SELECTION OF TRANSDUCED CD34(+) PROGENITORS AND ENZYMATIC CORRECTION OF CELLS FROM GAUCHER PATIENTS, WITH BICISTRONIC VECTORS

The gene transfer efficiency of human hematopoietic stem cells is stil l inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease, We constructed a bicistronic retroviral vector containing the human glucocerebrosida se (GC) cDNA and the human small cell surface antigen CD24 (243 bp). E xpression of both cDNAs was controlled by the long terminal repeat enh ancer/promoter of the Molony murine leukemia virus, The CD24 selectabl e marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalom yocarditis virus internal ribosomal entry site. Virus-producing GP+env AM12 cells were created by multiple supernatant transductions to creat e vector producer cells, The vector LGEC has a high titer and can driv e expression of GC and the cell surface antigen CD24 simultaneously in transduced MH 3T3 cells and Gaucher skin fibroblasts, These transduce d cells have been successfully separated from untransduced cells by fl uorescence-activated cell sorting, based on cell surface expression of CD24, Transduced and sorted NIH 3T3 cells showed higher GC enzyme act ivity than the unsorted population,demonstrating coordinated expressio n of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured, The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of norma l GC enzyme activity), Efficient transduction of CD34(+) hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of whic h (100%) were vector positive, The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-g ranulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix he matopoietic colonies, demonstrating their ability to differentiate int o these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared w ith untransduced CD34(+) progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that all ow isolation of transduced hematopoietic progenitors and cells that ha ve been metabolically corrected.