Hemophilia is a genetic disorder defined by either an inadequate supply or an inactive blood clotting factor. The two most common forms are hemophilia A and B, caused by genetic defects in clotting factors VIII and IX, respectively. Both are X-linked recessive disorders characterized by prolonged bleeding and hemorrhage, often into soft tissues and joints. Current treatments include protein replacement, bone marrow transplant, and gene therapy. We propose to develop a new therapeutic approach to the treatment of hemophilia A that will have direct applicability to other genetic diseases. We will perform gene editing in vivo on bone marrow stem cells and endothelial cells, as well as, many other cell types, with the aim of correcting the genetic errors that cause the disease. We believe that this is possible due to our development of nucleoside modified mRNA that can efficiently deliver proteins in vivo, our extensive experience with modified RNA, and the recent identification of the CRISPR system that uses a single enzyme (cas9) with guide RNAs to introduce chromosomal breaks at specific locations in mammalian DNA. The cas9-guide RNA system with short single stranded DNA homologous sequences was able to mediate homologous recombination mediated repair in embryonic stem cells with greater than 65% efficiency, ex vivo. We hypothesize that nucleoside modified mRNA will deliver cas9 protein that makes a break in the vicinity of the Factor VIII SNPs as directed by a guide RNA that will lead to homologous recombination mediated repair of defective genes. We also hypothesize that RNA can be delivered to stem cells in vivo by attaching ligands to the surface of lipid nanoparticles that will then target them to stem cells.
Four specific aims will be performed: 1) Targeting and repair of defective genes in cell lines and primary cells including bone marrow stem cells. 2) Development of delivery systems that target endothelial and bone marrow stem cells in vivo. 3) Treatment of rats containing SNPs that result in hemophilia A. 4) Treatment of sheep with hemophilia A. We will develop this new approach for treatment of genetic diseases using hemophilia A as a model, but once developed, the targeting of other genetic diseases will only require a new target sequence in the guide RNA. The ability to edit genes in vivo will greatly reduce the risks and cost of therapy that currently require ex vivo isolation and manipulation of stem cells or bone marrow transplantation.

Public Health Relevance

Current treatments for hemophilia A use treatments that result in significant morbidity, such as Factor replacement, bone marrow transplant or gene therapy. This study will use a new platform for therapeutic protein delivery based on nucleoside modified RNA to develop a safe and effective therapeutic for editing defective genes in vivo that are responsible for Hemophilia A.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Gene and Drug Delivery Systems Study Section (GDD)
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Warren, Ronald Q
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University of Pennsylvania
Internal Medicine/Medicine
Schools of Medicine
United States
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