Effects of busulfan dose escalation on engraftment of infant rhesus monkey hematopoietic stem cells after gene marking by a lentiviral vector.
OBJECTIVE
Non-myeloablative cytoreduction is used in clinical hematopoietic stem cell gene therapy trials to increase engraftment of gene-modified cells. We utilized an infant rhesus monkey model to identify an optimal dosage of busulfan that results in efficient long-term gene marking with minimal toxicities.


METHODS
Bone marrow (BM) was harvested, followed by a single 2-hour intravenous infusion of busulfan at escalating dosages of 0 to 160 mg/m(2). CD34(+) cells were immunoselected from BM, transduced overnight with a simian immunodeficiency virus-based lentiviral vector carrying a non-expressed marker gene, and injected intravenously 48 hours post-busulfan administration. Pharmacokinetics were assessed, as well as adverse effects and peripheral blood and BM gene marking.


RESULTS
Increasing dosages of busulfan resulted in increased area-under-the-curve (AUC) with some variability at each dosage level, suggesting interindividual variation in clearance. Blood chemistries were normal and no adverse effects were observed as a result of busulfan infusion. At 120 and 160 mg/m(2), transient neutropenia and thrombocytopenia were noted but not lymphopenia. Over the 6 months of study posttransplantation, a busulfan dosage-related increase in gene marking was observed ranging from undetectable (no busulfan) up to 0.1% gene-containing cells in animals achieving the highest busulfan AUC. This corresponds to a more than 100-fold increase in gene marking over the busulfan dosage range studied.


CONCLUSIONS
These data indicate that increased gene marking of hematopoietic stem cells can be achieved by escalating busulfan dosages from 40 to 160 mg/m(2) without significant toxicity in infant nonhuman primates.
Kahl
Christoph A
CA
Division of Research Immunology/Bone Marrow Transplantation, The Saban Research Institute of Childrens Hospital Los Angeles, CA 90027, USA.
Tarantal
Alice F
AF
Lee
Chang I
CI
Jimenez
Daniel F
DF
Choi
Christopher
C
Pepper
Karen
K
Petersen
Denise
D
Fletcher
Misty D
MD
Leapley
Alyssa C
AC
Fisher
Jennifer
J
Burns
Travis S
TS
Ultsch
Man-Ni
MN
Dorey
Frederick J
FJ
Kohn
Donald B
DB
D000818:Animals; D019540:Area Under Curve; D001483:Base Sequence; D002066:Busulfan / Q000493:pharmacokinetics / Q000494:pharmacology*; D002460:Cell Line; D017931:DNA Primers; D004305:Dose-Response Relationship, Drug; D005819:Genetic Markers; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008253:Macaca mulatta
D017931:DNA Primers; D005819:Genetic Markers; D002066:Busulfan