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经口给予重组2型腺相关病毒载体后向胃肠道的基因转移。

Gene transfer to the gastrointestinal tract after peroral administration of recombinant adeno-associated virus type 2 vectors.

作者信息

Shao Guohong, Greathouse Kristin, Huang Qin, Wang Chiou-Miin, Sferra Thomas J

机构信息

Center for Gene Therapy, Columbus Children's Research Institute, OH, USA.

出版信息

J Pediatr Gastroenterol Nutr. 2006 Aug;43(2):168-79. doi: 10.1097/01.mpg.0000228118.59853.ba.

Abstract

OBJECTIVES

The transfer of exogenous genetic material to cells within the gastrointestinal (GI) tract has many potential therapeutic applications. An attractive feature of the GI tract for gene transfer is its accessibility through the orogastric route. In this study, we evaluated the stability of recombinant adeno-associated virus type 2 (rAAV2) vectors within the GI tract and whether rAAV2-mediated gene transfer could be increased through manipulation of the intraluminal environment.

METHODS

The stability of rAAV2 vectors carrying beta-galactosidase and enhanced green fluorescence protein transgenes was determined in the presence of hydrochloric acid, pepsin, trypsin, chymotrypsin gastric fluid and intestinal fluid and after in vivo administration. For in vivo experiments, the rAAV2 vector carrying the beta-galactosidase transgene was administered perorally to FVB/NJ mice. Groups of mice received the vector alone or in combination with sodium bicarbonate and aprotinin. Gene transfer to the stomach and small intestine was evaluated by polymerase chain reaction and histochemical assays.

RESULTS

The stability of rAAV2 was reduced by hydrochloric acid, trypsin, chymotrypsin, gastric fluid and intestinal fluid. The vector was not stable within the lumen of the GI tract. Gastric acid neutralization with sodium bicarbonate and protease inhibition with aprotinin increased the in vivo stability of the vector and the level of gene transfer to the stomach and all regions of the small bowel. In both groups of mice (vector alone and vector plus sodium bicarbonate and aprotinin), transgene-derived protein expression (beta-galactosidase) was below the level of detection of the histochemical assay.

CONCLUSIONS

Recombinant AAV2 are adversely affected by physiological conditions within the proximal GI tract. Gastric acid neutralization and inhibition of intestinal protease activity improved rAAV2 stability and increased the level of gene transfer within the GI tract. Despite these changes, transduction of the GI tract after peroral rAAV2 administration remained low.

摘要

目的

将外源性遗传物质转移至胃肠道(GI)内的细胞有许多潜在的治疗应用。胃肠道用于基因转移的一个吸引人的特点是可通过经口胃途径进入。在本研究中,我们评估了重组2型腺相关病毒(rAAV2)载体在胃肠道内的稳定性,以及是否可以通过改变管腔内环境来增加rAAV2介导的基因转移。

方法

在盐酸、胃蛋白酶、胰蛋白酶、糜蛋白酶、胃液和肠液存在的情况下以及体内给药后,测定携带β-半乳糖苷酶和增强型绿色荧光蛋白转基因的rAAV2载体的稳定性。对于体内实验,将携带β-半乳糖苷酶转基因的rAAV2载体经口给予FVB/NJ小鼠。小鼠组单独接受载体或与碳酸氢钠和抑肽酶联合接受载体。通过聚合酶链反应和组织化学分析评估基因向胃和小肠的转移。

结果

盐酸、胰蛋白酶、糜蛋白酶、胃液和肠液降低了rAAV2的稳定性。该载体在胃肠道管腔内不稳定。用碳酸氢钠中和胃酸和用抑肽酶抑制蛋白酶可增加载体的体内稳定性以及基因向胃和小肠所有区域的转移水平。在两组小鼠(单独载体组和载体加碳酸氢钠和抑肽酶组)中,转基因衍生的蛋白质表达(β-半乳糖苷酶)均低于组织化学分析的检测水平。

结论

重组AAV2受到近端胃肠道内生理条件的不利影响。中和胃酸和抑制肠道蛋白酶活性可改善rAAV2的稳定性并增加胃肠道内的基因转移水平。尽管有这些变化,经口给予rAAV2后胃肠道的转导效率仍然很低。

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