Gene correction in hematopoietic progenitor cells by homologous recombination

Homologous recombination (gene targeting) has many desirable features for g ene therapy, because it can precisely correct mutant genes and restore thei r normal expression, and random nonhomologous integration of DNA is infrequ ent in cells in which homologous recombination has occurred. There are, how ever, no reports of attempts to use homologous recombination to correct mut ant genes in normal hematopoietic stem cells (HSCs), which are prime cells for therapy of a variety of hematological and other conditions, presumably because of their low abundance and uncertainty that homologous recombinatio n can occur at a usable frequency in these cells. The experiments reported here encourage optimism in this respect by demonstrating targeted correctio n of a defective hypoxanthine phosphoribosyltransferase gene in hematopoiet ic progenitor cells that can form colonies in methylcellulose culture. Thes e clonogenic cells are in the same lineage as HSCs but are more abundant an d more mature and so less pluripotent. Corrected colonies were identified b y their survival in selective medium after electroporation of correcting DN A into unfractionated mouse bone marrow cells and were confirmed by reverse transcription-PCR and sequencing. The observed frequency (4.4 +/- 3.3 x 10 (-5) per treated clonogenic cell) is the same as in embryonic stem cells (2 .3 +/- 0.4 x 10(-5)) with the same DNA and mutation. These data suggest tha t gene targeting to correct mutant genes eventually will prove feasible in HSCs capable of long-term bone marrow reconstitution.