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Principal Investigator: Patricia DEVAUX
Organization: MAYO CLINIC ROCHESTER
Fiscal Year: 2021
Award: $318,000
Funding agency: National Heart Lung and Blood Institute
Abstract.
The recent advances in induced pluripotent stem cells (iPSCs) and gene therapy tools have opened up a new
avenue to study and treat diseases, particularly of disorders with defective bone marrow. Bone marrow failure
syndromes (BMFS) are characterized by reduced blood cells due to a dysfunctional bone marrow cells.
Fanconi anemia (FA) is one such bone marrow failure syndrome where cellular reprogramming is inefficient,
owing to interference of the disease-related genes. To overcome this limitation, it is necessary to
fundamentally correct the abnormal gene (e.g.: FANCD1) during or prior to the reprogramming process. In the
past, obtaining genetically modified iPSC from the fibroblasts of these patients typically involved multiple steps.
But recent progress in the field has paved way for simultaneous reprogramming and gene targeting in a single
step using multiple episomal vectors. In this study we propose to transform the multiple vector-single step
procedure to a single vector-one step approach to obtain corrected iPSC from FA fibroblasts. Our single vector
is based on a non-integrating negative strand RNA virus, Measles virus (MV). The central hypothesis is that a
MV vectors can be designed to express all components in one genome, and lead to the generation of clinically
safe, corrected and functional iPSCs from FA fibroblasts. The rationale for the proposed research is that the
“one-cycle” MV vector, MV4F, expressing the four reprogramming factors, generate iPSC from human
fibroblasts and is quickly diluted and eliminated from the iPSC after reprogramming. Guided by strong
preliminary data, the specific aim of this particular application is to produce a set of one-cycle “all-in-one” MV
vectors, containing the four reprogramming factors plus Cas9-gRNA, and setup the protocol to concurrently
reprogram and edit the genome of human fibroblasts carrying a genetic mutation. The proposed work is
innovative, because it capitalizes on a new technology that relies on a single vector expressing the four
reprogramming factors (RFs) for the reprogramming of somatic cells into iPSC; and our group developed that
technology. Finally, the corrected iPSC will be tested for their ability to differentiate into hematopoietic stem
cells. The proposed work is significant because we develop a new single vector for the production of corrected
iPSC, that will be eliminated quickly from the established iPSC and that can be rapidly translated into the clinic,
as it is based on the safe measles vaccine strain. Finally, the proposed research is relevant to that part of
NIH’s mission that pertains to develop new treatments for inherited bone marrow failure syndromes,
hemoglobinopathies, immunodeficiencies, and other monogenetic disorders to reduce the burden of human
disease.
Terms: <Allogenic><BRCA2><BRCA2 gene><Birth Defects><Blood Cells><Blood Diseases><Blood Precursor Cell><Bone Marrow><Bone Marrow Blood-Deriving Cell><Bone Marrow Blood-Forming Cell><Bone Marrow Cells><Bone Marrow Reticuloendothelial System><Breast Cancer 2 Gene><Breast Cancer Type 2 Susceptibility Gene><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cas nuclease technology><Cell Body><Cells><Clinic><Clinical><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Congenital Abnormality><Congenital Anatomic Abnormality><Congenital Anatomical Abnormality><Congenital Defects><Congenital Deformity><Congenital Malformation><Congenital Pancytopenia><DNA Alteration><DNA Damage Repair><DNA Repair><DNA Sequence Alteration><DNA Therapy><DNA mutation><Data><Degenerative Disorder><Development><Diamond-Blackfan anemia><Disease><Disorder><Double-Stranded DNA><Early Onset Gene Breast Cancer 2><FANCD1><Failure><Fanconi Anemia><Fanconi Panmyelopathy><Fanconi dysplasia><Fanconi's Anemia><Fibroblasts><Gene Abnormality><Gene Targeting><Gene Transfer Clinical><Generations><Genes><Genetic Alteration><Genetic Change><Genetic Diseases><Genetic Intervention><Genetic defect><Genetic mutation><Genome><Goals><Guide RNA><HSC transplantation><Hematologic Diseases><Hematological Disease><Hematological Disorder><Hematopoietic Progenitor Cells><Hematopoietic Stem Cell Transplant><Hematopoietic Stem Cell Transplantation><Hematopoietic stem cells><Hemoglobinopathies><Hereditary><Hereditary Breast Cancer 2><Human><Immunodeficiency Disorder><Immunodeficiency Syndrome><Immunologic Deficiency Syndromes><Immunological Deficiency Syndromes><Inherited><Lead><Lentivirinae><Lentivirus><Malignant><Malignant - descriptor><Measles Vaccine><Measles virus><Mendelian disease><Mendelian disorder><Mendelian genetic disorder><Mission><Modern Man><Modification><Mutation><NIH><National Institutes of Health><Non-Polyadenylated RNA><Outcome><Pathway interactions><Patients><Pb element><Peripheral Blood Cell><Primary Erythroid Hypoplasia><Procedures><Process><Production><Progenitor Cell Transplantation><Protocol><Protocols documentation><RNA><RNA Gene Products><RNA Viruses><RNA vaccine><RNA-based vaccine><Regenerative Medicine><Research><Ribonucleic Acid><Risk><Sequence Alteration><Somatic Cell><Stem Cell Transplantation><Stem cell transplant><System><Technology><Testing><Translating><United States National Institutes of Health><Unscheduled DNA Synthesis><Viral><Viral Vector><Virus-Lenti><Work><alternative treatment><base><blood disorder><blood stem cell><bone marrow failure syndrome><brca 2 gene><c myc><c-myc Genes><clinical relevance><clinically relevant><cmyc><combination gene therapy><congenital aplastic anemia><degenerative condition><degenerative disease><design><designing><developmental><ds-DNA><dsDNA><gRNA><gene therapy><gene-based therapy><genetic condition><genetic disorder><genetic therapy><genome editing><genome mutation><genomic alteration><genomic editing><genomic therapy><heavy metal Pb><heavy metal lead><hematopoietic progenitor><hematopoietic stem progenitor cell><hemopoietic progenitor><hemopoietic stem cell><human disease><human pluripotent stem cell><hypoimmunity><iPS><iPSC><iPSC technology><iPSCs><immune deficiency disorder><immunodeficiency><induced pluripotent stem cell><induced pluripotent stem cell technology><innovate><innovation><innovative><loss of function><mRNA vaccine><mRNA-based vaccine><monogenic disease><monogenic disorder><new technology><next generation><novel><novel technologies><pathway><rougeole virus><rubeola virus><single-gene disease><single-gene disorder><stem cell based therapy><stem cell gene therapy><stem cell mediated therapy><stem cell therapeutics><stem cell therapy><stem cell treatment><stem cell-based treatment><tool><v-myc Avian Myelocytomatosis Viral Oncogene Cellular Homolog><vector><vector-induced>