FUNCTIONAL AND TARGETING POLYMERIC GENE CARRIERS

描述（由申请人提供）：细胞疗法正成为终末期心血管疾病患者广泛接受的治疗方法。可再注射的纯化的自体细胞是明显的选择，而原代成肌细胞已首先用于临床梗死后移植。一期试验已经确定了可行性和安全性，但这种方法的有效性仍然有些怀疑。这些原代成肌细胞已证实可移植并改善左心室功能，但有心律失常的不良事件。然而，心肌细胞保护和血管侧支不受主要影响。对注射的原代成肌细胞进行基因修饰，使其表达一种促血管生成、抗凋亡的分子，可以减少心肌损伤和瘢痕形成，并比未修饰的细胞产生更有效的反应。非病毒聚合物具有一些有利于缺血心肌细胞治疗的特性。由于只需要在短时间内表达，因此首选非病毒载体的瞬时表达。在细胞基因组中没有整合治疗基因来增加对无限制生长的恐惧。非病毒聚合物具有生物相容性和非免疫原性，因此在转染或随后的移植时几乎没有毒性。我们提出，与其他非病毒聚合物载体相比，这种新型生物可还原聚合物，聚（半胱胺双丙烯酰胺-二氨基己烷），聚（CBA-DAH）将以更高的效率和最小的毒性转染原代成肌细胞。这种新型的生物可还原聚合物在包括原代成肌细胞在内的几种不同细胞系中表现出高转染率和小毒性。这归因于聚合物一旦进入细胞质就具有可还原性。合成聚合物的物理化学性质将使用核磁共振（1H-NMR）和高效液相色谱（HPLC）等既定方法来确定。聚(CBA-DAH)/pCMV-VEGF多聚物将通过电泳迁移率转移测定、动态光散射（DLS）、zeta电位和细胞质降解动力学进行评估。将通过转基因原代成肌细胞体外和体内VEGF表达及其下游效应（qRT-PCR和Western blot分析，用于细胞凋亡和血管生成试验）来评估有效递送。在何处和多少细胞注射到梗死心肌的初步表征将有助于初步体内实验的表征。这将与免疫原性、毒性和安全性的生物分布研究相结合。该提案的完成将包括功能研究，包括磁共振成像，梗死大小，左心室壁厚度和大鼠模型中的白细胞浸润。公共卫生相关性：尽管冠状动脉疾病的细胞移植治疗取得了进展，但心脏缺血后的功能恢复仍然是发病率和死亡率的主要原因。目前，临床上尚无可靠的预防心肌缺血再灌注损伤的方法。在此应用中，我们建议开发一种新的生物可还原聚合物，将VEGF基因传递给原代成肌细胞，用于治疗急性心肌梗死，以解决目前治疗策略的这些不足。

DESCRIPTION (provided by applicant): Cellular therapy is becoming a widely accepted procedure for patients with end-stage cardiovascular disease. Purified autologous cells that can be re-injected are the obvious choice and primary myoblasts have been the first to be used in clinical post-infarction transplantation. Phase I trials have established feasibility and safety, but efficacy of this procedure remains somewhat skeptical. These primary myoblasts have demonstrated engraftment and improvement in left ventricular function but with adverse event of arrhythmia. However, cardiomyocyte protection and vessel collateralization of the myocardium is not primarily affected. Genetic modification of the injected primary myoblasts to express a pro-angiogenic, anti-apoptotic molecule may reduce myocardial damage and scar formation and elicit a more efficacious response than un-modified cells. Non-viral polymers possess several characteristics that are favorable for use in cellular therapy of ischemic myocardium. Since expression is only required for a short amount of time, the transient expression of non-viral carriers is preferred. There is no integration of the therapeutic genes within the cellular genome to raise the fear of unrestricted growth. Non-viral polymers are biocompatible and non-immunogenic so that little to no toxicity is present upon transfection or subsequent transplantation. We propose that the novel bioreducible polymer, poly(cystamine bisacrylamide-diaminohexane), poly(CBA-DAH) will transfect primary myoblasts with superior efficiency and minimal toxicity compared to other non-viral polymeric carriers. This novel bioreducible polymer has demonstrated high transfection rates and little toxicity in several different cell lines, including primary myoblasts. This has been attributed to the reducible nature of the polymer once it enters the cytosol. Physiochemical properties of the synthesized polymer will be ascertained using established methods such as nuclear magnetic resonance (1H-NMR) and high performance liquid chromatography (HPLC). The poly(CBA-DAH)/pCMV-VEGF polyplexes will be evaluated by electrophoretic mobility shift assay, dynamic light scattering (DLS), zeta potential, and cytosolic degradation kinetics. Effective delivery will be evaluated by in vitro and in vivo expression of VEGF by genetically modified primary myoblasts and its downstream effects (qRT-PCR and Western blot analysis, for apoptosis and angiogenesis assays). Primary characterization of where and how many cells to inject into infarcted myocardium will assist in characterizing initial in vivo experiments. This will be paired with immunogenicity, toxicity and biodistribution studies for safety. Completion of the proposal will include functional studies consisting magnetic resonance imaging, infarct size, left ventricular wall thickness, and leukocyte infiltration in rat model. PUBLIC HEALTH RELEVANCE: Despite advances in cellular transplantation therapies for coronary artery disease, functional recovery following cardiac ischemia remains a major cause of morbidity and mortality. At the present time, there is no clinically reliable means to prevent ischemia/reperfusion injury to the myocardium. In this application, we propose to develop a novel bioreducible polymer to deliver VEGF gene to primary myoblasts for the treatment of acute myocardial infarct to address these short- comings of current therapeutic strategies.