Brzezinski Margaret, Yanay Ofer, Waldron Lanaya, Barry Simon C, Osborne William R A
Department of Pediatrics, University of Washington, Seattle, WA 98195, USA.
J Gene Med. 2007 Jul;9(7):571-8. doi: 10.1002/jgm.1050.
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.
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.
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.
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.
针对以中性粒细胞计数<500个细胞/微升为特征的中性粒细胞减少症患者的基因治疗，我们在临床前大鼠模型中研究了慢病毒载体提供持续粒细胞集落刺激因子（G-CSF）递送以及允许在第二次病毒给药后进行转基因表达的能力。
给大鼠肌肉注射（IM）24×10⁶和9×10⁶感染单位（IU）的VSV-G假型自失活（SIN）慢病毒，该慢病毒编码大鼠G-CSF cDNA并包含中心多聚嘌呤核苷酸序列（cPPT）和增强子（PRE）元件。为了确定第二次病毒给药的有效性，对治疗的大鼠和未处理的大鼠肌肉注射促红细胞生成素（EPO）-慢病毒。监测大鼠的中性粒细胞和红细胞生成情况。通过病毒中和试验和酶联免疫吸附测定（ELISA）检测慢病毒抗体。
高剂量和低剂量病毒给药分别使中性粒细胞计数增加到5660±930个细胞/微升（平均值±标准差）和4010±850个细胞/微升，持续>17个月，且显著高于对照组的1890±570个细胞/微升（p≤0.0002）。用EPO-病毒治疗的大鼠产生了显著升高的血细胞比容（HCT）（63.1±4.3%对46.0±3.2%），对G-CSF-病毒介导的中性粒细胞生成没有影响。在血清稀释度≥1:10时，通过病毒中和或ELISA未检测到抗病毒抗体。对照动物和处理动物之间的淋巴细胞和血小板没有显著差异（p>0.1）。仅注射部位肌肉的基因组DNA对前病毒呈阳性，表明没有病毒传播。
肌肉注射G-CSF-慢病毒可使中性粒细胞计数升高并持续，后续注射EPO-慢病毒对其无影响。EPO-慢病毒给药后血细胞比容显著增加。这些数据支持通过肌肉注射给予定量慢病毒来治疗严重慢性中性粒细胞减少症患者。
