Fanconi anemia (FA) is an inherited recessive disorder caused by deficiency of one of several (up to 9) proteins involved in the regulation of DNA repair. Patients exhibit birth defects, suffer from bone marrow failure early in life, and are prone to develop cancers including leukemia and solid tumors. The only effective treatment for FA is allogeneic bone marrow transplant, but many patients lack a matched donor. We propose the treatment of FA by introduction and expression the FANC gene by combining the cell-loading technology of MaxCyte, Inc., with the DNA integrating technology of Discovery Genomics, Inc. (DGI).
In Specific Aim 1, we will first test the combination of these two technologies by using reporter genes to ascertain long-term gene transfer and expression in cultured hematopoietic cells. DGI will assemble transposons designed for introduction and expression of red fluorescent protein (dsRed). MaxCyte will then use its electroporation technology for high efficiency loading of the transposon DNA into several different hematopoietic cell lines (Jurkat, KB, K562) along with a source of transposase which mediates transposition from the newly-introduced plasmid DNA into chromosomal DNA. Gene expression will be assayed over time by flow cytometry, and molecular analyses (PCR, Southern blot) will be conducted for analysis of transposition into chromosomal targets.
In Aim 2, DGI will assemble transposons designed for expression of the human FANC-C and FANC-A genes. MaxCyte will then load these transposons into lymphoblastoid cell lines established from patients with Fanconi anemia type C or A, respectively, testing these cell populations for reduced sensitivity to the DNA damaging agent mitomycin C as well as for transposition by molecular genetic analysis. These Phase 1 studies will provide the basis for further preclinical development of the combined cell loading and DNA integrating technologies targeting hematopoietic stem cells (HSC) in Phase 2. These studies will be ground-breaking in that long-term expression of genes after non-viral introduction into HSC has yet to be reported, and will require the efficient cell loading and integrating capacities provided by our combined technologies. Initial development cell loading / DNA integration approach is proposed here for Fanconi anemia, subsequently providing the technical basis for treatment of other inherited (immunodeficiencies, hemoglobinopathies) or acquired (AIDS) diseases.
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