Retroviral mediated gene transfer in chronic myelogenous leukaemia

Abstract

Gene therapy for chronic myelogenous leukaemia (CML) may provide a therapeutic option for patients who are ineligible for bone marrow transplantation. To determine the feasibility of such an approach we evaluated the transduction efficiency of CML progenitor colonies from seven patients in chronic phase. Vector transduction was optimized using the CML-derived K562 cell line and applied to CML mononuclear cells. After vector exposure, optimal gene transfer was noted when CML mononuclear cell cultures contained stem cell factor, IL-3, GM-CSF and erythropoietin. The addition of IL-6 to this combination decreased transduction efficiency. Using these conditions, 20.4% +/- 2.4 (SE) of erythroid colonies (CFU-GEMM and BFU-E) and 20.2% +/- 4.7 of CFU-GM colonies were G418 resistant. This compares with a transduction efficiency of 5.9% +/- 1.1 and 6.4% +/- 1.5, respectively, for erythroid and CFU-GM colonies using marrow obtained from normal donors. Only a modest increase in gene transfer was noted when CML cells were stimulated with cytokines for the 24 h preceding vector exposure. Vector DNA in colonies expressing the BCR/ABL transcript was documented by performing PCR analysis on individual colonies. The relatively high gene transfer rate in CML suggests that this disease might be very suitable for gene therapy.

Assessment of Transduction Rates of Porcine Bone Marrow

“…2 and Table 1). These values appear to be more compatible to those reported from studies on whole BMC from humans (8,9,27) and other large animals (4,7,28).…”

“…G 418 r COLONY-FORMING UNIT (CFU) ASSAYS have been widely used in different human or large animal retrovirus gene transfer models to assess in vitro transduction efficiencies and to document the in vivo long-term expression of the proviral Neo r gene (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Discrepancies between the reading of these CFU assays and the effective presence of the Neo r gene in the targeted cells, as measured by PCR, have been previously reported (5,10,12).…”

Chronic myelogenous leukaemia CD34+ cells exit G0/G1 phases of cell cycle more rapidly than normal marrow CD34+ cells

“…Our laboratory group has been interested in studying the feasibility of gene therapy in CML, and has identified several factors that appear to suggest that CML may be well suited to gene therapy approaches to treatment. Transduction efficiencies, which we have shown to be inherently increased in CML progenitors compared to normals (Cornetta et al, 1994), were shown to be further increased through the use of the 30/35 kD chymotryptic fragment of fibronectin during retroviral-mediated gene transfer (RMGT) . Since cell division is essential for successful RMGT (Nolta & Kohn, 1990;Miller et al, 1990), it is likely that cells which respond rapidly to in vitro cytokine stimulation would possess an enhanced gene transfer rate compared to quiescent cells, providing a possible mechanism behind the ease of gene transfer in CML HPC.…”

“…The chimaeric state of CML marrow allows for potential treatment modalities, such as gene therapy, aimed at the exploitation of normal BCR/ABL(¹) haemopoiesis and/or the selective repression of leukaemic BCR/ABL(þ) haemopoiesis for CML patients ineligible for the only known cure for CML, namely allogeneic BM transplantation (Goldman & Lu, 1982;McGlave, 1990). We have investigated the feasibility of gene therapy in CML marrow cells (Cornetta et al, 1994;Traycoff et al, 1997;Veena et al, 1997;Braun et al, 1997) and have documented a three-fold higher transduction efficiency of unfractionated, unstimulated CML progenitors when compared to normal BM progenitor cells (Cornetta et al, 1994). These data suggest a possible inherent ability of CML cells to proliferate and enter active phases of cell cycle in vitro, a phenomenon essential for successful vector integration and retroviral-mediated gene transfer (RMGT) of target cells (Nolta & Kohn, 1990).…”

Antisense therapeutics in chronic myeloid leukaemia: the promise, the progress and the problems

“…61 ODN have also been introduced into cells by retroviral strategies. [62][63][64][65] Our group has demonstrated that following the introduction …”