FUNCTIONAL AND TARGETING POLYMERIC GENE CARRIERS

功能性和靶向性聚合基因载体

• 批准号: 8130654
• 负责人: SUNG WAN KIM
• 金额: $ 33.86万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-15 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8130654
• 关键词: Acute; Acute myocardial infarction; Address; Adverse event; Affect; Angiogenic Factor; Animal Model; Apoptosis; Apoptotic; Area; Arrhythmia; Autologous; Biodistribution; Biological Assay; Blood Vessels; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cell Line; Cells; Characteristics; Cicatrix; Clinical; Confocal Microscopy; Coronary Arteriosclerosis; Cystamine; Cytosol; Development; Electrophoretic Mobility Shift Assay; Engineering; Engraftment; Enzymes; Evaluation; Fluorescence-Activated Cell Sorting; Fright; Genes; Genetic; Genome; Growth; Health; Hemangioma; High Pressure Liquid Chromatography; Immune response; In Vitro; Infarction; Infiltration; Ischemia; Kinetics; Left; Left Ventricular Function; Leukocytes; Link; Magnetic Resonance Imaging; Measurement; Mediating; Methods; Modeling; Modification; Morbidity - disease rate; Myoblasts; Myocardial; Myocardial Ischemia; Myocardium; Natural regeneration; Nature; Nuclear Magnetic Resonance; Patients; Phase I Clinical Trials; Polymers; Procedures; Proliferating; Property; Rattus; Recovery of Function; Reperfusion Injury; Safety; Skeletal Muscle; Staging; Stem cells; Therapeutic; Thick; Time; Toxic effect; Transfection; Transplantation; Vascular Endothelial Growth Factors; Vascular blood supply; Ventricular; Viral; Viral Vector; Western Blotting; angiogenesis; biocompatible polymer; cytotoxicity; disulfide bond; gene therapy; immunogenicity; improved; in vivo; interest; light scattering; mortality; myogenesis; nephelometry; novel; prevent; research study; response; safety study; therapeutic angiogenesis; therapeutic gene; zeta potential

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.

描述(由申请人提供)：细胞治疗正在成为终末期心血管疾病患者广泛接受的程序。可以再次注射的纯化的自体细胞是显而易见的选择，原代成肌细胞已经成为临床脑梗塞后移植的第一个使用细胞。I期试验已经确定了可行性和安全性，但该程序的有效性仍有一些疑问。这些原代成肌细胞表现出植入和改善了左心功能，但有心律失常的不良事件。然而，心肌细胞的保护和心肌的血管侧支并不主要受到影响。对注射的原代成肌细胞进行基因修饰，表达一种促血管生成、抗凋亡的分子，可能会减少心肌损伤和疤痕形成，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特性，有利于用于缺血心肌的细胞治疗。因为只需要表达很短的时间，所以最好是非病毒载体的瞬时表达。治疗基因没有整合到细胞基因组中，从而引发了对不受限制的生长的恐惧。非病毒聚合物具有生物相容性和非免疫原性，因此在转基因或随后的移植中几乎没有毒性。我们认为，与其他非病毒载体相比，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低的毒性转入原代成肌细胞。这种新型的生物可还原聚合物在几种不同的细胞系中表现出高转染率和低毒性，包括原代成肌细胞。这归因于聚合物一旦进入细胞质就具有可还原的性质。合成聚合物的物理化学性质将使用现有的方法，如核磁共振(1H-核磁共振)和高效液相色谱(HPLC)来确定。通过凝胶迁移率改变分析、动态光散射(DLS)、Zeta电位和胞浆降解动力学对聚(CBA-DAH)/pCMV-VEGF多聚体进行了评价。通过转基因原代成肌细胞在体外和体内表达血管内皮生长因子及其下游效应(qRT-PCR和Western印迹分析，用于细胞凋亡和血管生成检测)来评估有效的传递。初步确定在哪里以及有多少细胞注入梗塞心肌将有助于初步的活体实验。这将与免疫原性、毒性和生物分布研究相结合，以确保安全性。该提案的完成将包括功能研究，包括磁共振成像，梗死面积，左室壁厚度，以及大鼠模型中的白细胞渗透。公共卫生相关性：尽管细胞移植疗法在治疗冠状动脉疾病方面取得了进展，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。目前尚无临床上可靠的预防心肌缺血/再灌注损伤的方法。在这一应用中，我们建议开发一种新型的可生物还原的聚合物，将血管内皮生长因子基因转移到原代成肌细胞，用于治疗急性心肌梗死，以解决目前治疗策略的这些不足。