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Delayed Targeting of Cytokine-Nonresponsive Human Bone Marrow
CD34+ Cells With Retrovirus-Mediated Gene Transfer Enhances
Transduction Efficiency and Long-Term Expression of Transduced
Genes
Ponnazhagan Veena,
Christie M. Traycoff,
David A. Williams,
Jon McMahel,
Susan Rice,
Ken Cornetta, and
Edward F. Srour
From the Division of Hematology/Oncology and Indiana Elks Cancer
Research Center, Department of Medicine, Department of Pediatrics,
Herman B Wells Center for Pediatric Research, Howard Hughes Medical
Institute, and Department of Microbiology and Immunology, Indiana
University School of Medicine, Indianapolis, IN.
Primitive hematopoietic progenitor cells (HPCs) are potential
targets for treatment of numerous hematopoietic diseases using retroviral-mediated gene transfer (RMGT). To achieve high efficiency of
gene transfer into primitive HPCs, a delicate balance between cellular
activation and proliferation and maintenance of hematopoietic potential
must be established. We have demonstrated that a subpopulation of human
bone marrow (BM) CD34+ cells, highly enriched for primitive
HPCs, persists in culture in a mitotically quiescent state due to their
cytokine-nonresponsive (CNR) nature, a characteristic that may prevent
efficient RMGT of these cells. To evaluate and possibly circumvent
this, we designed a two-step transduction protocol using
neoR-containing vectors coupled with flow
cytometric cell sorting to isolate and examine transduction efficiency
in different fractions of cultured CD34+ cells. BM
CD34+ cells stained on day 0 (d0) with the membrane dye
PKH2 were prestimulated for 24 hours with stem cell factor (SCF),
interleukin-3 (IL-3), and IL-6, and then transduced on fibronectin with
the retroviral vector LNL6 on d1. On d5, half of the cultured cells
were transduced with the retroviral vector G1Na and sorted on d6 into
cytokine-responsive (d6 CR) cells (detected via their loss of PKH2
fluorescence relative to d0 sample) and d6 CNR cells that had not
divided since d0. The other half of the cultured cells were first
sorted on d5 into d5 CR and d5 CNR cells and then infected separately
with G1Na. Both sets of d5 and d6 CR and CNR cells were cultured in
secondary long-term cultures (LTCs) and assayed weekly for transduced
progenitor cells. Significantly higher numbers of G418-resistant
colonies were produced in cultures initiated with d5 and d6 CNR cells
compared with respective CR fractions (P < .05). At week 2, transduction efficiency was comparable between d5 and d6 transduced CR
and CNR cells (P > .05). However, at weeks 3 and 4, d5 and
d6 CNR fractions generated significantly higher numbers of
neoR progenitor cells relative to the respective CR
fractions (P < .05), while no difference in transduction
efficiency between d5 and d6 CNR cells could be demonstrated.
Polymerase chain reaction (PCR) analysis of the origin of transduced
neoR gene in clonogenic cells demonstrated that
mature progenitors (CR fractions) contained predominantly LNL6
sequences, while more primitive progenitor cells (CNR fractions) were
transduced with G1Na. These results demonstrate that prolonged
stimulation of primitive HPCs is essential for achieving efficient RMGT
into cells capable of sustaining long-term in vitro hematopoiesis. These findings may have significant implications for the development of
clinical gene therapy protocols.
Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3693-3701
© 1998 by The American Society of Hematology.
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