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Blood, 1 October 2000, Vol. 96, No. 7, pp. 2432-2439
GENE THERAPY
Efficient retrovirus-mediated gene transfer to transplantable
human bone marrow cells in the absence of fibronectin
Burkhard Hennemann,
Il-Hoan Oh,
Jean Y. Chuo,
Christian P. Kalberer,
Patricia
D. Schley,
Stefan Rose-John,
R. Keith Humphries, and
Connie J. Eaves
From the Terry Fox Laboratory, British Columbia Cancer
Agency and the Departments of Medical Genetics and Medicine, University
of British Columbia, Vancouver, BC, Canada; Medizinische Klinik,
Abteilung Pathophysiologie, Johannes Gutenberg Universität,
Mainz, Germany.
The low frequency of transplantable hematopoietic stem cells in
adult human bone marrow (BM) and other differences from cord blood stem
cells have impeded studies to optimize the retroviral transduction of
stem cells from adult sources. To address this problem, first a
cytokine combination was defined that would both maximize the kinetics
of adult BM CD34+CD38 cell mitogenesis and
minimize the period of prestimulation required for the transduction of
these cells by a MSCV-GFP/neor virus in tissue culture
dishes in the absence of fibronectin. Three days of stimulation with
flt3-ligand, Steel factor, interleukin (IL)-3, and hyper-IL-6 proved
both necessary and sufficient to obtain 83% ± 2%
GFP+ CD34+CD38 cells,
75% ± 10% G418-resistant clonogenic progenitors, and
50% ± 20% transduced long-term culture-initiating cells as
recovered 48 hours after a single exposure to virus. Moreover, this was accompanied by a several-fold increase in viral receptor (pit-1) messenger RNA transcripts in the target cells. Using this
prestimulation protocol, repeated daily exposure to new virus (3×) did
not alter the proportion of transduced cells over that obtained with a
single exposure. Adult human BM cells able to engraft immunodeficient (NOD/SCID- 2M/) mice were also
efficiently transduced (10%-20% GFP+ human lymphoid and
myeloid cells present 6-8 weeks after transplant) using a 6-day
prestimulation and infection protocol. A clinically useful efficiency
of retrovirus-mediated gene transfer to transplantable adult human BM
stem cells can thus be obtained with a protocol that allows their
semisynchronous activation into cycle and concomitant increased
expression of virus receptor transcripts before virus exposure.
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