Stable integration of large (> 100 kb) PAC constructs in HaCaT keratinocytes using an integrin-targeting peptide delivery system

Transfer of large DNA constructs in gene therapy studies is being recognise d for its importance in maintaining the natural genomic environment of the gene of interest and providing tissue-specific regulation and control. Howe ver, methods used to deliver such constructs have been poorly studied. We u sed a receptor-mediated, integrin-targeting transfection system enhanced by liposomes, to deliver a 110 kb PAC (P1-based artificial chromosome) to HaC aT keratinocytes. The PAC contained the collagen VII locus, an EGFP (enhanc ed green fluorescent protein) reporter gene and the puromycin resistance ge ne (pac) to allow selection of stably transfected cells. Analysis of puromy cin resistant and EGFP-expressing colonies by Western blot showed that coll agen VII production increased dramatically after transfection, indicating s uccessful transfer of a large fully functional genomic locus. Fluorescent i n situ hybridisation (FISH) and Southern blot analysis revealed that the PA C had integrated as at least one copy per cell. EGFP expression has persist ed for 35 weeks, suggesting stable transgene expression. We conclude that t he integrin-targeting peptide method of gene delivery is an effective means of stably delivering large DNA constructs to human keratinocytes and could be of benefit for genomic gene therapy approaches.