Stable transduction of recombinant adeno-associated virus into hematopoietic stem cells from normal and sickle cell...

Stable introduction of genes into human hematopoietic stem cells with self-renewing potential is a necessary requirement for gene therapy strategies. We have developed an adeno-associated virus (AAV) vector and a partial packaging cell line that produces recombinant AAV at a titer of 10(8) transducing particles per milliliter. A high-titer viral stock containing the CMV/lacZ gene was used to transfer lacZ sequences into CD34+ Lin-Thy+ hematopoietic stem cells purified from normal and homozygous sickle cell patients. After infection, the cells were cultured in two ways. In the first set of experiments, the cell were expanded 300-fold in liquid culture for 21 days and plated in methylcellulose. Burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte/macrophage (CFU-GM) were then analyzed for lacZ sequences. In the second set of experiments, infected cells were cultured for 6 weeks under conditions that maintain long-term culture-initiating cells (LTC-IC). Progenitors were plated in methylcellulose, and BFU-E were analyzed for lacZ DNA. Stable transduction of lacZ sequences was observed in 25% of the colonies in both sets of experiments. These results demonstrate for the first time that LTC-IC can be transduced stably with a recombinant AAV vector. The results suggest that AAV may be a useful vector for genetic therapy of sickle cell disease and other hematopoietic disorders.