CD34(+++) STEM PROGENITOR CELLS PURIFIED FROM CRYOPRESERVED NORMAL CORD-BLOOD CAN BE TRANSDUCED WITH HIGH-EFFICIENCY BY A RETROVIRAL VECTORAND EXPANDED EX-VIVO WITH STABLE INTEGRATION AND EXPRESSION OF FANCONI-ANEMIA COMPLEMENTATION C-GENE/

A future possibility for treatment of genetic diseases may be gene the rapy using autologous cord blood (CB) stem/progenitor cells. This migh t require cryopreservation of CB stem/progenitor cells prior to purifi cation, gene transduction, and ex vivo expansion of cells. To address this possibility, nonadherent low density T-lymphocyte depleted (NALT( -)) cells from fresh or cryopreserved cord blood were sorted for CD34( +++) phenotype, transduced with a recombinant retroviral vector encodi ng Fanconi anemia complementation C (FACC) gene, and cells expanded ex vivo in suspension culture for 7 days with growth factors. The result s demonstrate: 1) high recovery of viable cells after thawing; 2) high efficiency purification of CD34(+++) cells from NALT(-) cells prior t o and after cryopreservation; 3) high degree of expansion of nucleated cells and immature progenitors from CD34(+++) cells before and after cryopreservation; 4) efficient transduction with stable integration an d expression of newly introduced genes in cryopreserved and then sorte d stem/progenitor cells, as detected prior to and after ex vivo expans ion; and 5) high efficiency transduction of single isolated CD34(+++) cells obtained from cryopreserved NALT(-) CB. This information should be of value for future studies evaluating the use of cryopreserved cor d blood for gene transfer/gene therapy.