347. Long-Term Gene Expression and Phenotypic Correction of FANCC Deficient Lymphoblastoid Cells Using the Sleeping Beauty Transposon System

Abstract

We are moving forward with development of our proprietary Sleeping Beauty Transposon (SBT) system for the treatment of Fanconi anemia (FA) via ex vivo electroporation of hematopoietic stem cells. FA is an inherited recessive disorder caused by deficiency in one of a number of genes whose products form a complex involved in DNA repair. The primary hematological hallmarks of the disease are aplastic anemia, bone marrow failure, and increased susceptibility to leukemias thought to be caused by defective cellular mechanisms for DNA repair. Allogeneic bone marrow transplantation is currently the only curative treatment for these aspects of the disease and despite improvements in clinical protocols over the years difficulties with allotransplants for FA still persist. The primary goal of this study is to evaluate the potential of SBT vectors carrying the FANCC gene to effectaffect transposition and establish long-term expression in FANCC-deficient cell populations after introduction by electroporation. Additionally, we show that integration of FANCC transposons can correct the sensitivity of lymphoblastoid cell lines (LCL) derived from FANCC patients to DNA damaging agents such as mytomycin C. We constructed a transposon containing a PGK promoter to regulate transcription of the normal human FANCC cDNA sequence. The vector containing the transposon also includes a ubiquitin promoter regulating expression of the SB transposase gene, encoded outside of the transposon. The SBT-FANCC vector was introduced via electroporation into FANCC deficient and normal control LCLs. Long-term maintenance of FANCC complementation in the FANCC-deficient cells was observed as a sustained reduction in sensitivity to mitomycin C similar to normal controls. In addition, the isolation and molecular characterization of clonal populations of these FANCC-complemented cells demonstrated FANCC transposon integration into the genome by SB mediated transposition. Preliminary results using SBT vectors in K562 cells suggest that a transposase provided on a separate plasmid may increase the efficiency of transposition. As a result, studies are also underway to characterize the efficacy of the SBT-FANCC vector described above compared to a similar vector in which the transposase is supplied either by a separate plasmid or by a SB-encoding mRNA. These studies demonstrate molecular feasibility of the SB transposon system in combination with electroporation-based cell loading technology for genetic therapy of Fanconi anemia.