Retroviruses are efficient in gene transfer, and hematopoietic stem cells (HSC) are an attractive target for therapeutic gene transfer, because one or very few of them can support the production of all blood cells. However, major problems have been encountered in obtaining reliable transfer and expression of tissue-specific genes; in addition, re-population of the bone marrow by rare transvected HSC may require ablative procedures that are not attractive for the therapy of inherited disorders that are not immediately life- threatening. We intend to develop new vectors that will enable us to exploit the power of housekeeping gene promoters for overcoming the problem of transcription silencing; and to adopt a system in which the expression of the transvected gene will provide self- selection of the successfully transvected cells. As a specific example we will use the X-linked glucose 6-phosphate dehydrogenase (G6PD) gene, because it offers several distinct advantages. (1) This gene has been thoroughly characterized as a prototype housekeeping gene, whose promoter is embedded in a GC island. (2) We have previously shown that the core promoter is competent for expression in a variety of cells and in transgenic mice. (3) Our previous studies of heterozygotes for severe G6PD deficiency, who are genetic mosaics as a result of X-chromosome inactivation, have shown that in the hematopoietic system there is a growth advantage for G6PD normal cells over severely G6PD deficient cells. (4) We have obtained murine G6PD-null embryonic stem cells (ESC) by targeted homologous recombination, and we already have mice that are heterozygous for G6PD deficiency. (5) In preliminary experiments we have constructed vectors in which the G6PD gene is driven either by a retroviral Long Terminal Repeat or by the G6PD promoter itself, and both are effective in the transfer and expression of enzymatically active G6PD. We intend to capitalize on the joint expertise of participants in this Program Project, particularly with respect to hematopoietic cell cultures (Project 2), in order to obtain gene transfer into HSC, and subsequently show that phenotypically corrected ESC and HSC grow preferentially in vitro and in vivo. This work may serve ultimately to provide treatment for cases. of G6PD deficiency that cause severe chronic hemolytic anemia, and also as a model for the transfer and expression, for therapeutic purposes, of other genes.
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