title: Preclinical studies of streptolysin-O in enhancing antisense oligonucleotide uptake in harvests from chronic myeloid leukaemia patients.
abstract: Antisense oligodeoxyribonucleotides (ODN) have been shown to produce a sequence-specific cleavage of BCR-ABL mRNA. They may therefore have clinical 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 intracytoplasmic 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 in stable chronic phase of CML. Samples studied were either fresh BM harvests 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 permeabilised 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 mononuclear fractions of harvests. In contrast, SL-O consistently significantly enhanced ODN uptake in samples which were first selected for CD34-positive cells, and this was achieved without either direct toxicity or inhibition 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 CD34-positive cells tended to achieve superior permeabilisation to CD34 positively selected BM harvests. SL-O can be used to safely enhance the intracellular uptake of antisense ODN. This is best achieved on harvests which are first selected for CD34-positive cells.
authors.affiliation: University Department of Haematology, Royal Liverpool University Hospital, UK.
authors.forename: C M
authors.initials: CM
authors.lastname: Broughton
authors.forename: D G
authors.initials: DG
authors.lastname: Spiller
authors.forename: N
authors.initials: N
authors.lastname: Pender
authors.forename: M
authors.initials: M
authors.lastname: Komorovskaya
authors.forename: J
authors.initials: J
authors.lastname: Grzybowski
authors.forename: R V
authors.initials: RV
authors.lastname: Giles
authors.forename: D M
authors.initials: DM
authors.lastname: Tidd
authors.forename: R E
authors.initials: RE
authors.lastname: Clark
mesh_terms: D000328:Adult; D018952:Antigens, CD34 / Q000032:analysis; D001426:Bacterial Proteins; D002463:Cell Membrane Permeability / Q000187:drug effects; D002469:Cell Separation; D003114:Colony-Forming Units Assay; D005260:Female; D016044:Fusion Proteins, bcr-abl / Q000235:genetics; D006410:Hematopoiesis; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000628:therapy*; D008297:Male; D008875:Middle Aged; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage*; D013301:Streptolysins / Q000627:therapeutic use*
chemical_list: D018952:Antigens, CD34; D001426:Bacterial Proteins; D016376:Oligonucleotides, Antisense; D013301:Streptolysins; C025964:streptolysin O; D016044:Fusion Proteins, bcr-abl