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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2271-2286
Efficient and Durable Gene Marking of Hematopoietic Progenitor
Cells in Nonhuman Primates After Nonablative Conditioning
M. Rosenzweig,
T.J. MacVittie,
D. Harper,
D. Hempel,
R.L. Glickman,
R.P. Johnson,
A.M. Farese,
N. Whiting-Theobald,
G.F. Linton,
G. Yamasaki,
C.T. Jordan, and
H.L. Malech
From New England Regional Primate Research Center, Harvard Medical
School, Southborough, MA; University of Maryland Greenebaum Cancer
Center, Baltimore, MD; Markey Cancer Center, University of Kentucky
Medical Center, Lexington, KY; Cell Genesys, Foster City, CA; and
Laboratory of Host Defenses, National Institute of Allergy and
Infectious Diseases, Bethesda, MD.
Optimization of mobilization, harvest, and transduction of
hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF)
mobilization of peripheral blood stem cells and retrovirus transduction
using hematopoietic growth factors to introduce a reporter gene, murine
CD24 (mCD24), into hematopoietic stem cells in nonhuman primates.
Rhesus macaques were treated with Flt3-L (200 µg/kg) and G-CSF (20 µg/kg) for 7 days and autologous CD34+ peripheral blood
stem cells harvested by leukapheresis. CD34+ cells were
transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence
of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321
(50 ng/mL) in serum-free medium. An important and novel feature of this
study is that enhanced in vivo engraftment of transduced stem cells was
achieved by conditioning the animals with a low-morbidity regimen of
sublethal irradiation (320 to 400 cGy) on the day of transplantation.
Engraftment was monitored sequentially in the bone marrow and blood
using both multiparameter flow cytometry and semi-quantitative DNA
polymerase chain reaction (PCR). Our data show successful and
persistent engraftment of transduced primitive progenitors capable of
giving rise to marked cells of multiple hematopoietic lineages,
including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% ± 32% (n = 4) of granulocytes
expressed mCD24 antigen at the cell surface. Peak in vivo levels of
genetically modified peripheral blood lymphocytes approached 35% ± 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks
posttransplantation. In addition, naïve (CD45RA+
and CD62L+) CD4+ and CD8+
cells were the predominant phenotype of the marked CD3+ T
cells detected at early time points. A high level of marking persisted
at between 10% and 15% of peripheral blood leukocytes for 4 months
and at lower levels past 6 months in some animals. A cytotoxic
T-lymphocyte response against mCD24 was detected in only 1 animal. This
degree of persistent long-lived, high-level gene marking of multiple
hematopoietic lineages, including naïve T cells, using a
nonablative marrow conditioning regimen represents an important step
toward the ultimate goal of high-level permanent transduced gene
expression in stem cells.
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