Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Medical Immunology, Augustenburger Platz 1, 13353, Berlin, Germany; Berlin Institute of Health at Charité - Universitätsmedizin Berlin, BIH Center for Regenerative Therapies (BCRT), Föhrer Straße 15, 13353, Berlin, Germany; Berlin-Brandenburg School for Regenerative Therapies (BSRT; Graduate School 203 of the German Excellence Initiative), Augustenburger Platz 1, 13353, Berlin, Germany.
Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Medical Immunology, Augustenburger Platz 1, 13353, Berlin, Germany; Berlin Institute of Health at Charité - Universitätsmedizin Berlin, BIH Center for Regenerative Therapies (BCRT), Föhrer Straße 15, 13353, Berlin, Germany.
Biomaterials. 2023 Mar;294:121971. doi: 10.1016/j.biomaterials.2022.121971. Epub 2022 Dec 26.
In vitro transcribed (IVT-)mRNA has entered center stage for vaccine development due to its immune co-stimulating properties. Given the widely demonstrated safety of IVT-mRNA-based vaccines, we aimed to adopt IVT-mRNA encoding VEGF for secretory phenotype modulation of therapeutic cells. However, we observed that the immunogenicity of IVT-mRNA impairs the endogenous secretion of pro-angiogenic mediators from transfected mesenchymal stromal cells, instead inducing anti-angiogenic chemokines. This inflammatory secretome modulation limits the application potential of unmodified IVT-mRNA for cell therapy manufacturing, pro-angiogenic therapy and regenerative medicine. To uncouple immunogenicity from the protein expression functionality, we immuno-engineered IVT-mRNA with different chemically modified ribonucleotides. 5-Methoxy-uridine-modification of IVT-mRNA rescued the endogenous secretome pattern of transfected cells and prolonged secretion of IVT-mRNA-encoded VEGF. We found that high secretion of IVT-mRNA-encoded protein further depends on optimized cell adhesion. Cell encapsulation in a collagen-hyaluronic acid hydrogel increased secretion of IVT-mRNA-encoded VEGF and augmented the endogenous secretion of supporting pro-angiogenic mediators, such as HGF. Integrating minimally immunogenic mRNA technology with predesigned matrix-derived cues allows for the synergistic combination of multiple dimensions of cell manipulation and opens routes for biomaterial-based delivery of mRNA-engineered cell products. Such multimodal systems could present a more biologically relevant way to therapeutically address complex multifactorial processes such as tissue ischemia, angiogenesis, and regeneration.
体外转录（IVT-）mRNA 因其免疫协同特性而成为疫苗开发的焦点。鉴于基于 IVT-mRNA 的疫苗已被广泛证明安全，我们旨在采用 IVT-mRNA 编码 VEGF 来调节治疗性细胞的分泌表型。然而，我们观察到 IVT-mRNA 的免疫原性会损害转染间充质基质细胞中内源性分泌促血管生成介质的能力，反而会诱导抗血管生成趋化因子。这种炎症性分泌组调节限制了未经修饰的 IVT-mRNA 用于细胞治疗制造、促血管生成治疗和再生医学的应用潜力。为了将免疫原性与蛋白表达功能分离，我们使用不同的化学修饰核苷酸对 IVT-mRNA 进行免疫工程改造。5-甲氧基尿嘧啶修饰的 IVT-mRNA 挽救了转染细胞的内源性分泌组模式，并延长了 IVT-mRNA 编码 VEGF 的分泌。我们发现，IVT-mRNA 编码蛋白的高分泌进一步取决于优化的细胞黏附。在胶原-透明质酸水凝胶中封装细胞可增加 IVT-mRNA 编码的 VEGF 的分泌，并增强支持性促血管生成介质（如 HGF）的内源性分泌。将最小免疫原性 mRNA 技术与预先设计的基质衍生信号整合在一起，允许对细胞进行多种维度的协同操作，并为基于生物材料的 mRNA 工程细胞产品的递呈开辟了途径。这种多模态系统可能为治疗复杂的多因素过程（如组织缺血、血管生成和再生）提供更具生物学相关性的方法。
