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A bicistronic therapeutic retroviral vector enables sorting of transduced
CD34+ cells and corrects the enzyme deficiency in cells from Gaucher
patients
JA Medin, M Migita, R Pawliuk, S Jacobson, M Amiri, S Kluepfel-Stahl, RO Brady, RK Humphries and S Karlsson
Developmental and Metabolic Neurology Branch, National Institute of
Neurological Disease and Stroke, National Institutes of Health, Bethesda,
MD, USA.
Corrective gene transfer for therapeutic intervention in metabolic and
hematopoietic disorders has been hampered by the relatively inefficient
transduction of human hematopoietic stem cells. To overcome this, a
bicistronic recombinant retrovirus has been generated that delivers both a
therapeutic glucocerebrosidase (GC) cDNA for the treatment of Gaucher
disease, and a small murine cell surface antigen (heat-stable antigen
[HSA]) as a selectable marker. An amphotropic retroviral- producing cell
clone was created, and filtered supernatant was used to transduce NIH 3T3
cells. Sorting of transduced cells by flow cytometry enabled separation
into populations based on cell surface fluorescence intensity derived from
the expressed HSA. Significant increases in GC enzyme activity were seen
for the transduced and especially the transduced and sorted cells.
Similarly, increases in GC specific activity were seen in transduced and
sorted skin fibroblasts from a patient with Gaucher disease. To streamline
future transfer and sorting protocols for hematopoietic cells, transformed
B-cell lines from Gaucher patients were created. Type I B cells were
transduced and sorted, and large increases in GC specific activity occurred
with concomitant increases in integrated retroviral copy numbers. In
addition, toward the goal of using this selectable approach for corrective
gene transfer to bone marrow stem cells, CD34+ cells were isolated from
normal BM donors, transduced, and sorted based on cell surface expression
of HSA. Proviral DNA was detected in approximately 40% of clonogenic
progenitor colonies derived from unsorted, transduced CD34+ cells,
demonstrating the high titer of the vector. However, after sorting, 100% of
the colonies had the corrective GC cDNA, demonstrating the efficiency of
this selective system for human hematopoietic progenitors. It is expected
that strategies based on this approach will allow sorting of transduced
cells of many types before implantation of transduced cells to animals or
patients. This vector system may also be used to simplify manipulations and
studies on retroviral-mediated gene delivery in vitro and for in vivo
models.
Volume 87,
Issue 5,
pp. 1754-1762,
03/01/1996
Copyright © 1996 by The American Society of Hematology
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