G-CSF-lentivirus administration in rats provided sustained elevated neutrophil counts and subsequent EPO-lentivirus administration increased hematocrits.
BACKGROUND
Towards gene therapy treatment of patients with neutropenia, characterized by neutrophil counts < 500 cells/microl, we investigated the ability of lentivirus vectors to provide sustained granulocyte colony-stimulating factor (G-CSF) delivery and to permit transgene expression from a second virus administration in a preclinical rat model.


METHODS
Rats were injected intramuscularly (IM) with 24 x 10(6) and 9 x 10(6) infectious units (IU) of a VSV-G-pseudotyped self-inactivating (SIN) lentivirus encoding rat G-CSF cDNA and containing cPPT and PRE elements. To determine the effectiveness of a second virus administration treated rats and a naïve rat received erythropoietin (EPO)-lentivirus IM. Rats were monitored for neutrophil and red blood cell production. Lentivirus antibodies were assayed by virus-neutralizing assay and ELISA.


RESULTS
High and low dose virus administration increased neutrophil counts to 5660 +/- 930 cells/microl (mean +/- SD) and 4010 +/- 850 cells/microl, respectively, that were sustained for > 17 months and were significantly higher than controls counts of 1890 +/- 570 cells/microl (p< or =0.0002). Rats treated with EPO-virus produced significantly increased hematocrits (HCT) (63.1 +/- 4.3% vs. 46.0 +/- 3.2%, p < 0.0001) without effect on G-CSF-virus-mediated neutrophil production. Antivirus antibodies were not detectable at serum dilutions > or =1:10 by virus neutralization or ELISA. Lymphocytes and platelets were not significantly different between control and treated animals (p > 0.1). Only genomic DNA from muscle at injection sites was positive for provirus suggesting lack of virus spread.


CONCLUSIONS
G-CSF-lentivirus administered IM provided elevated, sustained neutrophil counts that were unchanged by subsequent EPO-lentivirus administration. Significantly increased hematocrits were obtained following EPO-lentivirus delivery. These data support the treatment of patients with severe chronic neutropenia by dosed lentivirus delivery IM.
Brzezinski
Margaret
M
Department of Pediatrics, University of Washington, Seattle, WA 98195, USA.
Yanay
Ofer
O
Waldron
Lanaya
L
Barry
Simon C
SC
Osborne
William R A
WR
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D001772:Blood Cell Count; D004247:DNA / Q000378:metabolism; D004921:Erythropoietin / Q000235:genetics* / Q000378:metabolism*; D015316:Genetic Therapy; D016678:Genome / Q000235:genetics; D016179:Granulocyte Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism*; D006367:HeLa Cells; D006400:Hematocrit; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008297:Male; D009504:Neutrophils / Q000166:cytology*; D011533:Proviruses / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344
D000914:Antibodies, Viral; D004921:Erythropoietin; D016179:Granulocyte Colony-Stimulating Factor; D004247:DNA