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

项目主任/首席调查员(末位、第一位、中位)：关建军 项目摘要 糖尿病合并严重肢体缺血(CLI)的截肢率和死亡率明显较高。 CLI的特点是血液灌注量极低，骨骼肌退化。相应地，再生 血管系统和骨骼肌将挽救四肢。然而，内皮细胞和骨骼肌细胞的存活率很低， 糖尿病CLI在高血糖和缺血状态下的下层细胞功能损害肢体修复。 目前，尽管生长因子疗法是一种有希望的策略，但目前还没有有效的治疗方法。 然而，生长因子疗法在血管再生和骨骼再生方面的疗效相对较低。 肌肉，因为多种生长因子同时需要血管形成和肌肉生成，而这些不能 通过目前的方法可以很容易地实现。 在这个项目中，我们建议使用一种新的具有促血管生成和促肌生成特性的TRIM72蛋白 在糖尿病CLI中再生血管和骨骼肌。TRIM72的设计将使其拥有更长的 在缺血组织中的滞留时间(扩散速度较慢)，因此显示出更长的治疗效果。要传递 经过改造的TRIM72(ETRIM72)，它将被封装到缺血肢体靶向纳米颗粒中，随后 通过临床上有吸引力的静脉注射进行分娩。然后，纳米颗粒将主要积聚在缺血区 四肢并逐渐释放ETRIM72。释放的蛋白质将通过(1)促进血管形成和肌肉生成 通过细胞膜修复和细胞活化提高内皮细胞和骨骼肌细胞的存活率 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的高血糖和缺血状态。 在我们的初步研究中，我们通过将TRIM72与多肽CSTSMLKAC进行遗传融合而开发出ETRIM72 它针对的是缺血肢体的缺血环境。ETRIM72的第一个版本能够在缺血时保留 四肢明显长于TRIM72。在静脉注射缺血肢体靶向后，ETRIM72释放纳米粒， 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。TRIM72在高血糖和高血糖状态下促进血管生成和肌肉生成的作用 以前还没有关于缺血情况的报道。 根据我们的初步研究，我们假设ETRIM72的受控释放将同时增加 高血糖和缺血条件下内皮细胞和骨骼肌细胞的存活、迁移和形态发生 导致糖尿病缺血肢体血管和骨骼肌加速再生。 目标1将检验这样一个假设，即最优的ETRIM72释放谱将显著促进存活、迁移 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。AIM#2将测试 ETRIM72缓释纳米粒对糖尿病小鼠肢体缺血模型的疗效。 该项目具有创新性，因为它设计了一种新的前血管生成和前肌肉生成蛋白来 同时再生糖尿病缺血肢体的血管和骨骼肌。组织滞留时间越长 工程蛋白的时间以及局部和受控释放有望显著改善 治疗效果。 OMB编号0925-0001/0002(批准的第03/2020版至2023年2月28日)续页格式页

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