Preclinical optimization and design for manufacturability of immunoregulatory tissue-engineered vascular grafts

总结 尽管近年来出现了少数基于临床的小口径翻译公司 组织工程化血管移植物（TEVG），临床动脉搭桥术的黄金标准， 自体静脉或动脉移植物。我们的小组已经开发了，与以前的NIH资金，TEVG的基础上， 间充质干细胞/基质细胞（MSC）的促再生免疫调节能力， 管状可生物降解的支架。MSC是免疫调节性的，因为它们既募集宿主嗜中性粒细胞， 通过分泌因子激活巨噬细胞并将募集的细胞调节为耐受性和促再生表型。 我们还证明了MSCs可以刺激新的功能性血管细胞外基质的产生， 基质在体外和体内，MSC在我们的TEVG中发挥急性抗血栓形成作用。 我们发表的工作已经严格测试了人源性MSC诱导TEVG重塑的能力， 当作为大鼠主动脉间置移植物植入时，在最近未发表的作品中（禁运待决 IP保护），我们还成功地在相同的大鼠模型中测试了基于 MSC分泌的免疫调节因子。这些方法包括使用细胞因子和MSC分泌的细胞因子。 负载因子的微球和负载到TEVG支架中的MSC衍生的细胞外囊泡（EV）。 我们根据“有效载荷”（MSC，负载有尼古丁的细胞）的可行组合来定义我们的TEVG策略。 微球、MSC分泌因子加载的微球和MSC衍生的EV）加载到三种不同的微球中。 支架的类型（聚（酯氨基甲酸酯）脲（PEUU）、冻干丝素蛋白（LyoGel）和双层多孔丝）。 我们还开始扩大这些TEVG配置的制造工艺， 最终的大型动物试验，并已证明成功与绵羊植入试点研究，预计 这个提案提交。 我们提出的里程碑驱动的项目是这个两阶段催化赠款机制的理想选择， 该项目的完成将为我们的TEVG技术的临床转化带来重大进展。R61 第一阶段有两个目标和里程碑，这将使我们能够充分评估，在我们的完善和相对 高通量大鼠模型，所有可行TEVG配置。R61阶段的主要结局将是 鉴定用于TEVG构建体的免疫调节有效载荷和支架的最佳组合， 最佳地在体内重塑成天然样动脉。R33阶段有五个目标和里程碑， 首先展示成功制造的按比例放大版本的TEVG配置，满足 R61阶段的里程碑，然后对最佳（最多四个）配置进行大型动物测试。的 这一阶段的结果将是确定最佳的TEVG配置，以推进临床试验 和商业化，并解决设计的可制造性和发展的监管和 商业计划和人脉

SUMMARY Despite the emergence of a few companies in recent years based on the clinical translation of small diameter tissue-engineered vascular grafts (TEVGs), the gold standard for arterial bypass in the clinic continues to be autologous vein or artery grafts. Our group has developed, with previous NIH funding, TEVGs based on the documented pro-regenerative immunoregulatory potency of mesenchymal stem/stromal cells (MSCs) and tubular, biodegradable scaffolds. MSCs are immunoregulatory in that they both recruit host neutrophils and macrophages via secreted factors and modulate the recruited cells to a tolerant and pro-regenerative phenotype. We have also demonstrated that MSCs can stimulate the production of new functional vascular extracellular matrix both in-vitro and in-vivo and that MSCs play an acute antithrombogenic role in our TEVGs. Our published work has rigorously tested the ability of human-derived MSCs to induce remodeling of TEVG constructs when implanted as rat aortic interposition grafts. In very recent unpublished work (embargoed pending IP protection), we have also successfully tested in the same rat model cell-free TEVG strategies based on immunoregulatory factors secreted by MSCs. These have included the use of cytokine- and MSC secreted factor-loaded microspheres and MSC-derived extracellular vesicles (EVs) loaded into TEVG scaffolds. We define our TEVG strategies in terms of feasible combinations of “payload” (MSCs, cytokine-loaded microspheres, MSC secreted factor-loaded microspheres, and MSC-derived EVs) loaded into three different types of scaffolds (poly(ester urethane)urea (PEUU), lyophilized silk fibroin (LyoGel), and bilayered porous silk). We have also begun to scale-up the fabrication processes of these TEVG configurations in anticipation of eventual large animal testing and have demonstrated success with a sheep implant pilot study in anticipation of this proposal submission. Our proposed milestone-driven project is ideal for this two-phase Catalyze grant mechanism and successful completion will lead to significant progress toward the clinical translation of our TEVG technology. The R61 phase has two objectives with milestones that will allow us to fully evaluate, in our well-established and relatively high-throughput rat model, all feasible TEVG configurations. The primary outcome of the R61 Phase will be to identify the best combination(s) of immunoregulatory payload and scaffold for a TEVG construct that can most optimally remodel in-vivo into a native-like artery. The R33 phase has five objectives with milestones that will first demonstrate successful fabrication of scaled-up versions of the TEVG configurations that meet the milestones of the R61 phase, and then perform large animal testing of the best (up to four) configuration(s). The outcome of this phase will be to identify the optimal TEVG configuration to move forward toward clinical testing and commercialization, and also to address design for manufacturability and development of regulatory and business plans and connections.