GENE-TRANSFER INTO HEMATOPOIETIC STEM-CELLS - LONG-TERM MAINTENANCE OF IN-VITRO ACTIVATED PROGENITORS WITHOUT MARROW ABLATION

Adoptive transfer of genetically modified somatic cells will play an i ncreasingly important role in the management of a wide spectrum of hum an diseases. Among the most appealing somatic cells as potential gene transfer vehicles are hematopoietic cells, because of their wide distr ibution and their well-characterized capacities for proliferation, dif ferentiation, and self-renewal. Genes can be readily transferred into short-lived and lineage-restricted hematopoietic cells, but there rema ins a need to develop reliable methods for gene transfer into hematopo ietic stem cells in large animals. In this work, we used a gene transf er approach in which hematopoietic cells in long-term marrow cultures were exposed to the replication-defective retrovirus N2, bearing the r eporter gene neo, on multiple occasions during 21 days of culture. Gen etically marked cultured autologous cells were infused into 18 canine recipients in the absence of marrow-ablative conditioning. neo was det ected by Southern blotting and/or the polymerase chain reaction in the marrow, blood, marrow-derived granulocyte/macrophage and erythroid pr ogenitors, and cultured T cells in dogs after infusion. In most dogs, the proportion of long-term marrow culture cells contributing to hemat opoiesis rose during the first 3 months after infusion and peaked with in the first 6. The maximal levels attained were between 10% and 30% G 418-resistant (neo-positive) granulocyte/macrophage progenitors. At 12 months, five dogs maintained greater than 10% G418-resistant progenit ors, and for two of them this level exceeded 20%. Two dogs had greater than 5% G418-resistant hematopoietic progenitors at 24 months after i nfusion. Our data suggest that very primitive hematopoietic progenitor s are maintained in long-term marrow cultures, where they can be trigg ered into entering the cell cycle. In vivo, these activated cells like ly continue normal programs of proliferation, differentiation, and sel f-renewal. Their progeny can be maintained at clinically relevant leve ls for up to 2 years without the requirement that endogenous hematopoi esis be suppressed through chemo- or radiotherapy prior to adoptive tr ansfer. Long-term marrow culture cells may thus be ideal targets for g ene therapy involving adoptive transfer of transduced hematopoietic ce lls.