17.6 Extra chromosomal replicating units in transgenesis

DNA molecules that can be replicated and segregated to daughter cells in an autonomous, extrachromosomal form avoiding insertional mutagenesis can be developed as another method of transgenesis in gene therapy. DNA viruses such as SV40 and Epstein–Barr virus (EBV) normally replicate episomally in mammalian cells and transfer viral episomes to daughter cells during cell division. Episomal plasmid vectors with viral ori sequence and requisite viral proteins (like Tag and EPVNA1) have been generated that can sustain the expression of a therapeutic transgene. For example, EBV-based episomal plasmid with 115 kb hypoxanthine phosphoribosyl transferase ( HPRT ) locus that was stably expressed in HPRT -deficient human lung fibroblast cells. Unfortunately, use of such episomes in human gene therapy is restricted as viral proteins such as Tag, alter the function of other key host cell proteins interfering with the retinoblastoma and p53 tumour-suppressor pathways.

To overcome this problem ‘pEPI' plasmid containing an SV40-ori sequence and scaffold/matrix attachment region (S/MAR) from the human β-interferon gene cluster has been developed that can propagate episomally, for several hundred divisions in CHO cells and HeLa human cervical cancer cells. Mitotic stability in pEPI results from the interaction of the S/MAR element with components of the nuclear matrix which is crucial for the organization of ‘boundary elements' to protect their coding regions and to allow transcription factors to access enhancer and/or promoter sequences. Interaction of pEPI is mediated by binding of S/MAR to scaffold attachment factor A proteins, allows co-segregation of pPEPI with mitotic chromosomes and brings it closer to host replication machinery facilitating its own replication along with the host cell cycle. Transcription and replication are linked in pPEPI. Transcription of the AT rich S/MAR sequence is important in opening of chromatin structure to allow replication of pEPI and thus replication of this plasmid is not solely dependent on the SV ori. This property of chromosomal components like S/MAR can thus be exploited to develop novel episomally replicating plasmids for sustained expression of therapeutic genes in gene therapy.