PRECLINICAL STUDIES OF STREPTOLYSIN-O IN ENHANCING ANTISENSE OLIGONUCLEOTIDE UPTAKE IN HARVESTS FROM CHRONIC MYELOID-LEUKEMIA PATIENTS

Antisense oligodeoxyribonucleotides (ODN) have been shown to produce a sequence-specific cleavage of BCR-ABL mRNA. They may therefore have c linical potential for purging harvests from chronic myeloid leukaemia (CML) patients, prior to autografting. Whilst ODN are highly effective in cell-free systems, their uptake into intact cells is very poor. We have previously reported that reversible permeabilisation of CML cell lines with Streptolysin-O (SL-O) can dramatically increase intracytop lasmic and nuclear ODN uptake. In this study, we examined whether SL-O permeabilisation could be used to enhance ODN uptake into bone marrow (BM) and peripheral blood stem cell (PBSC) harvests from CML patients , without undue toxicity. All 19 harvests studied were from patients i n stable chronic phase of CML. Samples studied were either fresh BM ha rvests following leucoconcentration, fresh PBSC: collections, or from previously cryopreserved harvests. Cells were permeabilised by SL-O to load them with fluorescein-labelled ODN. The proportion of permeabili sed and viable cells was assessed by fluorescein uptake and propidium iodide exclusion, respectively, by flow cytometry. The effect of SL-O on ODN uptake and cell toxicity was unpredictable on simple mononuclea r fractions of harvests. In contrast, SL-O consistently significantly enhanced ODN uptake in samples which were first selected for CD34-posi tive cells, and this was achieved without either direct toxicity or in hibition of CFU-GM growth. The SL-O concentration required for optimal permeabilisation varied considerably from case to case, in line with previous data on cell lines. PBSC harvests positively selected for CD3 4-positive cells tended to achieve superior permeabilisation to CD34 p ositively selected BM harvests. SL-O can be used to safely enhance the intracellular uptake of antisense ODN. This is best achieved on harve sts which are first selected for CD34-positive cells.