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High-Efficiency Gene Transfer Into Ex Vivo Expanded Human
Hematopoietic Progenitors and Precursor Cells by Adenovirus Vectors
Beat M. Frey,
Neil R. Hackett,
Jeffrey M. Bergelson,
Robert Finberg,
Ronald G. Crystal,
Malcolm A.S. Moore, and
Shahin Rafii
From the Division of Hematology-Oncology, Cornell University Medical
College, New York; James Ewing Laboratory of Developmental
Hematopoiesis, Memorial Sloan-Kettering Cancer Center, New York;
Division of Pulmonary and Critical Care Medicine, The New York
Hospital-Cornell Medical Center, New York, NY; Dana-Farber Cancer
Institute, Laboratory of Infectious Diseases, Harvard Medical School,
Boston, MA; and Children's Hospital, Philadelphia, PA.
Replication-deficient adenoviral vectors (AdVec), which infect
cycling and noncycling cells with high efficiency, low toxicity, and
ease of delivery, provide ideal vehicles to study the expression of
regulatory genes controlling different stages of hematopoiesis. To
examine the infection efficiency of AdVec in hematopoietic precursor
and progenitor cells, we used a replication-deficient adenovector
expressing the humanized form of the cDNA for green fluorescent protein
(AdGFP), permitting assessment of infection efficiency and kinetics of
transgene expression in viable hematopoietic cells using flow cytometry
and fluorescence microscopy. Flow-cytometric analysis of ex vivo
expanded hematopoietic precursor cells infected with a multiplicity of
infection (MOI) of 100 of AdGFP show that 78% of megakaryocytic
(CD41a+ and CD42b+) cells, 82% of
dendritic (CD1a+) cells, 41% of RBC precursors
(glycophorin A+), and 32% of monocytic
(CD14+) cells expressed GFP. Nineteen percent ± 1%
of freshly isolated CD34+ cells from peripheral blood
leukapheresis products infected under the same conditions expressed
GFP. Morphologic evaluation of ex vivo expanded, AdGFP-infected
CD34+ cells showed normal maturation. The functional
capacity of AdGFP-infected CD34+ cells was analyzed by
quantifying clonogeneic efficiency and proliferative capacity.
Infection of CD34+ progenitor cells with MOIs of 1 to 100 did not impair clonogeneic efficiency of CD34+ cells.
However, MOI greater than 100 resulted in a significant inhibition of
colony-forming unit-granulocyte/granulocyte-macrophage (CFU-G/GM)
formation. In sequential dilution expansion over 3 weeks (Delta assay),
the cytokine-driven proliferative potential of CD34+
cells was not impaired following exposure to AdGFP at MOIs of 1 to
1,000. The GFP+ population expanded 10- to 15-fold at
high MOIs (500 to 1,000), indicating multiple copies of the transgene
in the initially infected CD34+ cells, which were
expressed in subsequent progenies. These data show that AdVec deliver
transgenes with high efficiency and low toxicity to hematopoietic
progenitor and precursor cells. Introduction of marker genes such as
GFP into hematopoietic cells by AdVec will provide a valuable system
for study of development, homing, and trafficking of hematopoietic
precursor and progenitor cells in vitro and in vivo. Furthermore, these
results provide insights into the design of gene therapy strategies for
treatment of hematologic disorders by AdVec.
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2781-2792
© 1998 by The American Society of Hematology.
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