Correction of a disease gene mutation by homologous recombination (HR) with a transfected DNA repair template represents an ideal form of gene therapy for many inherited hematological disorders. Therapeutic gene correction via HR in hematopoietic stem cells is unlikely to succeed, however, without major improvements in its efficiency. One promising approach is to use an engineered zinc finger endonuclease to specifically cleave the target gene and so promote HR between a dsDNA template and its chromosomal target (Urnov et al (2005) Nature 435:646-51). We are exploring an alternative approach based on the expression of Redβ, a single stranded (ss) DNA annealing protein from bacteriophage λ. In bacteria, Redβ promotes HR-mediated gene modification using templates with short regions of homology (e.g. 30–100nt). Such HR is independent of RecA-mediated host cell HR. For double stranded (ds) DNA templates, co-expression of λ exonuclease (the red α gene product) is required, but ssDNA templated modifications are particularly efficient and require only Redβ expression. Such properties could be useful for therapeutic gene correction if reproduced in human cells. Preliminary experiments in mouse embryonic stem cells suggested that Redβ can function in mammalian cells (Zhang Y. et al, (2003) BMC Mol. Biol. 4). To explore this further, we have generated a human cell line in which large amounts of Redβ can be expressed inducibly in the nucleus. In contrast to the over-expression of endogenous HR proteins Rad51 (RecA homologue) or Rad52 (ssDNA annealing protein), sustained Redβ over-expression is well tolerated, with no impairment of cell proliferation or viability. We find that Redβ does indeed promote HR in human cells. Surprisingly, dsDNA and ssDNA templated gene modification are stimulated to similar extents by Redβ. Stimulations are modest, but similar to those achieved by human Rad51 over-expression. The effects of co-expressing Red α are under investigation. We have also shown that ssDNA-templated gene correction is stimulated by transient, RNAi-mediated, depletion of the mismatch repair protein MSH2, and that such stimulation is additive with Redβ-stimulated gene correction.