LYOPHILIZATION OF NONVIRAL GENE DELIVERY SYSTEMS

更有效的合成基因传递系统将继续开发并在人体临床试验中进行测试。虽然在改进非病毒载体方面取得了很大进展，但一些关键的药物问题仍未得到解决。例如，非病毒载体聚集的趋势限制了该技术广泛使用的潜力。这种不稳定性导致了产品质量的变化，并且不允许在使用前对制剂进行广泛的测试。因此，人们对开发稳定的冻干制剂产生了极大的兴趣，这种制剂可以在室温下运输和储存。初步研究清楚地表明，冷冻干燥过程显著改变了非病毒载体的物理特性，降低了其生物活性。利用经验方法，这些研究还表明，在急性冻干胁迫下，双糖能够保护合成的递送载体，但尚未解决在储存过程中稳定或保存的机制。先前关于脂质体和蛋白质的研究提出了两种相互竞争的机制来解释糖提供的保护作用。然而，目前尚不清楚这些机制是否适用于非病毒载体的稳定。本提案中描述的工作将测试辅料在干燥状态下稳定其他生物药物的机制是否适用于用于基因传递的大分子复合物。认识到许多不同类型的非病毒载体正在被优化用于基因治疗，我们研究的主要目标是制定合理的配方指南，这些指南通常适用于在冷冻干燥和储存期间稳定dna大分子复合物。除了设计赋形剂配方来稳定冻干制剂外，这些研究将有助于更好地表征合成基因传递系统的“天然状态”。

More efficient synthetic gene delivery systems continue to be developed and tested in human clinical trials. While much progress has been made in improving non-viral vectors, some critical pharmaceutical problems remain unresolved. For example, the tendency of non-viral vectors to aggregate limits the potential for widespread use of this technology. This instability introduces variability in product quality and does not allow preparations to be extensively tested prior to use. As a result, there is tremendous interest in developing stable lyophilized formulations that could be shipped and stored at ambient temperatures. Preliminary studies have clearly demonstrated that the freeze-drying process significantly alters physical characteristics and reduces biological activity of non-viral vectors. Utilizing an empirical approach, these studies have also shown that disaccharides are capable of protecting synthetic delivery vehicles during acute lyophilization stress, but have yet to address the mechanism of stabilization or preservation during storage. Previous work with liposomes and proteins has suggested two competing mechanisms to explain protection provided by sugars. However, it is unclear whether either of these mechanisms is applicable to the stabilization of non-viral vectors. The work described in this proposal will test whether the mechanisms employed by excipients to stabilize other biopharmaceuticals in the dried state are applicable to macromolecular complexes used in gene delivery. Recognizing that many different types of non-viral vectors are being optimized for gene therapy, the primary goal of our study is to develop rational formulation guidelines that are generally applicable to the stabilization of DNA-based macromolecular complexes during freeze- drying and storage. In addition to designing excipient formulations to stabilize lyophilized preparations, these studies will help to better characterize the "native state" of synthetic gene delivery systems.