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.

糖衣我们的转基因间充质干细胞之路：糖萼启发的细胞培养基质为间充质干细胞提供聚阳离子介导的 pDNA 传递。

• 批准号: 10647120
• 负责人: RAMYA KUMAR
• 金额: $ 40.39万
• 依托单位: COLORADO SCHOOL OF MINES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-11 至 2025-08-10
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10647120
• 关键词: Address; Adhesions; Adoption; Angiogenic Factor; Autoimmune Diseases; Binding; Biocompatible Materials; Biomanufacturing; Carbohydrates; Cell Adhesion; Cell Culture Techniques; Cell Nucleus; Cell Proliferation; Cell Therapy; Cells; Charge; Chemicals; Chondroitin Sulfates; Clinical; Cost Control; Cues; Engineering; Environment; Gene Delivery; Genes; Genetic; Genetic Engineering; Glucosamine; Glucose; Glycocalyx; Glycosaminoglycans; Goals; Growth Factor; Heart Diseases; Heparitin Sulfate; Human; Human Engineering; Immobilization; In Vitro; Learning; Maintenance; Malignant Neoplasms; Mediating; Mesenchymal Stem Cells; Modification; Molecular; Multipotent Stem Cells; Nuclear; Nuclear Envelope; Nuclear Import; Nucleic Acids; Pattern; Plasmids; Polymers; Polysaccharides; Polystyrenes; Production; Proliferating; Property; Publications; Quality Control; Regenerative Medicine; Reproducibility; Research; Spatial Distribution; Structure; Sulfate; Therapeutic; Thick; Toxic effect; Viral; Viral Vector; access disparities; carboxylate; carboxylation; clinical application; copolymer; cost; cytotoxicity; design; genetic payload; immunoregulation; improved; interfacial; intervertebral disk degeneration; manufacture; manufacturing cost; marginalization; mimetics; novel; polycation; scale up; self-renewal; stem cell differentiation; stem cell expansion; stem cell proliferation; stem cell self renewal; stem cell therapy; sugar; tissue culture; tissue repair; transgene expression; uptake

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.

项目概要 人类间充质干细胞 (hMSC) 在治疗自身免疫性疾病方面已显示出前景，可以- cer、心脏病和椎间盘退变通过分泌治疗促进组织修复。 免疫调节因子和促血管生成因子等抽动生物分子的遗传修饰在疾病方面很有价值。 尽管病毒载体在遗传上是有效的，但研究 hMSC 分泌组并增强治疗效力。 修饰 hMSC、放大和制造挑战限制了广泛的临床应用。 聚阳离子基因载体是可以替代病毒载体的合成材料，是用途最广泛、规模最大的合成材料之一。 对于介导 hMSC 中高转基因表达的聚阳离子载体，我们提供了可行且经济的选择。 必须解决递送效率、细胞毒性和 hMSC 分化维持之间的权衡 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展 hMSC。 我们假设，坎模拟细胞培养基质的灵感来自于糖萼的组成和组织。 估计碳水化合物残基的多价呈现——让人想起糖胺聚糖（GAG）—— hMSC-基质界面上的 hMSC 会通过隔离生长因子 (GF) 介导来刺激 hMSC 增殖 hMSC 粘附和增殖。多价 GAG 模拟聚合物刷将固定的 GF 呈现给 hMSC 具有高局部浓度，导致 hMSC 相对于未修饰的组织增殖更快 当 hMSC 包膜增殖增强时，细胞核会更频繁地溶解， 与生物不同，促进有效负载的核摄取，并促进聚阳离子介导的转基因表达。 逻辑上衍生的 GAG，如硫酸乙酰肝素或硫酸软骨素，GAG 模拟聚合物刷是化学- 定义明确、经济且可重复，使我们能够在不同批次之间建立联系 基质界面特性、hMSC 自我更新和聚阳离子介导的转基因表达。 含有中性、硫酸化或羧化 β-葡萄糖/葡萄糖胺混合物的三元共聚物上浆刷 残基，我们将了解硫酸盐/羧酸盐基序的空间分布和多价呈现 聚糖残基（由刷子厚度控制）指导粘附、增殖和细胞命运决定 此外，我们将通过以下方式鉴定可增强聚阳离子介导的基因传递的 GAG 模拟底物。 与之前的方法不同，促进多聚阳离子穿梭质粒 (pDNA) 在 hMSC 细胞核内的导入。 尝试（但失败）通过专注于优化聚阳离子来增强 hMSC 中的转基因表达 结构中，我们采用整体概念框架，平等考虑 hMSC 底物线索 以及部署 GAG 模拟细胞培养基质，或者- 胸骨 hMSC 自我更新和有效的聚阳离子介导的 pDNA 传递，我们将获得遗传修饰 我们的研究结果可用于降低产量。 化成本、减轻监管负担并扩大 hMSC 治疗的可及性。