USE OF SYNTHETIC OLIGONUCLEOTIDES IN BONE MARROW PURGING

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This project sought to improve the delivery of oligodeoxynucleotides (ODNs) in a clinical model. A series of investigations designed to increase the efficiency of cell uptake led to the use of electroporation (EP). We characterized EP mediated ODN uptake, and demonstrated that: transfection rates of 100% were typically attained, overall cell uptake was increased by up to l0 fold, uptake was immediate, and ODN localized primarily to the nucleus and cytoplasm (sites of ODN pharmacologic action). Increased efficacy was demonstrated by showing that suppression of c-myc protein by c-myc antisense ODNs was seen in a variety of cell lines within 90 minutes. Sequence-dependent decreases in cell viability within 24 hours were also demonstrated. Using the U937 human lymphoma cell line in a murine xenograft model, prolonged animal survival was demonstrated for mice receiving cells preloaded with c-myc antisense ODN. A thorough analysis of EP upon normal hematopoiesis demonstrated little to no effect upon normal hematopoietic activity. These tests included: CFU-GM and CFU-S colony forming assays, and competitive bone marrow repopulation assays in murine systems, and CFU-GM assays with human bone marrow cells. By performing a mixing assay in which normal human bone marrow was contaminated with U937 cells, we demonstrated that c-myc antisense, introduced into cells by EP, had no significant effect upon normal CFU-GM activity, while colony forming activity of U937 cells was decreased by 87%.