17.5 Safe integration of corrective genes to human genome

The problem of insertional mutagenesis in gene therapy can be avoided by directing transgene into safe genomic locations that are not associated with cell proliferation or tumor suppression. Site specific recombination used by prokaryotic viruses may be helpful in this regard. Prokaryotic viruses express site specific recombinases (SSRs) that recognize unique genomic sequences called recombination sites within pathogen and host genome and mediate recombination at those sites. SSRs recognize defined but degenerate sequences and thus can be manipulated for integration into eukaryotic genome. Thus combination of SSR and non viral vectors can be useful in making safe integrations resulting in permanent expression of the therapeutic gene. For proper integration SSR must recognize specific recombination sites, for example loxP in case of bacteriophage Cre recombinase. Thus for use in human gene therapy these SSRs must be able to recognize endogenous sites in human genome called pseudosites that resemble phage recombination sites. Mammalian genomes contain several pseudo sites, e.g, bacteriophage Cre recombinase is capable of integrating between phage loxP site and pseudo loxP site in human genome albeit with 4 fold lower efficiency.

Figure 17.1 Site specific recombination by Cre recombinase:

SSRs from phages (Cre ) and from yeast (Flp ) catalyses both excision and integration in equilibrium and because the excision reaction is favored this results in inefficient integration and subsequent expression. Other SSR like phage γ integrase create hybrid recombination sites and require separate excisionase for its function. Some phage SSR like those from λ, HXO22 require cofactors like IHF for recombination whereas other SSR like those from phage R4, TP901 and ΦC31 donot require cofactors and are capable of stable integration.

ΦC31 SSR is the most efficient non mammalian SSR for human gene therapy. This recombinase recognizes pseudosite psA in human genome and mspA and mspL1 in mice and is capable of treating recessive genetic disorders. ΦC31 is capable of integrating large gene like alpha typeVII collagen (COL7A1) and dystropin gene to be used in the treatment of RDEB and Duchenne muscular dystrophy .