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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2263-2270
In Vivo Marking of Rhesus Monkey Lymphocytes by Adeno-Associated Viral
Vectors: Direct Comparison With Retroviral Vectors
Yutaka Hanazono,
Kevin E. Brown,
Atsushi Handa,
Mark E. Metzger,
Dominik Heim,
Gary J. Kurtzman,
Robert E. Donahue, and
Cynthia E. Dunbar
From the Hematology Branch, National Heart Lung and Blood Institute,
National Institutes of Health, Bethesda, MD; and Avigen Inc, Alameda,
CA.
We have compared adeno-associated virus (AAV)-based and
retrovirus-based vectors for their ability to transduce primary T lymphocytes in vitro and then tracked the persistence of these genetically marked lymphocytes in vivo, using the rhesus monkey model.
To avoid the complication of immune rejection of lymphocytes transduced
with xenogeneic genes in tracking studies primarily designed to
investigate transduction efficiency and in vivo kinetics, the vectors
were designed without expressed genes. All vectors contained
identically mutated  -galactosidase gene (-gal) and neomycin resistance gene (neo) DNA sequences separated by
different length polylinkers, allowing simple differentiation by
polymerase chain reaction (PCR). Each of 2 aliquots of peripheral blood
lymphocytes from 4 rhesus monkeys were transduced with either AAV or
retroviral vectors. The in vitro transduction efficiency (mean vector
copy number/cell) after the ex vivo culture was estimated by PCR at 0.015 to 3.0 for AAV, varying depending on the multiplicity of infection (MOI) used for transduction, and 0.13 to 0.19 for the retroviral transductions. Seven days after transduction,
Southern blot analysis of AAV-transduced lymphocytes showed
double-stranded and head-to-tail concatemer forms but failed to show
integration of the AAV vector. AAV and retroviral aliquots were
reinfused concurrently into each animal. Although the retrovirally
marked lymphocytes could be detected for much longer after infusion, AAV transduction resulted in higher short-term in vivo marking efficiency compared with retroviral vectors, suggesting possible clinical applications of AAV vectors in lymphocyte gene therapy when long-term vector persistence is not required or desired.
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