Sleeping Beauty-mediated correction of Fanconi anemia type C

Background: The Sleeping Beauty (SB) transposon system can insert defined sequences into chromosomes to direct the extended expression of therapeutic genes. Our goal is to develop the SB system for nonviral complementation of Fanconi anemia (FA), a rare autosomal recessive disorder accompanied by progressive bone marrow failure. Methods: We used a CytoPulse electroporation system (CytoPulse, Glen Burnie, MD, USA) to introduce SB transposons into human lymphoblastoid cells (LCL) derived from both Fanconi anemia type C (FA-C) defective and normal patients. Correction of the FA-C defect was assessed by resistance to mitomycin C, a DNA-crosslinking agent. Results: Culture of both cell types with the antioxidant N-acetyl- l-cysteine improved cell viability after electroporation. Co-delivery of enhanced green fluorescent protein (GFP) transposon with SB100X transposase-encoding plasmid supported a 50- to 90-fold increase in stable GFP expression compared to that observed in the absence of SB100X for normal LCL, but in FA-C defective LCL SB100X enhancement of stable GFP-expression was a more moderate five- to 13-fold. SB-mediated integration and expression of the FA-C gene was demonstrated by the emergence of a mitomycin C-resistant population bearing characteristic transposon-chromosome junction sequences and exhibiting a mitomycin dose response identical to that of normal LCL. Conclusions: The SB transposon system achieved stable expression of therapeutic FA-C genes, complementing the genetic defect in patient-derived cells by nonviral gene transfer.