17.8 Maintenance of transgene expression in host system

With evolution eukaryotic systems have developed mechanisms to maintain genome integrity and prevent foreign gene expression. Transient expression of transgenes introduced by viral or non viral vectors and episomal plasmids is in part due to de novo cytosine methylation by DNA methyltransferases that transcriptionally silent chromatin structures. Transgene expression is more favored by viral promoters as compared to human housekeeping promoters, but cytosines in viral promoters are main targets for cellular methyltransferases. Thus promoter development has been focused by mix-matching transcription factor binding sites for strong and long term expression. Also the addition of S/MAR sequence can provide position independent transcription and inhibit transgene methylation. In nonviral gene therapy the bacterial plasmid backbone used also contributes to transgene silencing. To overcome this problem phage recombination system have been used to form minicircle DNA that can be maintained as episomes in vivo and facilitate 560 fold higher transgene expression. mRNA splicing is another barrier to stable transgene expression that can be reduced by altered codon used to remove splice junctions. Moreover, immune response against accumulated foreign proteins in host circulation might restrict transgene stability which can be reduced by employing specific T cells, but further study is required in this aspect.

17.9 Future prospects

Non viral procedures of gene therapy mimic the basic viral mechanisms to achieve long term expression of the corrective gene without disturbing host gene expression and signaling pathways. The efficiency of targeted delivery is the key to clinical success of non viral gene therapy. The effectiveness and difficulties of such delivery can now be well determined in terms of quantification, magnitude and duration of reporter gene expression, using small animal imaging technologies in vivo . Safe and long term transgene expression is the ultimate requirement of human gene therapy. Today gene therapy is not only restricted in proving for a missing gene but being explored in various aspects like anti-cancer gene therapy etc. and tailoring non-viral gene delivery strategies might help in realization of the potentials of human gene therapy.