Gene Delivery of P53 in a Tumor-bearing Mouse Model

P53 在荷瘤小鼠模型中的基因传递

• 批准号: 6773406
• 负责人: ARCHIBALD JAMES MIXSON
• 金额: $ 27.4万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-05 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6773406
• 关键词: MCF7 cell; angiogenesis inhibitors; athymic mouse; biomaterial evaluation; biotechnology; clinical research; confocal scanning microscopy; disease /disorder model; fluorimetry; gene delivery system; gene expression; gene therapy; genetic transcription; human tissue; immunocytochemistry; in situ hybridization; laboratory mouse; liposomes; lung neoplasms; metastasis; neoplasm /cancer therapy; p53 gene /protein; polymers; skeletal neoplasm; terminal nick end labeling; transfection

DESCRIPTION (provided by applicant): Systemic gene delivery of p53 with cationic liposomes has been shown to reduce tumor growth in pre-clinical models. Although gene therapy with p53 may include several antiturnor mechanisms, antiangiogenesis is one important mechanism by which p53 inhibits tumor growth. Similar to other genes encoding antiangiogenic proteins, p53 gene therapy delivered by cationic liposomes is hampered by inadequate transfection in vivo. Recently our laboratory has developed linear and branched HK (histidine-lysine) polymers that in combination with liposomes significantly increased transfection in the presence or absence of serum. To test the overall hypothesis that further understanding of HK enhancement of transfection will facilitate the development of HK carriers for in vivo antiangiogenic gene therapy, the following specific aims are planned. Aim 1 will compare liposomes to HK/liposomes as gene delivery carriers of p53 for their ability to inhibit tumor growth. Since the tumor vasculature is readily accessible to systemic therapy, targeting tumor endothelial cells increases the likelihood that gene therapy will be successful. Two promising HK/liposome/p53 plasmid complexes will be compared to a liposome/p53 plasmid complex for their ability to reduce tumor angiogenesis and growth in vivo. To enhance specificity and antitumor efficacy of p53 in vivo, these HK-containing carriers will be modified with a vascular specific cyclic peptide (-NGR-) and a hydrophilic shield (PEG). Because the tumor endothelium is a primary target of complexes in vivo, we will test our hypothesis that the addition of HK polymers to liposome/p53 plasmid complexes will significantly improve gene therapy resulting in greater tumor inhibition. Since instability of the complexes in serum limits in vivo efficacy, Aim 2 is designed to clarify the mechanism by which the HK polymer enhances stability of liposome:DNA complexes in the presence of serum. In contrast to poly-L-lysine, the presence of HK maintains high level of gene expression and markedly enhances resistance of the liposome:DNA complex to serum, even with prolonged incubations. Whether hydrogen bonding or hydrophobic properties of histidine stabilize the HK:liposome:DNA complexes in the presence of serum will be tested. In Aim 3 the role of binding of HK to DNA in transfection will be examined to test the hypothesis that the interplay between different affinities of these polymers with DNA and the cell's endosomal pH accounts for variations in transfection efficiency. The ability to alter the histidine and lysine ratio and the complexity of the HK polymer is likely to change endosomal pH, which may affect binding and transfection efficiency. Understanding these properties that govern DNA release from the HK/liposome carrier within acidic endosomes will enable the development of improved carriers. Aim 4 is designed to use information gathered in the above aims to compare the derivatives of HK for their abilities to inhibit tumor growth. Based on the mechanistic studies determined in the previous aims, improved designs of the HK carrier will be developed. In combination with modified liposomes, several HK polymers will be compared with the optimal carrier in Aim 1 for their ability to augment the antitumor activity of p53 in vivo. The modified HK-containing carriers of p53 will be investigated for their antitumor activity of lung metastases and orthotopically implanted tumors.

描述（由申请人提供）：在临床前模型中，用阳离子脂质体全身性基因递送p53已被证明可以减少肿瘤生长。尽管 p53 基因治疗可能包括多种抗肿瘤机制，但抗血管生成是 p53 抑制肿瘤生长的重要机制之一。与编码抗血管生成蛋白的其他基因类似，通过阳离子脂质体递送的p53基因治疗因体内转染不足而受到阻碍。最近，我们的实验室开发了线性和支化 HK（组氨酸-赖氨酸）聚合物，与脂质体结合可显着提高存在或不存在血清的情况下的转染。为了检验进一步了解 HK 增强转染将促进 HK 载体用于体内抗血管生成基因治疗的开发的总体假设，计划了以下具体目标。目标 1 将比较脂质体与 HK/脂质体作为 p53 基因递送载体抑制肿瘤生长的能力。由于肿瘤血管系统很容易进行全身治疗，因此靶向肿瘤内皮细胞增加了基因治疗成功的可能性。将比较两种有前途的 HK/脂质体/p53 质粒复合物与脂质体/p53 质粒复合物减少体内肿瘤血管生成和生长的能力。为了增强p53在体内的特异性和抗肿瘤功效，这些含有HK的载体将用血管特异性环肽（-NGR-）和亲水盾（PEG）进行修饰。由于肿瘤内皮是体内复合物的主要靶标，因此我们将检验我们的假设，即在脂质体/p53质粒复合物中添加HK聚合物将显着改善基因治疗，从而产生更大的肿瘤抑制作用。由于复合物在血清中的不稳定性限制了体内功效，因此目的 2 旨在阐明 HK 聚合物在血清存在下增强脂质体：DNA 复合物稳定性的机制。与聚-L-赖氨酸相比，即使在长时间孵育的情况下，HK 的存在也能维持高水平的基因表达，并显着增强脂质体：DNA 复合物对血清的耐受性。将测试组氨酸的氢键或疏水特性是否在血清存在下稳定 HK:脂质体:DNA 复合物。在目标 3 中，将检查 HK 与 DNA 结合在转染中的作用，以检验以下假设：这些聚合物与 DNA 的不同亲和力和细胞内体 pH 之间的相互作用解释了转染效率的变化。改变组氨酸和赖氨酸比例以及 HK 聚合物复杂性的能力可能会改变内体 pH 值，从而影响结合和转染效率。了解这些控制酸性内体中 HK/脂质体载体 DNA 释放的特性将有助于开发改进的载体。目标 4 旨在使用上述目标中收集的信息来比较 HK 衍生物抑制肿瘤生长的能力。根据先前目标确定的机械研究，将开发香港航母的改进设计。与修饰的脂质体相结合，几种 HK 聚合物将与目标 1 中的最佳载体进行比较，以确定它们增强 p53 体内抗肿瘤活性的能力。将研究修饰后的含 HK 的 p53 载体对肺转移瘤和原位植入肿瘤的抗肿瘤活性。