ABSTRACT
Abstract-Stem cells have both self-renewing and homing/differentiative capabilities and thereby provide for life-long cell replacement in tissues and organs. They are therefore the ideal targets for most gene therapy protocols, i.e. both for long-term and transient gene expression protocols where they are either the long term carriers of the therapeutic gene (inherited/degenerative disease) or the mobilized/recruted targets of a transient regenerative process such as the formation of new blood vessels. Long-term gene therapy is thus amenable to synergistic combinations where ex vivo/in vivo genetic engineering of stem cells can be associated with in vivo transient topical expression of minigenes encoding various factors such as homing, regenerative and differentiative ones. Such a strategy is still hampered by many hurdles of which random integration of therapeutic DNA is a major safety concern. Emerging technologies are however aimed at efficient site-specific integration of therapeutic transgenes and at endogenous gene repair/modification. Promising site-specific integration vectors are at the pre-clinical stage and rely on an adeno-associated virus (AAV) rep platform or on phage phiC31 integrase. However, unlike these approaches, gene targeting is driven by homologous recombination and has thus target flexibility. It mediates DNA exchanges between chromosomal DNA and transfecting/transducing DNA, thereby providing the means to modify at will the sequence of target chromosomal DNA. Gene targeting stands thus as the ultimate process both for gene repair/alteration and targeted (i.e. site-specific) transgene integration. Such a process is however highly inefficient unless target chromosomal DNA is struck by a double-strand break (DSB). In addition, it is overwhelmed by random integration. In order to increase gene targeting frequency and eliminate random integration, we devised an approach that relies on the transfer into target cells of premade presynaptic filaments, i.e. the very complexes between recombinase protein and single-stranded DNA (ssDNA) that mediate the key reaction from homologous recombination (homologous DNA pairing-strand exchange with double-stranded [ds] DNA). Upon publication of the enzymatic properties of recombinase RAD51, we invented chimeric presynaptic filaments with a dsDNA core, and shifted from gene conversion to 'true' gene targeting. In these dsDNA-cored presynaptic filaments, therapeutic heterologous sequences and repetitive elements are comprised in the recombination-locked dsDNA core, and homologous recombination is driven by both recombinationactivated ssDNA tails that are 100% identical to their respective target chromosomal sequences.
