Tailored Polymers for Delivery of Gene Medicines

分子疗法为治疗多种遗传性疾病提供了最好的希望。与目前的药物治疗相比，永久修复缺陷基因的能力是可取的，目前的药物治疗通常对根除疾病的根本原因几乎没有起到什么作用。成功的分子治疗包括精确了解疾病中的基因缺陷，然后输送核酸来调节基因表达。通过递送质粒DNA(PDNA)来增强基因表达，或者通过递送短干扰RNA(SiRNA)来沉默基因表达是可能的。但治疗的成功完全取决于基因载体的使用，因为核酸不能被细胞内化。阳离子聚合物是用于这一目的的安全传递载体，但它们的转染率低且毒性高。这一发现项目的目标是创造能够与核酸自组装的“工程”聚合物，从而创造出理想的细胞输送纳米颗粒。我们一直在设计一类由疏水性脂类和阳离子聚合物构建的两亲性聚合物。我们的工作使我们找到了低分子量(0.6-2.0 kDa)的聚乙烯亚胺(PEI)作为最佳的起始材料。疏水部分被发现在自组装的纳米颗粒之间建立了桥梁，通过脂-脂结合稳定了结构，并将货物运送到细胞中的能力提高了10倍。前一个供资期间没有解决的一个具体问题是货物的受控交付和释放，这是实现最佳功能成果所必需的。拟议的项目旨在解决这一问题。我们的总体假设是：两亲性聚合物可以由阳离子聚合物和脂类制成，用于控制核酸在细胞中的传递和释放，最终导致由核酸介导的功能结果的改善。将探讨结构与功能的关系，以更好地了解核酸输送的限制障碍和克服这些障碍的方法。该项目的具体目标是：AIM-1。设计一个小分子量(&lt；2 kDa)两亲性聚合物文库，其具有改进的脂类取代基，可用于内体逃逸。我们以前的工作探索了有限范围的脂类，并建立了关于内体逃逸的功能特征的初步信息。这一努力将通过探索其他具有有前景的功能特性的类脂取代基来扩大。将对聚合物库的相关物理化学性质以及核酸的细胞递送进行表征。AIM-2。通过不稳定的键将有效的脂质偶联到聚合物上。利用短的聚合物骨架，选定的脂类将通过可切割的连接固定在聚合物骨架上。聚合物的稳定性以及生成的纳米颗粒在细胞内的运输将被表征。目的：确定用于核酸输送的聚合物的功能性能。我们预测聚合物载体和核酸之间形成的自组装复合体的性质来决定功能结果。这一目标将在体外探索相关细胞系统的功能结果。拟议项目的结果将对人类疾病的分子疗法的发展产生重大影响。将通过探索现有运载工具(即病毒)的更安全替代品(即聚合物)的有效性，为聚合物引导的遗传剂输送奠定基础。在探索将分子组装成纳米粒子的同时，该项目的成功完成将开发出有效和安全地应用于分子治疗的功能材料。

Molecular therapy offers the best promise of a cure for a wide range of genetic disorders. The ability to permanently ‘fix’ defective genes is desirable over current drug therapy, which typically do little to eradicate the underlying cause of the disease. Successful molecular therapy involves a precise knowledge of a gene defect in a disease, followed by delivery of nucleic acids to modulate gene expression. It is possible to enhance gene expression by delivering plasmid DNAs (pDNA) or silence gene expression by delivering short interfering RNA (siRNA). But therapeutic success is absolutely dependent on the use of gene carriers since nucleic acids cannot be internalized by cells. Cationic polymers are safe delivery vehicles for this purpose but they suffer from low transfection efficiencies and high toxicities. This Discovery Project aims to create ‘engineered’ polymers that can self-assemble with nucleic acids to create nanoparticles ideal for cellular delivery. We have been engineering a class of amphiphilic polymers constructed from hydrophobic lipids and cationic polymers. Our work has led us to low molecular weight (0.6 – 2.0 kDa) polyethylenimines (PEI) as the optimal starting material. Hydrophobic moieties were found to create bridges among self-assembled nanoparticles, stabilizing the structure via lipid-lipid associations and enhancing delivery of the cargo into cells by as much as 10-fold. A specific issue not addressed in the previous funding period was controlled delivery and release of the cargo, which is needed for optimal functional outcomes. The proposed project is intended to address this issue. Our overall hypothesis is: amphiphilic polymers can be engineered from cationic polymers and lipids for controlled delivery and release of nucleic acids in cells, ultimately leading to improved functional outcomes mediated by nucleic acids. Structure-function relationships will be pursued to better understand limiting barriers for delivery of nucleic acids and approaches to overcome these barriers. Specific aims of this project are:Aim-1. To design a library of small molecular weight (<2 kDa) amphiphilic polymers with improved lipid substituents for endosomal escape. Our previous work explored a limited range of lipids and established preliminary information on functional features for endosomal escape. This effort will be expanded by exploring other lipid substituents with promising functional features. The relevant physicochemical properties of the polymer library will be characterized as well as cellular delivery of nucleic acids. Aim-2. To conjugate effective lipids to polymers via labile linkages. Using short polymeric backbones, the chosen lipids will be anchored onto polymer backbones via cleavable linkages. The stability of the polymers will be characterized as well as the intracellular trafficking of resultant nanoparticles.Aim-3. To determine functional performance of polymers for nucleic acid delivery. We anticipate the properties of self-assembled complexes formed between polymeric carriers and nucleic acids to dictate the functional outcomes. This aim will probe the functional outcomes in relevant cellular systems in vitro. The outcome of the proposed project will have significant impact on development of molecular therapies for human diseases. A foundation for polymer-guided delivery of genetic agents will be established by exploring the effectiveness of safer alternatives (i.e., polymers) to current delivery vehicles (i.e., viruses). While exploring molecular assembly into nanoparticles, functional materials for effective and safe application of molecular therapy will be developed as a result of successful completion of this project.