Figure 17.2 Mechanism of integration of therapeutic gene in human genome using site specific recombinases:

Prokaryotic origin of SSRs limits their application in eukaryotic hosts. Efficient transgene expression might be improved by using exogenous nuclear localization signal. Moreover, directed evolution is another approach to improve transgenesis by prokaryotic SSRs. In this technique the SSRs are subjected to multiple rounds of random mutagenesis and screening. Flpe, a derivative of Flp recombinase from yeast was developed using directed evolution. Various high throughput methods have now been developed to improve SSR activity that includes FACS and PCR based methods.

An attractive alternative of prokaryotic SSRs is Adeno Associated Virus (AAV). AAVs are non pathogenic mammalian virus and used viral encoded Rep protein to target transgene into specific sites called AAVS1, present on chromosome 19 in humans. The rep and cap genes in wild type AAV are bounded by 145 bp inverted terminal repeats (ITR). Rep is a replication protein with auxiliary recombinase function. Of the five alternatively spliced rep products only Rep68 and Rep78 catalyze site specific recombination by forming complexes with AAV ITRs and AAVS1 locus, followed by DNA strand cleavage and non homologous recombination.