FUNCTIONAL AND TARGETING POLYMERIC GENE CARRIERS

功能性和靶向性聚合基因载体

• 批准号: 8268422
• 负责人: SUNG WAN KIM
• 金额: $ 33.52万
• 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 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8268422
• 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治疗方法。纯化的自体细胞可以再次注射是明显的选择，原代成肌细胞已首先被用于临床梗死后移植。I期临床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植入并改善左心室功能，但伴有心律失常的不良事件。然而，心肌细胞保护和心肌的血管侧支化并未受到主要影响。对注射的原代成肌细胞进行基因修饰以表达促血管生成、抗凋亡分子可以减少心肌损伤和瘢痕形成，并引起比未修饰细胞更有效的反应。非病毒聚合物具有几个有利于用于缺血心肌细胞治疗的特性。由于表达仅需要很短的时间，因此优选非病毒载体的瞬时表达。治疗基因在细胞基因组中没有整合，不会引起对无限制生长的恐惧。非病毒聚合物是生物相容的和非免疫原性的，使得在转染或随后的移植时几乎不存在毒性。我们提出，新型生物可还原聚合物，聚（胱胺双丙烯酰胺-二氨基己烷），聚（CBA-DAH）将以上级效率和最小毒性与其他非病毒聚合物载体相比，抑制原代成肌细胞。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胞质溶胶就具有可还原性。将使用已建立的方法如核磁共振（1H-NMR）和高效液相色谱（HPLC）来确定合成聚合物的理化性质。将通过电泳迁移率变动分析、动态光散射（DLS）、zeta电位和胞质降解动力学评价聚（CBA-DAH）/pCMV-VEGF复合物。将通过遗传修饰的原代成肌细胞的VEGF的体外和体内表达及其下游效应（用于细胞凋亡和血管生成测定的qRT-PCR和蛋白质印迹分析）来评价有效递送。将细胞注射到梗死心肌中的位置和数量的初步表征将有助于表征初始的体内实验。这将与安全性的免疫原性、毒性和生物分布研究配对。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，但心肌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，还没有临床上可靠的方法来防止心肌缺血/再灌注损伤。在本申请中，我们提出开发一种新的生物可还原聚合物以将VEGF基因递送至原代成肌细胞用于治疗急性心肌梗死，以解决当前治疗策略的这些不足。

项目成果

Synergistically combined gene delivery for enhanced VEGF secretion and antiapoptosis.

• DOI: 10.1021/mp400178m
• 发表时间: 2013-10-07
• 期刊: Molecular pharmaceutics
• 影响因子: 4.9
• 作者: Won YW;Lee M;Kim HA;Nam K;Bull DA;Kim SW
• 通讯作者: Kim SW

Polymer transfected primary myoblasts mediated efficient gene expression and angiogenic proliferation.

• DOI: 10.1016/j.jconrel.2009.09.021
• 发表时间: 2010-02-25
• 期刊: JOURNAL OF CONTROLLED RELEASE
• 影响因子: 10.8
• 作者: Ou, Mei;Kim, Tae-il;Yockman, James W.;Borden, Bradley A.;Bull, David A.;Kim, Sung Wan
• 通讯作者: Kim, Sung Wan

Bioreducible polymers for therapeutic gene delivery.

• DOI: 10.1016/j.jconrel.2014.04.012
• 发表时间: 2014-09-28
• 期刊: Journal of controlled release : official journal of the Controlled Release Society
• 作者: Lee YS;Kim SW
• 通讯作者: Kim SW

Bioreducible polymers for gene delivery.

• DOI: 10.1016/j.reactfunctpolym.2010.11.016
• 发表时间: 2011-03-01
• 期刊: REACTIVE & FUNCTIONAL POLYMERS
• 影响因子: 5.1
• 作者: Kim, Tae-il;Kim, Sung Wan
• 通讯作者: Kim, Sung Wan

Bioreducible polymer-transfected skeletal myoblasts for VEGF delivery to acutely ischemic myocardium.

• DOI: 10.1016/j.biomaterials.2010.09.061
• 发表时间: 2011-01
• 期刊: BIOMATERIALS
• 影响因子: 14
• 作者: McGinn, Arlo N.;Nam, Hye Yeong;Ou, Mei;Hu, Norman;Straub, Catherine M.;Yockman, James W.;Bull, David A.;Kim, Sung Wan
• 通讯作者: Kim, Sung Wan