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，而目前的药物治疗通常对根除疾病的根本原因作用甚微。成功的分子治疗包括精确了解疾病中的基因缺陷，然后递送核酸以调节基因表达。可以通过递送质粒DNA（pDNA）来增强基因表达或通过递送短干扰RNA（siRNA）来沉默基因表达。但是，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此目的的安全递送载体，但它们具有低转染效率和高毒性。这个发现项目的目的是创造“工程”聚合物，可以与核酸自组装，以创造理想的细胞输送纳米粒子。 我们一直在设计一类由疏水脂质和阳离子聚合物构建的两亲聚合物。我们的工作使我们选择低分子量（0.6 - 2.0 kDa）聚乙烯亚胺（PEI）作为最佳起始材料。发现疏水部分在自组装纳米颗粒之间产生桥梁，通过脂质-脂质缔合稳定结构，并将货物递送到细胞中提高多达10倍。上一个供资期间没有处理的一个具体问题是货物的控制下交付和放行，这是取得最佳功能成果所必需的。拟议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质工程化，用于在细胞中控制核酸的递送和释放，最终导致由核酸介导的改善的功能结果。将追求结构-功能关系，以更好地理解限制核酸递送的障碍和克服这些障碍的方法。该项目的具体目标是：目标1。设计具有用于内体逃逸的改进的脂质取代基的小分子量（<2kDa）两亲聚合物的文库。我们以前的工作探讨了有限范围的脂质，并建立了内体逃逸功能特征的初步信息。这一努力将通过探索具有有前途的功能特征的其他脂质取代基来扩展。将表征聚合物文库的相关物理化学性质以及核酸的细胞递送。目标二通过不稳定的键将有效的脂质结合到聚合物上。使用短的聚合物主链，所选择的脂质将通过可裂解的键锚定到聚合物主链上。将表征聚合物的稳定性以及所得纳米颗粒的细胞内运输。目标三确定用于核酸递送的聚合物的功能性能。我们预期聚合物载体和核酸之间形成的自组装复合物的性质决定功能结果。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。将通过探索更安全的替代品（即，聚合物）到当前的递送载体（即，病毒）。在探索分子组装成纳米颗粒的同时，该项目的成功完成将开发出有效和安全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.