New Polymeric Carriers for Pulmonary Gene Delivery

描述(申请人提供)：基因疗法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传递技术，其临床应用尚未实现。目前的阳离子聚合物具有低的转染率，并且由于过度的电荷和不可生物降解的性质而对肺部有毒。此外，存在于质粒DNA中的未甲基化的CpG基团在与载体形成络合物时可能会引发炎症反应。我们假设，通过设计具有最小尺寸和低含量未甲基化CpG基团的多功能、更生物相容的载体和基因序列，可以获得更安全和更有效的非病毒基因递送系统。我们描述了一条新的合成路线，该路线合成了一类电荷密度可调、间距基团可变、受阻氮基含量高、pKa较低的多羟基亚胺。DNA/聚合物复合体对细胞和呼吸道的转染率是聚乙烯亚胺/DNA复合体的数倍。新聚合物的毒性比聚乙烯亚胺要低得多。更重要的是，可还原的二硫键和疏水基团可以添加到骨架上，使这些聚合物分别可生物降解和更有效。我们建议通过调节聚合物的性质，如DNA缩合、膜活性和生物降解性来识别最活跃和毒性最低的聚合物。我们还建议通过对这些聚合物的物理化学性质、转染机制以及体内和体外代谢谱的详细表征来更好地了解这种新的基因传递系统。此外，还将测试覆盖有ICAM-1特异性抗体的聚合物/DNA复合体的效果，以降低聚合物/DNA复合体的表面电荷，并通过受体介导的内吞作用触发特定的细胞摄取。最后，含有一个小尺寸功能基因表达盒的DNA片段将在体内进行测试，以减少炎症反应。结合这些创新，我们将建立一个安全、高效的非病毒基因输送系统，适合于呼吸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.