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Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 100-108
Adenoviral vector-mediated gene transfer to primitive human
hematopoietic progenitor cells: assessment of transduction and toxicity
in long-term culture
Karen L. MacKenzie,
Neil R. Hackett,
Ronald G. Crystal, and
Malcolm A. S. Moore
From the James Ewing Laboratory of Developmental Hematopoiesis,
Memorial Sloan-Kettering Cancer Center, New York, NY, and the Division
of Pulmonary and Critical Care Medicine, Weill Medical College of
Cornell University-New York Presbyterian Hospital, New York, NY.
Adenoviral gene transfer to primitive hematopoietic progenitor cells
(HPCs) would be useful in gene therapy applications where transient,
high-level transgene expression is required. In the present
investigations, we have used an adenoviral vector expressing the green
fluorescent protein (AdGFP) to quantify transduction of primitive HPCs
and assess adenoviral-associated toxicity in long-term culture. Here we
show that a cytokine cocktail protects mass populations of
CD34+ cells and primary colony forming unit-cultures
(CFU-Cs) from toxicity, enabling transduction
of up to 79% of CD34+ cells. Transduction of CFU-Cs and
more primitive HPCs was quantified following fluorescence activated
cell sorting for green flourescence protein expression. Our results
demonstrate transduction of 45% of primary CFU-Cs, 33% of week-5
cobblestone area forming cells (CAFCs), and 18% of week-5 CFU-Cs.
However, AdGFP infection inhibited proliferation of more primitive
cells. Although there was no apparent quantitative change in week-5
CAFCs, the clonogenic capacity of week-5 AdGFP-infected cells was
reduced by 40% (P < .01) when compared with mock-infected
cells. Adenoviral toxicity specifically affected the transduced
subset of primitive HPCs. Transduction of primitive cells is therefore
probably underestimated by week-5 CFU-Cs and more accurately indicated
by week-5 CAFCs. These studies provide the first functional and
quantitative evidence of adenoviral transduction of primitive HPCs.
However, further investigations will be necessary to overcome
adenoviral toxicity toward primitive HPCs before adenoviral vectors can
be considered a safe option for gene therapy.
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