Transduction of primitive human marrow and cord blood-derived hematopoietic progenitor cells with adeno-associated virus vectors

We evaluated the capacity of adeno-associated virus (AAV) vectors to transd uce primitive human myeloid progenitor cells derived from marrow and cord b lood in long-term cultures and long term culture-initiating cell (LTC-IC) a ssays. Single-colony analyses showed that AAV vectors transduced CD34(+) an d CD34(+)38(-) clonogenic cells in long-term culture. Gene transfer was rea dily observed in LTC-ICs derived from 5-; 8-, and 10-week cultures. Recombi nant AAV (rAAV) transduction was observed in every donor analyzed, although a wide range of gene transfer frequencies (5% to 100%) was noted. AAV tran sduction of LTC-ICs was stable, with week-8 and -10 LTC-ICs showing compara ble or better transduction relative to week-5 LTC-ICs. Fluorescence in situ hybridization (FISH) analyses performed to determine the fate of AAV vecto rs in transduced cells showed that 9% to 28% of: CD34(+) and CD34(+)38(-) c ells showed stable vector integration as evidenced by chromosome-associated signals in metaphase spreads. Comparisons of interphase and metaphase FISH suggested that a fraction of cells also contained episomal vector at early time points after transduction. Despite the apparent loss of the episomal forms with continued culture, the number of metaphases containing integrate d vector genomes remained stable long term. Transgene transcription and pla cental alkaline phosphatase (PLAP) expression was observed in CD34(+), CD34 (+)38(-) LTC-ICs in the absence of selective pressure. These results sugges t that primitive myeloid progenitors are amenable to genetic modification w ith AAV vectors. (C) 1999 by The American Society of Hematology.