Sugar-coating our way to genetically modified mesenchymal stem cells: Glycocalyx-inspired cell culture substrates that prime mesenchymal stem cells for polycation-mediated pDNA delivery.

项目摘要 人骨髓间充质干细胞（hMSCs）已被证明在治疗自身免疫性疾病的承诺，可以- cer、心脏病和椎间盘退变。hMSCs通过分泌疗法促进组织修复 生物分子如免疫调节因子和促血管生成因子。基因改造在泰- 修饰hMSC分泌蛋白并提高治疗效力。虽然病毒载体在遗传上是有效的， 修饰hMSC、放大和制造挑战限制了广泛的临床应用。在众多的 合成材料，可以取代病毒载体，聚阳离子基因载体是其中最通用的，规模， 经济实惠的选择。对于聚阳离子载体介导hMSCs中的高转基因表达，我们 必须解决递送效率、细胞毒性和维持hMSC分化之间的权衡 容量为了缓解聚阳离子介导的基因传递的瓶颈，我们将在糖胺聚糖上扩增hMSCs， 由糖萼的组成和组织启发的仿罐细胞培养基质。我们假设- 估计碳水化合物残基的多价呈现-使人联想到糖胺聚糖（GAG）- 在hMSC-基质界面上，将通过隔离生长因子（GF）介导的 hMSC粘附和增殖。多价GAG模拟聚合物刷将呈现固定的GF， 局部高浓度的hMSCs，导致hMSCs相对于未修饰的组织增殖更快- 培养聚苯乙烯。当hMSC增殖增强时，核被膜将更频繁地溶解， 促进有效载荷的核摄取，并促进聚阳离子介导的转基因表达。与生物- 逻辑衍生的GAG如硫酸乙酰肝素或硫酸软骨素，GAG模拟聚合物刷是化学衍生的， 标准化、经济、可批量复制，使我们能够在 基质界面性质、hMSC自我更新和聚阳离子介导的转基因表达。通过合成- 带有中性、硫酸化或羧化β-葡萄糖/葡糖胺混合物的三元共聚物刷上浆 残基，我们将学习如何硫酸/羧酸基序的空间分布和多价呈现 聚糖残基的分布（由刷状厚度控制）指导细胞的粘附、增殖和细胞命运决定。 hMSCs。此外，我们将确定GAG模拟底物，增加聚阳离子介导的基因传递， 促进多聚阳离子穿梭质粒（pDNA）进入hMSC核内。与以往的做法不同的 他们试图（但失败了）通过狭隘地关注优化聚阳离子来提高hMSCs中的转基因表达 结构，我们拥抱一个整体的概念框架，提供了平等的考虑hMSC基板线索 以及聚阳离子基因载体的分子设计。部署模拟GAG的细胞培养基质，或- 诱导hMSC自我更新和有效的聚阳离子介导的pDNA递送，我们将获得遗传修饰的hMSC。 fied hMSCs使用负担得起的和可扩展的生物材料平台。我们的研究结果可以部署到降低产量- 降低成本，减轻监管负担，拓宽hMSC治疗的途径。

PROJECT SUMMARY Human mesenchymal stem cells (hMSCs) have demonstrated promise in treating auto-immune disorders, can- cer, cardiac diseases, and intervertebral disc degeneration. hMSCs facilitate tissue repair by secreting therapeu- tic biomolecules such as immunomodulatory and pro-angiogenic factors. Genetic modification is valuable in tai- loring the hMSC secretome and boosting therapeutic potency. Although viral vectors are effective in genetically modifying hMSCs, scale-up and manufacturing challenges limit broad clinical application. Among the plethora of synthetic materials that can replace viral vectors, polycationic gene carriers are among the most versatile, scal- able, and economical options. For polycationic carriers to mediate high transgene expression in hMSCs, we must resolve the tradeoff between delivery efficiency, cellular toxicity, and maintenance of hMSC differentiation capacity. To ease bottlenecks in polycation-mediated gene delivery, we will expand hMSCs on glycosaminogly- can-mimetic cell culture substrates inspired by the composition and organization of the glycocalyx. We hypoth- esize that the multivalent presentation of carbohydrate residues—reminiscent of glycosaminoglycans (GAGs)— at hMSC–substrate interfaces will stimulate hMSC proliferation by sequestering growth factors (GFs) mediating hMSC adhesion and proliferation. Multivalent GAG-mimetic polymer brushes will present immobilized GFs to hMSCs with high local concentrations, causing hMSCs to proliferate more rapidly relative to unmodified tissue- culture polystyrene. When hMSC proliferation is enhanced, the nuclear envelope will dissolve more frequently, facilitating the nuclear uptake of payloads, and boosting polycation-mediated transgene expression. Unlike bio- logically derived GAGs such as heparan sulfate or chondroitin sulfate, GAG-mimetic polymer brushes are chem- ically defined, economical, and reproducible from batch to batch, enabling us to weave connections between substrate interfacial properties, hMSC self-renewal, and polycation-mediated transgene expression. By synthe- sizing ternary copolymer brushes bearing a mixture of neutral, sulfated, or carboxylated β-glucose/glucosamine residues, we will learn how the spatial distribution of sulfate/carboxylate motifs and the multivalent presentation of glycan residues (governed by brush thickness) directs the adhesion, proliferation, and cell fate decisions of hMSCs. Further, we will identify GAG-mimetic substrates that augment polycation-mediated gene delivery by facilitating the import of polycation-shuttled plasmids (pDNA) within hMSC nuclei. Unlike previous approaches that tried (and failed) to boost transgene expression in hMSCs by focusing narrowly on optimizing polycation structure, we embrace a holistic conceptual framework that offers equal consideration to hMSC substrate cues and the molecular design of polycationic gene carriers. Deploying GAG-mimetic cell culture substrates that or- chestrate hMSC self-renewal and efficient polycation-mediated pDNA delivery, we will obtain genetically modi- fied hMSCs using affordable and scalable biomaterial platforms. Our findings can be deployed to lower produc- tion costs, lighten regulatory burden, and broaden access to hMSC therapeutics.