzi, Federico; High, Katherine A (2013) Immune responses to AAV vectors: overcoming barriers to successful gene therapy. Blood 122:23-36
Anguela, Xavier M; Sharma, Rajiv; Doyon, Yannick et al. (2013) Robust ZFN-mediated genome editing in adult hemophilic mice. Blood 122:3283-7
Buchlis, George; Podsakoff, Gregory M; Radu, Antonetta et al. (2012) Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer. Blood 119:3038-41
Mingozzi, Federico; High, Katherine A (2011) Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat Rev Genet 12:341-55

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