Long-term in vivo gene expression via delivery of PEI-DNA condensates from porous polymer scaffolds
Long-term in vivo gene expression via delivery of PEI-DNA condensates from porous polymer scaffolds
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DOI：
10.1089/hum.2005.16.609
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发表时间：
2005-05-01
影响因子：
4.2
通讯作者：
Mooney, DJ
中科院分区：
文献类型：
--
作者：
Huang, YC;Riddle, K;Mooney, DJ
非病毒载体在组织工程基因治疗中有着广泛的应用前景，但存在转染效率低、表达时间短等缺点。我们假设，从三维可生物降解支架中持续递送聚（乙烯亚胺）（PEI）凝聚的DNA，鼓励细胞浸润，可以大大增强基因表达。为了验证这一假设，使用气体发泡工艺将编码β-半乳糖苷酶的PEI-浓缩质粒掺入多孔聚（丙交酯-共-乙交酯）（PLG）支架中。检查了四个条件：包封到PLG支架中的浓缩DNA和未浓缩DNA、空白支架以及浓缩DNA的团注递送与PLG支架的植入的组合。将含有浓缩β-半乳糖苷酶质粒的支架植入大鼠皮下组织，在整个15周的实验期间内导致高水平的基因表达，在外表面上观察到的β-半乳糖苷酶基因表达的广泛阳性染色就是例证。和整个外植支架的横截面。在8周和15周时间点，在对照条件下，在外表面或横截面上均未观察到阳性染色。此外，在15周时，掺入浓缩DNA的支架内的高百分比（55-60%）的细胞证明了DNA的表达，证实了包封的质粒DNA的持续摄取和表达。β-半乳糖苷酶基因表达的定量分析显示，在2周时间点，掺入凝聚DNA的支架中的表达水平比其他条件下的表达水平高一个数量级，在8周和15周时间点，比对照条件下的表达水平高近两个数量级。这项研究表明，PEI凝聚的质粒DNA从PLG支架的持续递送导致在体内长期和高水平的基因表达，并且该系统可能在骨组织工程等领域中找到应用。
Nonviral delivery vectors are attractive for gene therapy approaches in tissue engineering, but suffer from low transfection efficiency and short-term gene expression. We hypothesized that the sustained delivery of poly(ethylenimine) (PEI)-condensed DNA from three-dimensional biodegradable scaffolds that encourage cell infiltration could greatly enhance gene expression. To test this hypothesis, a PEI-condensed plasmid encoding beta-galactosidase was incorporated into porous poly(lactide-co-glycolide) (PLG) scaffolds, using a gas foaming process. Four conditions were examined: condensed DNA and uncondensed DNA encapsulated into PLG scaffolds, blank scaffolds, and bolus delivery of condensed DNA in combination with implantation of PLG scaffolds. Implantation of scaffolds incorporating condensed beta-galactosidase plasmid into the subcutaneous tissue of rats resulted in a high level of gene expression for the entire 15-week duration of the experiment, as exemplified by extensive positive staining for beta-galactosidase gene expression observed on the exterior surface and throughout the cross-sections of the explanted scaffolds. No positive staining could be observed for the control conditions either on the exterior surface or in the cross-section at 8- and 15-week time points. In addition, a high percentage (55-60%) of cells within scaffolds incorporating condensed DNA at 15 weeks demonstrated expression of the DNA, confirming the sustained uptake and expression of the encapsulated plasmid DNA. Quantitative analysis of beta-galactosidase gene expression revealed that expression levels in scaffolds incorporating condensed DNA were one order of magnitude higher than those of other conditions at the 2-week time point and nearly two orders of magnitude higher than those of the control conditions at the 8- and 15-week time points. This study demonstrated that the sustained delivery of PEI-condensed plasmid DNA from PLG scaffolds led to an in vivo long-term and high level of gene expression, and this system may find application in areas such as bone tissue engineering.