Strategies for Personalized Gene Repair using I22I Severe Hemophilia-A as a Model

DESCRIPTION provided by applicant Major benefits to hemophilia patients of endogenous expression of coagulation factors include the avoidance of potentially crippling joint bleeds and importantly induction of immunological tolerance for hemophilia patients who develop antibodies against replacement coagulation factors About of all individuals with hemophilia HA develop antibodies against Factor VIII FVIII but almost half of African American HA patients experience this very deleterious complication Patients with anti FVIII antibodies have a poorer prognosis than HA patients able to be effectively treated with replacement FVIII Current approaches to impart endogenous expression of functional coagulation factors in hemophilia patients rely on the use of viral elements In a recent clinical trial six hemophilia B HB patiets experienced an improvement from severe to mild or moderate hemophilia following intravenous infusion of a therapeutic adeno associated virus AAV vector bearing a fully functional Factor IX gene F The therapeutic F was targeted to liver cells where it was incorporated and expressed Use of viral elements has drawbacks however that may not be overcome Many patients cannot be treated with AAV vectors because they have antibodies against it based on prior exposure to similar viruses Also the AAV capsid is immunogenic at doses used in the HB trial This is problematic especially since data from studies in the validated canine model of HA show that much higher doses of AAV would be required for efficacy in human HA A key goal of this project is to develop a viral free means to restore endogenous expression of FVIII We plan to repair the most common F mutation that causes severe HA the intron inversion F I I The F I I provides for expression of much of the FVIII protein on a contiguous polypeptide Only the last portion of FVIII is not expressed by the F I I It has been appreciated that F I I may be susceptible to gene repair yet the molecular tools suitable to enable a safe and effective repair have only recently emerged The F I I mutation that occurs in man also causes HA in dogs thus providing a model system for preclinical development that would be readily translatable to humans The blood outgrowth endothelial cell BOEC is an ideal cell in which to repair the F I I because wild type BOECs secrete FVIII are accessible from a simple blood draw and can expanded up to cells Prior work shows that limited numbers of BOECs home to and persist in the liver We propose to develop i the tools for CRISPR associated nuclease CasN mediated gene repair of F I I in model cells lines and ii techniques for manipulation of BOECs These tools and techniques will be utilized to repair the F I I mutation in autologous canine BOECs for testing in vivo in HA dogs in subsequent Phase studies Our proposed proof of concept studies allow us to take the first steps in the preclinical development of a safe viral free CasN mediated method to restore FVIII secretion in the majority of patients with severe HA PUBLIC HEALTH RELEVANCE Hemophilia A HA is a genetic bleeding disorder resulting in the inability to stop bleeding normally People with HA need infusions of a replacement protein called FVIII We are working on a safe way to repair HA patientsandapos own cells to make FVIII on their own so that these patients avoid the complications of the disease