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Principal Investigator: DREW WEISSMAN
Organization: UNIVERSITY OF PENNSYLVANIA
Fiscal Year: 2019
Award: $679,764
Funding agency: National Heart Lung and Blood Institute
Project Summary
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.
Terms: <Aberrant Chromosome><Animal Model><Animal Models and Related Studies><Antihemophilic Factor><Archaea><Archaebacteria><Archaeobacteria><Archaeon><Autoprothrombin II><Bacteria><Binding><Biologic Models><Biological Models><Bleeding><Blood Clotting><Blood Coagulation Disorders><Blood Coagulation Factor><Blood Coagulation Factor IX><Blood Coagulation Factor VIII><Blood coagulation><Bone Marrow Grafting><Bone Marrow Stem Cell><Bone Marrow Transplant><Bone Marrow Transplantation><CRISPR><CRISPR/Cas system><Cell Body><Cell Line><Cell Nucleus><CellLine><Cells><Christmas Disease><Christmas Factor><Chromosomal Aberrations><Chromosomal Abnormalities><Chromosomal Alterations><Chromosomal Breaks><Chromosome Aberrations><Chromosome Alterations><Chromosome Anomalies><Chromosome Break><Chromosome abnormality><Chronic Disease><Chronic Illness><Clinical><Clustered Regularly Interspaced Short Palindromic Repeats><Coagulation Disorder><Coagulation Factor IX><Coagulation Factor VIII><Coagulation Factor VIIIc><Coagulation Factors><Coagulopathy><Common Rat Strains><Cytogenetic Aberrations><Cytogenetic Abnormalities><DNA><DNA Damage Repair><DNA Repair><DNA Sequence><DNA Therapy><Deoxyribonucleic Acid><Development><Disease><Disorder><EC 3.4.21.22><ES cell><Embryo><Embryonic><Endothelial Cells><Endothelium><Ensure><Enzyme Gene><Enzymes><Extracellular Protein><Factor IX><Factor IX Complex><Factor IX Deficiency><Factor IX Fraction><Factor VIII><Factor VIII Deficiency><Factor VIII F8B><G Protein-Complex Receptor><G Protein-Coupled Receptor Genes><G-Protein-Coupled Receptors><GPCR><Gene Targeting><Gene Transfer Clinical><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic Diseases><Genetic Intervention><Genetic defect><Goals><Graft Material><Guide RNA><HG38><Half-Life><Hemophilia><Hemophilia A><Hemophilia B><Hemorrhage><Homologous Sequences><Joints><Kinetics><LGR5><LGR5 gene><Lead><Leucine-Rich Repeat><Ligand Binding><Ligands><Link><Location><Mammalian Cell><Marrow Transplantation><Mediating><Messenger RNA><Methods><Model System><Modeling><Modification><Molecular Interaction><Morbidity><Morbidity - disease rate><Mutation><NHEJ><Non-Homologous End Joining><Non-Polyadenylated RNA><Non-homologous DNA End Joining><Nonhomologous DNA End Joining><Nonhomologous End Joining><Nucleosides><Nucleotides><Nucleus><Oligo><Oligonucleotides><Other Genetics><Ovis><Patients><Pb element><Phase 3 Clinical Trials><Phase III Clinical Trials><Plasma Thromboplastin Component><Polymers><Procoagulant Component><Progenitor Cells><Proteins><RNA><RNA Gene Products><RNA Sequences><Rat><Rats Mammals><Rattus><Ribonucleic Acid><Right-Handed Beta-Alpha Superhelix><Risk><Sequence Homologs><Sheep><Single-Stranded DNA><Site><Stem cells><Strains Cell Lines><Surface><System><TAL effector nuclease><TAL endonuclease><TALE nuclease><TALEN technology><TALENs><Thromboplastinogen><Tissue Transplantation><Transplanted tissue><Treatment Efficacy><Unscheduled DNA Synthesis><Variant><Variation><WNT Signaling Pathway><WNT signaling><Zinc Finger Domain><Zinc Finger Motifs><Zinc Fingers><adaptive immune response><adult stem cell><antihemophilic factor A><antihemophilic factor B><base><bleeding disorder><blood loss><cell type><chromosomal defect><chromosome defect><chronic disorder><clotting disorder><clotting factor><complex Blood-coagulation factor VIII><cost><cultured cell line><developmental><disability><embryonic stem cell><experience><gRNA><gene therapy><gene transplantation><gene transplantation for gene therapy><gene-based therapy><genetic condition><genetic disorder><genetic therapy><genome mutation><genomic therapy><heavy metal Pb><heavy metal lead><high reward><homologous recombination><human disease><iPS><iPSC><iPSCs><immunogenicity><improved><in vivo><induced pluripotent stem cell><intervention efficacy><lipid nanoparticle><mRNA><model of animal><model organism><mutant><nano particle><nano-sized particle><nanoparticle><nanosized particle><native protein drug><new approaches><new therapeutic approach><new therapeutic intervention><new therapeutic strategies><new therapy approaches><novel><novel approaches><novel strategies><novel strategy><novel therapeutic approach><novel therapeutic intervention><novel therapy approach><nuclease><oligos><pharmaceutical protein><phase 3 trial><phase III protocol><platelet cofactor I><pre-clinical><preclinical><prophylactic><protein drug agent><repair><repaired><site targeted delivery><soft tissue><stem cell of embryonic origin><targeted delivery><therapeutic efficacy><therapeutic protein><therapeutically effective><therapy efficacy><thromboplastinogen A><thromboplastinogen B><transcription activator-like effector nucleases>