Co-infection of HSV-1 amplicons containing the gene and a human artificial chromosome vector into primary XPC deficient fibroblast cells.

Gene therapy for xeroderma pigmentosum (XP), a rare, recessive DNA repair disease, has been considered since defects in XP genes result in severe and debilitating symptoms. Mutations in the DNA repair gene result in a more that 1000-fold increased sensitivity to sunlight-induced skin cancer. The gene is large (33 Kb) and the entire genomic locus is a difficult candidate for many gene therapy vectors to incorporate into their system by conventional cloning. Artificial chromosome vectors were developed to accommodate large genes and their regulatory sequences to allow full gene expression in cells. The HSV-1 human artificial chromosome (HAC) vectors we previously generated incorporated genes up to 100 Kb in a single vector. Subsequently, we modified the system to allow larger (>100 Kb) DNA gene sequences to be introduced by simultaneously infecting cells with two separate HSV-1 vector particles, one containing DNA required for HAC formation and the other with the desired gene. Following transduction, recombination of DNA formed a gene expressing HAC . The dual transduction system was successful for introduction and expression of the HPRT gene in human 3D engineered tissues and stem cells. In this study, we report the gene delivery and transient gene expression via the dual transduction system in human cultured fibrosarcoma (HT1080) and primary XPC deficient patient cells.