High-Efficiency Retroviral Gene Transfer into Murine High-Proliferative-Potential Cells Cycle-Activated by Cytosine Arabinoside

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Abstract
We investigated cytosine arabinoside (Ara-C) as a potential agent for in vivo cycle activation of hematopoietic progenitors for the purpose of retroviral-mediated gene transfer. C57BI mice were treated intraperitoneally with one of three regimens of Ara-C: a single 1,750 mg/kg dose (regimen 1), a 1,750 mg/kg dose on day 0, and a 1,500 mg/kg dose on day 2 (LD50) (regimen 2), or a 1,750 mg/kg dose on day 0 and a 1,500 mg/kg dose on day 3 (regimen 3). The high-proliferative-potential cells (HPPC)/105 cells were 47.0 ± 7.5 pretreatment. The post-treatment HPPC cloning efficiencies were 40.6 ± 3.4, 83.6 ± 6.1, and 20.4 ± 3.2 HPPC/105 cells on days 1, 2, and 4, respectively, with regimen 1; 60.0 ± 7.9, 194.0 ± 9.6, and 103.0 ± 11.0 HPPC/105 cells 1, 2, and 4 days after the second Ara-C dose, respectively, with regimen 2; and 266 ± 13.4, 132 ± 23.9, and 118.0 ± 5.7/105 cells 1, 2, and 4 days after the second Ara-C dose, respectively, with regimen 3. The transduction efficiency of HPPC from untreated animals with N2 viral supernatant was 4.9 ± 5.8%. The post-treatment HPPC transduction efficiencies were 22.4 ± 8.4%, 22.7 ± 11.7%, and 0.4 ± 0.6%, 1, 2, and 4 days after treatment with Ara-C, respectively, with regimen 1; 41.9 ± 21.8%, 54.8 ± 11.5%, and 0% 1, 2, and 4 days after the second Ara-C dose, respectively, with regimen 2; and 22.3 ± 3.8%, 33.3 ± 12.4% and 0% 1, 2, and 4 days after a second Ara-C dose, respectively, with regimen 3. In vitro Ara-C susceptibility assays showed substantial cycle activation of HPPC 24–48 hr after the last Ara-C doses of the different regimens. These preliminary in vitro data suggest that Ara-C administration may be useful for cycle activating early hematopoietic progenitors to increase the efficiency of retroviral transduction. These results set the stage for in vivo animal studies that may lead to useful applications in future human gene therapy protocols. The utility of the in vivo administration of cytosine arabinoside (Ara-C) to cycle activate high-proliferative-potential colony precursors (HPPC) to improve the efficiency of retroviral gene transfer was investigated in a murine model. Animals received either a single intraperitoneal dose of Ara-C 1.75 gram/kg or two doses, with the initial dose followed by a 1.5-gram/kg dose either 2 or 3 days later. HPPC were maximally cycle activated 48 hr after the single dose or after the second dose if it was given on day 2, or 24 hr after the second dose if it was delayed to 3 days after the initial one. The transduction efficiency of HPPC increased from 4.9 ± 5.8% pretreatment to 22.7 ± 11.7% 2 days later with a single treatment, to 54.8 ± 11.5% 2 days after the second dose when it was given on day 2, or to 33.3 ± 12.4% 2 days after the second dose when it was given on day 3. These data suggest that Ara-C is potentially useful for improving the efficiency of retroviral gene transfer to bone marrow progenitors and will be useful in designing in vivo studies.

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