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.

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Heart, Lung, and Blood Program Project Review Committee (HLBP)
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Children's Hospital of Philadelphia
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