To
solve the problem of uncontrolled therapeutic gene integration,
which is a critical drawback of retroviral vectors for gene therapy,
the integration sites of exogenous genes should be precisely controlled
not to perturb endogenous gene expression. To accomplish this, we
explored the possibility of site-specific integration using two six-finger
artificial zinc-finger proteins (AZPs) tandemly conjugated via a flexible
peptide linker (designated “Tandem AZP”). A Tandem AZP
in which two AZPs recognize specific 19 bp targets in a donor and
acceptor DNA was expected to site-specifically recruit the donor DNA
to the acceptor DNA. Thereafter, an exogenously added integrase was
expected to integrate the donor DNA into a specific site in the acceptor
DNA (as it might be in the human genome). We demonstrated in vitro
that in the presence of Tandem AZP, ΦC31 integrase selectively
integrated a donor plasmid into a target acceptor plasmid not only
at 30 °C (the optimum temperature of the integrase) but also
at 37 °C (for future application in humans). We expect that with
further improvement of our current system, a combination of Tandem
AZP with integrase/recombinase will enable site-specific integration
in mammalian cells and provide safer gene therapy technology.
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