Formulation of Synthetic Vectors as Lyophilized Products

描述（由申请人提供）：这些胶体悬浮液作为液体配方的极端不稳定性阻碍了合成多核苷酸递送系统的发展。这种不稳定性也会影响基础研究，因为它迫使科学家们使用容易出现批次间显著差异的媒介制剂进行实验。因此，人们有兴趣开发方法，将合成载体配制成稳定的脱水制剂，可以批量制备，并在室温下运输和储存。本文提出的工作重点是开发合成载体的脱水方法，使其在急性冻干胁迫和长期储存期间保持物理特性和生物活性。先前对这些问题的研究表明，在急性冻干应激期间，通过使用大量的糖可以实现稳定。不幸的是，稳定所需的糖量与肌内或皮下注射的渗透性不相容；许多应用的首选给药方法，例如DNA疫苗。我们建议进行机制研究，以确定急性冻干胁迫下载体聚集的致病效应（损伤的主要机制），并寻求在等渗渗透压下实现稳定的策略。这些初步研究的结果将应用于研究两种提高储存稳定性的方法的实验。在第一种方法中，将直接评估配方在储存过程中减少活性氧积累的能力。这些实验利用了一种新颖的荧光技术，这是第一次允许在干燥的制剂中产生氧自由基进行监测。第二种方法与最近的报告一致，并质疑固态稳定性领域的传统教条，即“越干越好”。最终，这些方法的结果将在一项为期2年的完全优化配方的存储稳定性研究中结合起来。认识到不同类型的合成载体正在被优化用于基因/多核苷酸递送，这些实验采用不同的模型载体（例如，脂丛和多丛），以便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大分子复合物（例如，疫苗、病毒、纳米颗粒）。

DESCRIPTION (provided by applicant): The development of synthetic polynucleotide delivery systems is hampered by the extreme instability of these colloidal suspensions as liquid formulations. This instability also affects basic research by forcing scientists to conduct experiments with vector preparations that are prone to significant batch-to-batch variability. As a result, there is interest in developing methods that would allow synthetic vectors to be formulated as stable, dehydrated preparations that could be prepared in large batches, and shipped and stored at ambient temperatures. The work proposed here focuses on developing methods of dehydrating synthetic vectors such that their physical characteristics and biological activity are maintained during both acute lyophilization stress and prolonged storage. Previous work on these problems has demonstrated that stabilization can be achieved during acute lyophilization stress by employing high amounts of sugars. Unfortunately, the amount of sugar needed for stabilization is not osmotically compatible with intramuscular or subcutaneous injection; the preferred method of administration for many applications, e.g., DNA vaccines. We propose mechanistic studies to determine the causative effects of vector aggregation (the major mechanism of damage) during acute lyophilization stress, and pursue strategies that achieve stability at isotonic osmolalities. The findings from these initial studies will be applied to experiments investigating two approaches to enhance storage stability. In the first approach, formulations will be directly assessed for their ability to reduce the accumulation of reactive oxygen species during storage. These experiments utilize a novel fluorescence technique that, for the first time, allows the generation of oxygen radicals in dried preparations to be monitored. The second approach is consistent with recent reports and questions the conventional dogma in the field of solid-state stability that "drier is better". Ultimately, the results from these approaches will be combined in a 2-year storage stability study on fully-optimized formulations. Recognizing that different types of synthetic vectors are being optimized for gene/polynucleotide delivery, these experiments employ different model vectors (e.g., lipoplexes and polyplexes) in order to develop rational formulation guidelines that are generally applicable to the stabilization of macromolecular complexes (e.g., vaccines, viruses, nanoparticles) during freeze-drying and storage.