Gene Delivery of P53 in a Tumor-bearing Mouse Model

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传递p53基因已被证明可减少肿瘤生长。虽然P53基因治疗可能包括几种抗肿瘤机制，但抗血管生成是P53抑制肿瘤生长的重要机制之一。与其他编码抗血管生成蛋白的基因一样，阳离子脂质体提供的p53基因治疗也因体内转染率不足而受阻。最近，我们实验室开发了线性和支化的HK(组氨酸-赖氨酸)聚合物，与脂质体相结合，在有或没有血清的情况下显着增加了转染率。为了验证这样一个假设，即进一步了解HK增强转染将促进HK载体的发展，用于体内抗血管生成基因治疗，计划实现以下具体目标。目的1比较脂质体和HK/脂质体作为p53基因载体对肿瘤生长的抑制作用。由于肿瘤血管很容易获得系统治疗，靶向肿瘤内皮细胞增加了基因治疗成功的可能性。两种有前景的HK/脂质体/P53质粒复合体将与脂质体/P53质粒复合体进行比较，以研究它们在体内减少肿瘤血管生成和生长的能力。为了提高p53在体内的特异性和抗肿瘤效果，这些含HK的载体将被血管特异性环肽(-NGR-)和亲水性屏蔽物(PEG)修饰。由于肿瘤内皮是体内复合体的主要靶点，我们将检验我们的假设，即在脂质体/p53质粒复合体中加入HK聚合物将显著改善基因治疗，从而产生更大的肿瘤抑制作用。由于络合物在血清中的不稳定性限制了体内的疗效，AIM 2旨在阐明在有血清存在的情况下，香港聚合物增强脂质体DNA络合物稳定性的机制。与多聚L赖氨酸相比，HK的存在保持了高水平的基因表达，并显著增强了脂质体：DNA复合体对血清的抵抗力，即使在长时间孵育的情况下也是如此。在血清存在下，组氨酸的氢键或疏水性质是否稳定HK：脂质体：DNA复合体将被测试。在目标3中，将研究HK与DNA的结合在转染中的作用，以检验这些聚合物与DNA的不同亲和力与细胞内体pH之间的相互作用是导致转染率变化的原因。改变组氨酸和赖氨酸比例的能力以及HK聚合物的复杂性可能会改变内体的pH，这可能会影响结合和转染率。了解这些控制DNA从HK/脂质体载体释放的性质将有助于改进载体的开发。AIM 4旨在利用上述目的收集的信息来比较HK的衍生物抑制肿瘤生长的能力。根据先前目标中确定的机械研究，将开发改进的香港航母设计。将几种HK聚合物与目标1中的最佳载体相结合，比较它们在体内增强p53的抗肿瘤活性的能力。我们将研究经修饰的含HK的P53载体对肺转移瘤和原位移植瘤的抗肿瘤活性。

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.