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 20% 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 (F9). The therapeutic F9 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 F8 mutation that causes severe HA, the intron-22-inversion (F8I22I). The F8I22I provides for expression of much of the FVIII protein on a contiguous polypeptide. Only the last portion of FVIII is not expressed by the F8I22I. It has been appreciated that F8I22I may be susceptible to gene repair, yet the molecular tools suitable to enable a safe and effective repair have only recently emerged. The F8I22I 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 F8I22I because wild-type BOECs secrete FVIII, are accessible from a simple blood draw, and can expanded up to 1019 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 F8I22I in model cells lines and (ii) techniques for manipulation of BOECs. These tools and techniques will be utilized to repair the F8I22I mutation in autologous canine BOECs for testing in vivo in HA dogs in subsequent Phase 2 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 Statement: 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 patients' own cells to make FVIII on their own so that these patients avoid the complications of the disease.
Project Terms: adeno-associated viral vector; Affect; African American; Alleles; Animals; Antibodies; antibody inhibitor; arthropathies; Autologous; base; Bioinformatics; Biological Assay; Biological Models; Blood; Blood Coagulation Factor; Blood typing procedure; Canis familiaris; Capsid; Cell Line; cell type; Cells; cellular transduction; Clinic; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Complementary DNA; Complication; Data; Dependovirus; design; Development; Disease; Dose; Elements; Endogenous Factors; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Event; Exons; experience; Exposure to; F8 gene; F9 gene; Factor IX; Factor VIII; Gene Expression; gene repair; gene therapy; Genetic; Genetic Transcription; Genome; Genomics; Goals; health disparity; Hemophilia A; Hemophilia B; Hemorrhage; Hepatocyte; Hepatotoxicity; Home environment; Human; human reference genome; Immune response; Immune Tolerance; immunogenic; immunogenicity; In Vitro; in vivo; Individual; Infusion procedures; inhibitor/antagonist; Intervention; Intravenous infusion procedures; Introns; Joints; lentiviral-mediated; Life; Limb structure; Link; Liver; Location; lymphoblastoid cell line; man; Mediating; Mendelian disorder; Methods; minority health; Modeling; Molecular; Muscle; Mutation; nuclease; outcome forecast; Parvovirus; Patients; peripheral blood; Persons; Pharmaceutical Preparations; Phase; phase 2 study; Plasma; Plasmids; polypeptide; Population; pre-clinical; Preparation; Procedures; Process; Production; programs; Promotor (Genetics); Proteins; public health relevance; repaired; Reverse Transcriptase Polymerase Chain Reaction; Site; Small Business Innovation Research Grant; Stem cells; System; Techniques; Testing; Therapeutic; tool; Transfection; Viral; Virus; Work
