Reversing Electrostatic Interactions for Improved Gene Delivery

描述(申请人提供)：拟议工作的总体目标是利用分子间力的原理来设计用于基因传递的改进的阳离子两亲性/DNA超分子组件。具体地说，我们将设计、合成和评估用于基因传递的新型电荷反转两亲分子。这些功能性两亲分子在细胞内经历了从阳离子到阴离子的静电转变，从而从超分子组装中释放出DNA。我们假设，这种与DNA的静电相互作用的变化将转化为提高基因转染效率。一项详细的机理研究被提出，这项为期四年的提案需要以下三个具体目标：目的1.确定两亲分子的关键分子特征，1)DNA结合和从超分子组装中释放DNA，2)脂双层的不稳定。目的2.表征电荷反转两亲性和两亲性/DNA超分子组装体。目的3.体外评价电荷反转两亲性DNA超分子组装体与细胞的功能相互作用，并将P53基因导入乳腺癌细胞。这些目标的完成将提供：(1)鉴定一个或多个能将DNA输送到细胞的电荷反转两亲体；(2)用这些电荷反转两亲体递送核酸的机制；(3)了解这些两亲体在特定细胞类型中递送基因的优势和局限性；(4)展示一种与目前的基因递送载体概念不同的新方法；(5)将p53基因递送到乳腺癌细胞；以及(6)潜在地确定基因递送中限速步骤的性质。乳腺癌是一种将受益于改进或替代治疗方案的疾病。今天，转移性乳腺癌没有标准的治疗标准；所有一线联合疗法都被认为是同样有效的，缓解率约为60%。因此，我们把重点放在肿瘤抑制基因(P53)的传递上，用于乳腺癌的治疗。

DESCRIPTION (provided by applicant): The overall goal of the proposed effort is to use the principles of intermolecular forces to design improved cationic amphiphile/DNA supramolecular assemblies for gene delivery. Specifically, we will design, synthesize, and evaluate new charge-reversal amphiphiles for gene delivery. These functional amphiphiles undergo an electrostatic transition from cationic to anionic in cells to release DNA from the supramolecular assembly. We hypothesize that this change in electrostatic interactions with DNA will translate to enhanced gene transfection efficiency. A detailed mechanistic investigation is proposed which entails the following three specific aims for this four-year proposal: Aim 1. Determine the key molecular characteristics of the amphiphile required for 1) DNA binding and release of DNA from the supramolecular assembly and 2) destabilization of lipid bilayers. Aim 2. Characterize the charge-reversal amphiphiles and amphiphile/DNA supramolecular assemblies. Aim 3. Evaluate functional interactions of charge-reversal amphiphile/DNA supramolecular assemblies with cells in vitro and deliver the p53 gene to breast cancer cells. Completion of these aims will afford: (1) the identification of one or more charge-reversal amphiphiles that can deliver DNA to cells; (2) the mechanism of nucleic acid delivery with these charge-reversal amphiphiles; (3) an understanding of the advantages and limitations of these amphiphiles for gene delivery in a given cell type; (4) the demonstration of a new approach that is a conceptual departure from the current gene delivery vectors; (5) delivery of the p53 gene to breast cancer cells; and (6) potentially identify the nature of the rate- limiting step in gene delivery. Breast cancer is one disease that would benefit from improved or alternative treatment options. Today, there is no standard of care for metastatic breast cancer; all of the first-line combination therapies are regarded as equally efficacious at about a 60% response rate. Thus, we are focusing on the delivery of the tumor suppressor gene (p53) for the treatment of breast cancer.