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.

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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.