Department of Surgery, University of Tennessee, Graduate School of Medicine, Knoxville, Tennessee 37920, USA.
J Surg Res. 2012 Sep;177(1):178-84. doi: 10.1016/j.jss.2012.05.037. Epub 2012 Jun 1.
Gene therapy shows promise in the treatment of vascular disease. However, traditional transfection methods commonly used in the laboratory are poorly translatable to in vivo conditions, primarily due to the immune response to viral vectors, the cellular toxicity of chemical transfection, and the technical impracticality of electroporation. Biodegradable polymers have shown promise as a safe, predictable, and nontoxic alternative, relying on endocytosis of synthetic polymeric carriers, which are bioconjugated to the targeted genetic material of choice. However, to date most of the feasibility studies have been exclusively performed in stem cells. Differentiated cell types would be prime targets for therapeutic gene modulation in the prevention of various disease processes. We aim to establish polymeric transfection as a method for gene therapy in cells of vascular origin. Here we compared the efficiency of polymeric transfection with chemical transfection agents routinely used in a laboratory setting in vascular smooth muscle cells.
Human aortic smooth muscle cells (HASMC) were transfected with fluorescently labeled GAPDH siRNA or negative control (NC) siRNA. Transfection methods included poly(B-amino ester) polymer (StemFECT) bioconjugates, DharmaFECT2 complexes, and Santa Cruz complexes. Conjugate endocytosis was confirmed by fluorescent microscopy, and GAPDH gene silencing was assayed by qPCR normalized to 18S.
Santa Cruz reagent complexes were the least efficient, with the maximum achievable gene silencing using a 9 μL reagent : 70 pmol siRNA/mL complex (59% ± 6%; n = 3). Maximum GADPH gene silencing using DharmaFECT2 was achieved with a 1.5 μL reagent : 100 pmol siRNA/mL complex (19% ± 1% expression versus NC; n = 4). Equivalent silencing was achieved using a comparable StemFECT bioconjugate of 1.3 μL polymer : 100 pmol siRNA/mL (25% ± 3% expression versus NC; n = 4; P = NS versus DharmaFECT2). By increasing the StemFECT bioconjugate to 1.95 μL polymer : 100 pmol siRNA/mL, gene silencing was significantly increased (10% ± 1% expression versus NC; n = 6; P < 0.05 versus DharmaFECT2 and StemFECT 1.3:100).
HASMCs were efficiently transfected using polymeric bioconjugates in a manner comparable to and exceeding other transfection agents routinely used in vitro. This proof of concept establishes polymeric transfection as a viable method for in vitro investigation of differentiated vascular cells. Future studies will expand on this method of gene therapy for ex vivo transfection of whole vessel segments and in vivo transfection in animal models of vascular disease. Our long-term goal is to deliver molecular inhibitors of genes thought to play a role in intimal hyperplasia, restenosis, and vessel graft failure.
基因治疗在血管疾病的治疗中显示出前景。然而，传统的实验室常用的转染方法在体内条件下效果不佳，主要是由于病毒载体的免疫反应、化学转染的细胞毒性以及电穿孔的技术不切实际。可生物降解的聚合物作为一种安全、可预测和无毒的替代物具有前景，依赖于合成聚合物载体的内吞作用，该载体与所选靶向遗传物质进行生物共轭。然而，迄今为止，大多数可行性研究仅在干细胞中进行。分化细胞类型将是各种疾病过程中治疗性基因调节的主要靶标。我们旨在建立聚合物转染作为血管来源细胞基因治疗的一种方法。在这里，我们比较了聚合物转染与实验室常规使用的化学转染剂在血管平滑肌细胞中的效率。
用荧光标记的 GAPDH siRNA 或阴性对照（NC）siRNA 转染人主动脉平滑肌细胞（HASMC）。转染方法包括聚（B-氨基酯）聚合物（StemFECT）生物缀合物、DharmaFECT2 复合物和 Santa Cruz 复合物。通过荧光显微镜证实了共轭内吞作用，并用 qPCR 对 GAPDH 基因沉默进行了测定，结果归一化为 18S。
Santa Cruz 试剂复合物的效率最低，使用 9 μL 试剂：70 pmol siRNA/mL 复合物（59%±6%；n=3）可实现最大基因沉默。使用 DharmaFECT2 可实现最大 GADPH 基因沉默，使用 1.5 μL 试剂：100 pmol siRNA/mL 复合物（与 NC 相比，19%±1%的表达；n=4）。使用等效的 StemFECT 生物缀合物 1.3 μL 聚合物：100 pmol siRNA/mL 可实现等效的沉默（与 NC 相比，25%±3%的表达；n=4；P=NS 与 DharmaFECT2 和 StemFECT 1.3:100）。通过将 StemFECT 生物缀合物增加到 1.95 μL 聚合物：100 pmol siRNA/mL，基因沉默显著增加（与 NC 相比，10%±1%的表达；n=6；P<0.05 与 DharmaFECT2 和 StemFECT 1.3:100 相比）。
使用聚合物生物缀合物以与体外常规使用的其他转染剂相当的方式有效地转染 HASMC，甚至超过了其他转染剂。这一概念验证确立了聚合物转染作为体外研究分化血管细胞的可行方法。未来的研究将扩展这种基因治疗方法，用于对整个血管段进行离体转染，并在血管疾病的动物模型中进行体内转染。我们的长期目标是递送被认为在内膜增生、再狭窄和血管移植物失败中起作用的基因的分子抑制剂。
