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Biochemical investigation of the intracellular trafficking of non-viral and hybrid gene therapy vectors.

机译：非病毒和杂交基因治疗载体在细胞内运输的生化研究。

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The development of gene therapy promises improved treatment for a variety of medical conditions. New techniques present the opportunity to treat both inherited and acquired diseases more effectively, attacking the disorder at its genetic source. However, the lack of safe and efficient means for delivering therapeutic genes to target tissues remains problematic. Viruses, adapted to form highly efficient gene delivery vectors, are difficult (and expensive) to produce and purify, and have displayed immunogenic and oncogenic side effects in clinical trials. Synthetic vectors, developed to deliver genes without the drawbacks observed in viruses, are too inefficient for clinical use.;To improve synthetic vectors, we must understand the intracellular barriers the vectors must overcome. We have studied the cellular mechanisms synthetic vectors utilize to facilitate gene delivery. Using polyethylenimine (PEI) as a model vector, we have employed a series of small-molecule inhibitors of intracellular processes to investigate their role in synthetic vector delivery. Actin microfilament depolymerizer cytochalasin D (Cyt D) significantly decreased gene delivery in transfected cells, but caused only a minimal decrease in complex uptake. Microtubule depolymerization with colchicine resulted in a significant decrease in gene expression, while microtubule stabilization with paclitaxel considerably increased transgene expression. Transgene expression decreased in cells treated with dynein inhibitors erythro-9-[3-(2-hydroxynonyl)] adenine (EHNA) and sodium orthovanadate (Na3VO4), as well as in cells treated with kinesin inhibitors. Inhibition of endosome acidification with bafilomycin A1 (Baf A1) also resulted in decreased transgene delivery, a finding that supports the proton sponge hypothesis and is in agreement with results reported elsewhere.;To further improve synthetic vector efficiency, we have developed hybrid gene delivery vectors composed of envelope-free virus-like particles (VLP) from the murine leukemia virus (MLV) complexed with PEI. Joshua Ramsey previously used these hybrid vectors to successfully transduce cultured cells. We have expanded upon Ramsey's work by developing a VLP quantification assay using reverse transcriptase quantitative PCR (RT-qPCR), growing VLP in alternative media, and further characterizing the physical and infective properties of VLP and PEI:VLP complexes.;VLP grown in serum-free media (VLP-SF and VLP-OM grown in serum-free DMEM and Opti-MEMRTM I, respectively) required less PEI for optimal complexing, and yielded transfection efficiencies significantly higher than VLP-FB (grown in DMEM + 10% FBS). All PEI:VLP complexes were very large, with PEI:VLP-SF/OM being 50% larger than PEI:VLP-FB. Cytotoxicity was directly related to total PEI content, and uptake did not vary significantly with PEI:VLP ratio. VLP remained stable for a few weeks when stored at 4°C, but infectivity diminished rapidly at physiological temperatures. VLP infectivity was decreased by freeze-thaw and ultracentrifugation, but samples concentrated following ultracentrifugation showed increased expression. PEI:VLP vectors remained unable to produce long-term expression, even when using serum-free VLP and lower PEI concentrations.;As with polyplexes, we used cellular inhibitors to study hybrid vector processing. PEI:VLP were also compared with MLV with amphotropic (MLV-A) and vesicular stomatitis virus-G (MLV-V) envelope proteins. Actin depolymerization was shown to significantly decrease PEI:VLP gene delivery (MLV were relatively unaffected), while depolymerization of microtubules with colchicine caused no decrease in PEI:VLP- and MLV-mediated gene delivery. Microtubule stabilization, inhibition of dynein, and inhibition of endosome acidification all resulted in decreased gene delivery by PEI:VLP and MLV-V (MLV-A were relatively unaffected). This suggests that active transport of endosome-trafficked MLV-V and hybrid vectors may occur by a microtubule-independent mechanism. PEI:VLP vectors appear to be trafficked similarly to polyplexes as far as the early endosome, while acting more like endocytically-trafficked MLV-V in endosome escape and cytoplasmic processing.;We also used reverse transcriptase inhibitor 3 '-azido-3'-deoxythymidine (AZT) applied of post-transfection time increments to determine the timeframe of reverse transcription, thereby estimating endosomal escape of VLP. MLV were observed to achieve reverse transcription rapidly, with more than half of infecting viruses being reverse transcribed within 8 hours. PEI:VLP were delayed approximately 4 hours (relative to viruses) in having their RNA reverse transcribed. This suggests VLP experience delayed endosomal escape, unpackaging, or release of the capsid/reverse transcription complex from the lipid bilayer. This delay likely accounts for much of the inefficiency observed in PEI:VLP relative to MLV. (Similar experiments attempted with integrase inhibitors did not yield useful results.) Improvement of hybrid vectors probably depends on expediting the escape of VLP from the endolysosomal network and the VLP lipid bilayer. This might be accomplished by developing polymers with stronger endosomolytic properties and/or by the incorporation of fusogenic peptides to assist in endosome and bilayer escape.
机译：基因疗法的发展有望改善对各种医学状况的治疗。新技术为更有效地治疗遗传性和后天性疾病提供了机会，可以从遗传源上攻击疾病。然而，缺乏将治疗性基因递送至靶组织的安全有效的手段仍然存在问题。适于形成高效基因传递载体的病毒难以（且昂贵）产生和纯化，并且在临床试验中显示出免疫原性和致癌性副作用。为了提供基因而没有在病毒中观察到的缺点而开发的合成载体，对于临床使用而言效率太低。；要改进合成载体，我们必须了解载体必须克服的细胞内障碍。我们已经研究了合成载体用于促进基因递送的细胞机制。使用聚乙烯亚胺（PEI）作为模型载体，我们采用了一系列细胞内过程的小分子抑制剂来研究其在合成载体递送中的作用。肌动蛋白微丝解聚剂细胞松弛素D（Cyt D）显着降低了转染细胞中的基因传递，但仅引起最小的复合物吸收降低。秋水仙碱的微管解聚作用导致基因表达显着下降，而紫杉醇的微管稳定作用显着增加了转基因表达。用动力蛋白抑制剂erythro-9- [3-（2-羟基壬基）]腺嘌呤（EHNA）和原钒酸钠（Na3VO4）处理的细胞，以及用驱动蛋白抑制剂处理的细胞中的转基因表达降低。 bafilomycin A1（Baf A1）抑制内体酸化也导致转基因传递减少，这一发现支持质子海绵假说，并且与其他地方报道的结果相符。为了进一步提高合成载体的效率，我们开发了杂交基因传递载体由不含鼠白血病病毒（MLV）的无包膜病毒样颗粒（VLP）与PEI复合而成。约书亚·拉姆齐（Joshua Ramsey）以前曾使用这些杂种载体成功转导培养的细胞。我们通过开发使用逆转录酶定量PCR（RT-qPCR）的VLP定量测定法，在替代培养基中生长VLP并进一步表征VLP和PEI：VLP复合物的物理和感染特性来扩展Ramsey的工作。无培养基（分别在无血清DMEM和Opti-MEMRTM I中生长的VLP-SF和VLP-OM）所需的PEI较少，以实现最佳复合效果，并且转染效率明显高于VLP-FB（在DMEM + 10％FBS中生长） ）。所有PEI：VLP复合物都非常大，PEI：VLP-SF / OM比PEI：VLP-FB大50％。细胞毒性与总PEI含量直接相关，并且摄取量随PEI：VLP比值无显着变化。在4°C储存时，VLP在数周内保持稳定，但在生理温度下，传染性迅速降低。冻融和超速离心可降低VLP的感染力，但超速离心后浓缩的样品显示表达增加。即使使用无血清VLP和降低PEI浓度，PEI：VLP载体仍然无法产生长期表达。;与多聚体一样，我们使用细胞抑制剂来研究杂交载体的加工。也将PEI：VLP与带有两性（MLV-A）和水泡性口炎病毒G（MLV-V）包膜蛋白的MLV进行了比较。肌动蛋白的解聚作用可显着降低PEI：VLP的基因传递（MLV相对不受影响），而秋水仙碱的微管解聚不会导致PEI：VLP和MLV介导的基因传递下降。微管稳定，动力蛋白的抑制和内体酸化的抑制都导致PEI：VLP和MLV-V（MLV-A相对不受影响）的基因传递减少。这表明可能通过微管独立机制发生了内体运输的MLV-V和杂种载体的主动转运。 PEI：VLP载体似乎在早期内体中的转运与多聚体相似，而在内体逃逸和细胞质加工中的作用更像内吞的MLV-V。我们还使用了逆转录酶抑制剂3'-azido-3'-转染后时间的脱氧胸苷（AZT）的增加确定逆转录的时间框架，从而估计VLP的内体逃逸。观察到MLV迅速实现逆转录，超过一半的感染病毒在8小时内被逆转录。 PEI：VLP的RNA逆转录大约延迟了4个小时（相对于病毒）。这表明VLP经历了内体逃逸延迟，未包装或从脂质双层释放衣壳/逆转录复合物。相对于MLV，这种延迟可能是PEI：VLP中观察到的效率低下的主要原因。 （尝试用整合酶抑制剂进行的类似实验未获得有用的结果。）杂交载体的改进可能取决于加快VLP从溶酶体网络和VLP脂质双层的逸出。这可以通过开发具有更强的内溶酶特性的聚合物和/或通过融合融合肽来协助内体和双层逃逸来实现。

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