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The Effects of Conditioning and Lentiviral Vector Pseudotype on Short- and Long-Term Airway Reporter Gene Expression in Mice
Human Gene Therapy ( IF 4.2 ) Pub Date : 2021-08-17 , DOI: 10.1089/hum.2021.031
Chantelle Carpentieri 1, 2, 3 , Nigel Farrow 1, 2, 3 , Patricia Cmielewski 1, 2, 3 , Nathan Rout-Pitt 1, 2, 3 , Alexandra McCarron 1, 2, 3 , Emma Knight 4, 5 , David Parsons 1, 2, 3 , Martin Donnelley 1, 2, 3
Affiliation  

A gene addition therapy into the conducting airway epithelium is a potential cure for cystic fibrosis lung disease. Achieving sustained lung gene expression has proven difficult due to the natural barriers of the lung. The development of lentiviral (LV) vectors pseudotyped with viral envelopes that have a natural tropism to the airway has enabled persistent gene expression to be achieved in vivo. The aims of this study were to compare the yields of hemagglutinin (HA) and vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped HIV-1 vectors produced under the same conditions by our standard LV vector production method. We then sought to measure gene expression in mouse airways and to determine whether lysophosphatidylcholine (LPC) conditioning enhances short- and long-term gene expression. C57Bl/6 mouse airways were conditioned with 10 μL of 0.1% LPC or saline control, followed 1 h later by a 30 μL dose of an HA or VSV-G pseudotyped vector carrying either the LacZ or luciferase reporter genes. LacZ expression was assessed by X-gal staining after 7 days, while lung luminescence was quantified regularly for up to 18 months by bioluminescent imaging. The HA pseudotyped vectors had functional titers 25 to 60 times lower than the VSV-G pseudotyped vectors. Conditioning the lung with LPC significantly increased the total number of LacZ-transduced cells for both pseudotypes compared to saline control. Regardless of LPC conditioning, the VSV-G pseudotype produced higher initial levels of gene expression compared to HA. LPC conditioning did not increase the number of transduced basal cells for either pseudotype compared to saline, and was not required for long-term gene expression. Both pseudotyped vectors effectively transduced the upper conducting airways of wild-type mice. The use of LPC conditioning before vector delivery was not required in mouse lungs to produce long-term gene expression, but did improve short-term gene expression.

中文翻译:

调节和慢病毒载体假型对小鼠短期和长期气道报告基因表达的影响

对导气道上皮细胞进行基因添加疗法是治疗囊性纤维化肺病的潜在方法。由于肺的天然屏障,实现持续的肺基因表达已被证明是困难的。慢病毒 (LV) 载体的开发具有对气道具有天然趋向性的病毒包膜假型,从而能够在体内实现持久的基因表达. 本研究的目的是比较我们的标准 LV 载体生产方法在相同条件下生产的血凝素 (HA) 和水疱性口炎病毒-糖蛋白 (VSV-G) 假型 HIV-1 载体的产量。然后,我们试图测量小鼠气道中的基因表达,并确定溶血磷脂酰胆碱 (LPC) 调节是否能增强短期和长期基因表达。C57Bl/6 小鼠气道用 10 μL 0.1% LPC 或盐水对照进行调节,1 小时后用 30 μL 剂量的 HA 或 VSV-G 假型载体携带LacZ荧光素酶报告基因。7 天后通过 X-gal 染色评估 LacZ 表达,而通过生物发光成像定期定量肺发光长达 18 个月。HA 假型载体的功能滴度比 VSV-G 假型载体低 25 至 60 倍。与盐水对照相比,用 LPC 调理肺显着增加了两种假型的 LacZ 转导细胞的总数。无论 LPC 条件如何,与 HA 相比,VSV-G 假型产生的基因表达初始水平更高。与生理盐水相比,LPC 调节不会增加任一假型的转导基底细胞数量,并且不需要长期基因表达。两种假型载体都有效地转导了野生型小鼠的上导气道。
更新日期:2021-08-20
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