Reversing Electrostatic Interactions for Improved Gene Delivery

描述（由申请人提供）：拟议工作的总体目标是使用分子间力的原理来设计用于基因递送的改进的阳离子两亲物/DNA超分子组装体。具体来说，我们将设计，合成和评估新的电荷反转两亲基因传递。这些功能性两亲物在细胞中经历从阳离子到阴离子的静电转变，从超分子组装体中释放DNA。我们假设这种与DNA静电相互作用的变化将转化为增强的基因转染效率。提出了一个详细的机制调查，这需要以下三个具体目标，这四年的建议：目标1。确定1）DNA结合和DNA从超分子组装体中释放以及2）脂质双层不稳定所需的两亲物的关键分子特征。目标2.表征电荷反转两亲物和两亲物/DNA超分子组装体。目标3。评价电荷反转两亲物/DNA超分子组装体与体外细胞的功能相互作用并将p53基因递送至乳腺癌细胞。这些目标的完成将提供：（1）鉴定一种或多种可以将DNA递送到细胞的电荷反转两亲物;（2）用这些电荷反转两亲物递送核酸的机制;（3）理解这些两亲物在给定细胞类型中用于基因递送的优点和局限性;（4）展示了一种新的方法，该方法在概念上偏离了当前的基因递送载体;（5）将p53基因递送至乳腺癌细胞;以及（6）潜在地鉴定了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。目前，转移性乳腺癌没有标准治疗;所有一线联合治疗都被认为同样有效，缓解率约为60%。因此，我们专注于肿瘤抑制基因（p53）的递送用于治疗乳腺癌。

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.