Gene correction in hematopoietic progenitor cells by homologous recombination

Abstract

Homologous recombination (gene targeting) has many desirable features for gene therapy, because it can precisely correct mutant genes and restore their normal expression, and random nonhomologous integration of DNA is infrequent in cells in which homologous recombination has occurred. There are, however, no reports of attempts to use homologous recombination to correct mutant 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 recombination can occur at a usable frequency in these cells. The experiments reported here encourage optimism in this respect by demonstrating targeted correction of a defective hypoxanthine phosphoribosyltransferase gene in hematopoietic progenitor cells that can form colonies in methylcellulose culture. These clonogenic cells are in the same lineage as HSCs but are more abundant and more mature and so less pluripotent. T he ideal form of gene therapy would correct a mutant gene directly without causing changes elsewhere in the genome (1). Many of the problems associated with gene therapy would thereby be greatly reduced or eliminated, including lack of adequate expression, extinction of expression, and the mutagenesis associated with integrating the correcting sequences into random sites in the genome. Homologous recombination has the necessary prerequisites for use in this context, because it is capable of precisely correcting mutant genes (2), and random nonhomologous integration of targeting DNA into the genome is infrequent in cells in which homologous recombination has occurred (3). However, we find no reported attempts of using homologous recombination to correct mutant genes in normal hematopoietic stem cells (HSCs; ref. 4), which are prime cells for therapy of a variety of hematological and other conditions (5). The likely reasons appear to be the low abundance of these cells combined with uncertainty that homologous recombination can occur in them at a usable frequency. The experiments we report here were designed to test the possibility of using homologous recombination to correct a mutant gene in hematopoietic progenitor cells that can form colonies in tissue culture. These colony-forming cells (CFCs) are in the same lineage as HSCs but are more abundant and more mature, and therefore less pluripotent. We demonstrate that the hypoxanthine phosphoribosyltransferase (HPRT) gene, which is mutated in humans with Lesch-Nyhan disease (6, 7), can be corrected by homologous recombination in CFCs at frequencies equivalent to those seen in embryonic stem (ES) cells, encouraging optimism that homologous recombination to correct mutant genes in pluripotent stem cells capable of long-term hematopoietic repopulation eventually will prove feasible. . The mixtures were plated in standard methylcellulose medium (MethoCult GF M3434; StemCell Technologies, Vancouver) containing the following recombinant cytokines: mouse stem cell factor (50...

Lack of neighborhood effects from a transcriptionally active phosphoglycerate kinase–neo cassette located between the murine β-major and β-minor globin genes

“…The observation reported here, however, suggests that it may be possible to position a selectable marker cassette near the human ␤-globin gene in a manner that does not disrupt its expression; direct experiments to test this hypothesis using ES cells containing a copy of the human ␤-globin gene cluster should now be performed. The recent observation by Hatada et al 42 that hematopoietic progenitor cells have the machinery required to perform homologous recombination further supports this approach. However, significant obstacles remain.…”

Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells

“…However, although site-specific homologous recombination has been achieved by using hematopoietic progenitor cells in one study (23), it has not been possible to achieve this goal using tissue-specific stem cells. The present study demonstrates that it is possible to apply the concept and techniques of ES cell manipulation to spermatogonial stem cells (24).…”

“…There has been uncertainty that homologous recombination can occur at a usable frequency in tissue-specific stem cells (23). Several studies have indicated a low mutation frequency in spermatogonial stem cells.…”

Theoretical mechanisms in targeted and random integration of transgene DNA

“…Hatada et al [17] used gene targeting to correct a defective hypoxanthine phosphoribosyltransferase (HPRT) gene in hematopoietic progenitor cells. The approach was similar to gene targeting with ES cells or NT, in that selection was used to enrich for targeted outcomes.…”