New Polymeric Carriers for Pulmonary Gene Delivery

描述（由申请人提供）：基因治疗具有治疗各种肺部疾病的潜力。然而，由于缺乏安全有效的基因递送技术，其临床应用尚未实现。目前的阳离子聚合物具有低的转染效率，并且由于过量的电荷和不可生物降解的性质而对肺有毒。此外，质粒DNA中存在的未甲基化CpG基团在与载体复合递送时可能引发炎症反应。我们假设，一个更安全，更有效的非病毒基因传递系统可以通过设计多功能，更生物相容性的载体和基因序列，具有最小的尺寸和低含量的非甲基化的CpG基团。我们在这里描述了一种新的合成路线，导致一类多羟基亚烷基胺与可调的电荷密度，可变间距基团和高含量的受阻氮与降低pKa。使用DNA/聚合物复合物的细胞和气道转染显示出比使用聚乙烯亚胺/DNA复合物的转染活性高几倍。新的聚合物显示出比聚乙烯亚胺低得多的毒性。更重要的是，可还原的二硫键和疏水基团可以添加到主链上，分别使这些聚合物生物可降解和更有效。我们建议通过调整聚合物的性质，如DNA缩合，膜活性和生物降解性，以确定最活跃和毒性最小的聚合物。我们还建议更好地了解这种新的基因传递系统，通过详细表征的理化性质，转染机制和代谢概况，这些聚合物在体外和体内。此外，将测试用ICAM-1特异性抗体修饰的聚合物/DNA复合物减少聚合物/DNA复合物表面电荷和通过受体介导的内吞作用触发特异性细胞摄取的效果。最后，将在体内测试含有具有最少未甲基化CpG含量的小尺寸功能性基因表达盒的DNA片段以减少炎症反应。结合这些创新，我们将建立一个安全，有效的非病毒基因传递系统，适用于气道输送和其他器官的潜在应用。

DESCRIPTION (provided by applicant): Gene therapy has the potential for treating various lung diseases. However, its clinical use is unrealized because of the lack of a safe and efficient gene delivery technology. Current cationic polymers have low transfection efficiencies and are toxic to the lungs due to excessive charges and non-biodegradable nature. Also, unmethylated CpG groups that exist in plasmid DNA could trigger an inflammatory response when it is delivered as complex with a carrier. We hypothesize that a safer and more efficient nonviral gene delivery system can be derived by designing multifunctional, more biocompatable carriers and gene sequences that have minimal size and low content of unmethylated CpG groups. We described here a new synthetic route that led to a class of polyhydroxylalkyleneamine with adjustable charge densities, variable spacing groups and a high content of hindered nitrogens with lowered pKa. Cell and airway transfection using DNA/polymer complexes showed several fold higher transfection activity than those using polyethylenimine/DNA complexes. The new polymers showed much reduced toxicity than polyethylenimine. More importantly, reducible disulfide bonds and hydrophobic groups can be added to backbones to make these polymers biodegradable and more potent, respectively. We propose to identify most active and least toxic polymers by adjusting polymer properties such as DNA condensation, membrane activity and biodegradability. We also propose to better understand this new gene delivery system through detailed characterization of physicochemical properties, mechanism of transfection and metabolism profiles of these polymers in vitro and in vivo. In addition, polymer/DNA complexes dressed with antibodies specific to ICAM-1 will be tested for their effect to reduce the surface charges of the polymer/DNA complex and to trigger specific cell uptake via receptor-mediated endocytosis. Lastly, a DNA fragment containing a small-sized functional gene expression cassette with minimal unmethylated CpG contents will be tested in vivo to reduce inflammation response. Combining these innovations, we will establish a safe and efficient non-viral gene delivery system suitable for the airway delivery and potential applications for other organs.