Targeted delivery of a proangiogenic and promyogenic protein for regeneration of diabetic ischemic limbs

项目负责人/主要研究者（末、首、中）：关健军 项目摘要 糖尿病患者并发严重肢体缺血（CLI）的截肢率和死亡率显著增高。 CLI的特点是极低的血液灌注和骨骼肌退化。因此，再生 血管和骨骼肌就能挽救四肢然而，内皮细胞和骨骼肌细胞的存活率很低， 并且在糖尿病CLI的高血糖和缺血条件下较差的细胞功能损害肢体修复。 目前，没有有效的治疗方法，尽管生长因子治疗是一种有前途的策略。 然而，生长因子疗法在再生血管和骨骼中具有相对低的治疗功效。 肌肉，因为血管化和肌生成同时需要多种生长因子，而这些生长因子不能 通过当前的方法很容易实现。 在这个项目中，我们建议使用一种新的TRIM72蛋白，具有促血管生成和促肌生成的特性 在糖尿病CLI中再生血管和骨骼肌。TRIM72将被设计成具有更长的 在缺血组织中的保留时间（较慢的扩散速率），因此表现出更长的治疗效果。交付 工程化TRIM72（ETRIM72），它将被封装到缺血肢体靶向纳米颗粒中， 通过具有临床吸引力的静脉注射给药。然后，纳米颗粒将主要积聚在缺血的组织中。 四肢逐渐释放ETRIM72。释放的蛋白质将通过以下方式促进血管形成和肌生成：（1） 通过修复细胞膜，激活细胞，提高内皮细胞和骨骼肌细胞的存活率， 存活激酶;和（2）刺激内皮细胞和骨骼肌细胞迁移和形态发生， 糖尿病CLI的高血糖和缺血状况。 在我们的初步研究中，我们通过将TRIM 72与肽CSTSMLKAC基因融合来开发ETRIM 72 靶向缺血肢体的缺血环境。ETRIM72的第一个版本能够保留在缺血性 四肢明显长于TRIM72。在静脉注射缺血肢体靶向ETRIM 72释放纳米颗粒后， 释放的ETRIM72显著促进糖尿病患者血管和骨骼肌的再生， 肢体缺血TRIM 72在高血糖和高血糖条件下促进血管形成和肌生成的作用 缺血性病症以前没有报道。 基于我们的初步研究，我们假设ETRIM 72的控释将同时增加 高血糖和缺血条件下内皮和骨骼肌细胞的存活、迁移和形态发生 条件下，导致加速再生的血管和骨骼肌在糖尿病缺血肢体。 目的#1将检验最佳ETRIM 72释放曲线将显著促进存活、迁移和/或细胞增殖的假设。 以及在高糖和缺血条件下内皮细胞和成肌细胞的形态发生。目标#2将测试 使用糖尿病鼠肢体缺血模型的ETRIM 72释放纳米颗粒的功效。 这个项目是创新的，因为它设计了一种新的促血管生成和促肌生成蛋白， 同时再生血管和骨骼肌。组织滞留时间越长 工程蛋白的时间，连同局部和控制释放，预计将显着改善 疗效 OMB编号0925 - 0001/0002（修订版03/2020批准至02/28/2023）页码继续格式页码

Program Director/Principal Investigator (Last, First, Middle): GUAN, JIANJUN Project Summary Diabetic patients with critical limb ischemia (CLI) have significantly high rates of limb amputation and mortality. CLI is featured by extremely low blood perfusion and degenerated skeletal muscle. Accordingly, regeneration of vasculature and skeletal muscles will salvage the limbs. Yet the poor endothelial and skeletal muscle cell survival, and inferior cell functions under the hyperglycemia and ischemic conditions of diabetic CLI impair the limb repair. Currently, there is no effective treatment available although growth factor therapy represents a promising strategy. However, growth factor therapy has relatively low therapeutic efficacy in regenerating both vasculature and skeletal muscles, as multiple growth factors are simultaneously needed for vascularization and myogenesis, and these cannot be readily delivered by current approaches. In this project, we propose to use a novel TRIM72 protein with both pro-angiogenic and pro-myogenic properties to regenerate vasculature and skeletal muscles in diabetic CLI. The TRIM72 will be engineered to have longer retention time (slower diffusion rate) in ischemic tissue, thus exhibiting longer therapeutic effect. To deliver the engineered TRIM72 (ETRIM72), it will be encapsulated into ischemic limb-targeting nanoparticles, followed by delivering via clinically attractive IV injection. The nanoparticles will then predominantly accumulate in the ischemic limbs and gradually release ETRIM72. The released protein will promote vascularization and myogenesis by (1) improving the survival of endothelial cell and skeletal muscle cell through cell membrane repair, and activation of cell survival kinase; and (2) stimulating endothelial cell and skeletal muscle cell migration and morphogenesis under the hyperglycemia and ischemic conditions of diabetic CLI. In our preliminary studies, we have developed ETRIM72 by genetically fusing TRIM72 with peptide CSTSMLKAC that targets ischemic environment of ischemic limbs. This first version of ETRIM72 was able to retain in the ischemic limbs significantly longer than TRIM72. After IV injection of ischemic limb-targeting, ETRIM72-releasing nanoparticles, the released ETRIM72 significantly promoted regeneration of both vasculature and skeletal muscles in diabetic ischemic limbs. The function of TRIM72 in promoting vascularization and myogenesis under hyperglycemia and ischemic conditions has not been reported before. Based on our preliminary studies, we hypothesize that controlled release of ETRIM72 will simultaneously increase endothelial and skeletal muscle cell survival, migration and morphogenesis under hyperglycemia and ischemic conditions, leading to accelerated regeneration of both vasculature and skeletal muscles in diabetic ischemic limbs. Aim #1 will test the hypothesis that optimal ETRIM72 release profiles will significantly promote survival, migration and morphogenesis of endothelial cells and myoblasts under high glucose and ischemic conditions. Aim #2 will test efficacy of the ETRIM72-releasing nanoparticles using diabetic murine limb ischemia model. This project is innovative because it engineers a novel proangiogenic and promyogenic protein to simultaneously regenerate vasculature and skeletal muscles in diabetic ischemic limbs. The longer tissue retention time of the engineered protein, together with localized and controlled release are expected to significantly improve therapeutic efficacy. OMB No. 0925-0001/0002 (Rev. 03/2020 Approved Through 02/28/2023) Page Continuation Format Page