HIGH-LEVEL GENE-TRANSFER TO CORD-BLOOD PROGENITORS USING GIBBON APE LEUKEMIA-VIRUS PSEUDOTYPE RETROVIRAL VECTORS AND AN IMPROVED CLINICALLYAPPLICABLE PROTOCOL

The best methods for transducing hematopoietic progenitor cells usuall y involve either direct co-cultivation with virus-producing cells or h uman stromal supportive cells. However, these methods cannot be safely or easily applied to clinical use, Therefore, we aimed at improving r etrovirus-mediated gene transfer into hematopoietic progenitors derive d from cord blood CD34(+) cells using viral supernatant to levels achi eved at It:ast with direct co-cultivation and under conditions that ar e suitable for clinical applications, In a first set of experiments, C D34(+) cells were infected with supernatant containing amphotropic ret roviral particles carrying the nls-lacZ reporter gene and the effects of centrifugation, cell adhesion to fibronectin, and Polybrene on the transduction of both clonogenic progenitors (CFC) and long-term cultur e initiating cells (LTC-IC) were studied, Transduction efficiency was evaluated on the percentage and total number of progenitors expressing the P-galactosidase activity, Results show that a 48-hr infection of CD34(+) cells with viral supernatant combining centrifugation at 1000 x g for 3 hr followed by adhesion to fibronectin allows transduction l evels for both CFC and LTC-IC to be reached that are as good as using direct co-cultivation, In a second set of experiments, CD34(+) cells w ere infected using this optimized protocol with pseudotyped retroviral particles carrying the gibbon ape leukemia virus (GALV) envelope prot ein, Under these conditions, between 50 and 100% of CFC and LTC-IC wer e transduced. Thus, we have developed a protocol capable of highly tra nsducing cord blood progenitors under conditions suitable for a therap eutical use.