Cell Penetration Profiling for Biotherapeutics

对于许多类别的肽、蛋白质和核酸药物来说，细胞内递送仍然是主要障碍 在药物开发中。这个障碍一直具有挑战性，因为许多方法用于测量 “细胞渗透”实际上测量的是总细胞摄取，包括捕获在细胞表面或细胞内部的物质。 内体。很长一段时间以来，测量细胞质渗透的方法无法获得，这导致了 大大减缓了几类生物治疗药物的发展。此外，在没有基本了解的情况下 以时间分辨、细胞类型特异性的方式进行细胞质渗透，我们永远不会超越试验- 和错误作为开发针对特定组织中特定疾病目标的生物疗法的手段。 在之前的工作中，我们开发了氯烷渗透测定法（CAPA）来解决一些问题 以前的方法。 CAPA已被大量学术和工业实验室采用， 成为测量细胞质渗透的新“金标准”。在此更新提案中，我们描述了 解决生物治疗开发中具有挑战性的问题的新机会。第一个是如何 测量不同细胞类型的细胞质渗透。在目标 1 中，我们描述了使用腺相关 病毒 (AAV)，可在任何感兴趣的细胞类型中进行多功能 CAPA 测定。这解锁了令人兴奋的新功能 有机会比较数十种细胞系（包括原代细胞）的胞质渗透，并允许 测量对不同亚细胞区室的渗透。第二个重要问题是 了解细胞质渗透的动力学。在目标 2 中，我们通过引入新的“开启”来解决这个问题 CAPA 版本，应该更灵敏并且允许实时测量。最后，在目标 3 中，我们 设想一个氯烷渗透筛选（CAPS），它可以筛选数千个库 一次有数百万个分子。我们将把这种新的筛选应用于大型环肽库和 反义寡核苷酸。这些数据将代表我们对结构理解的巨大飞跃—— 这些类别的分子的渗透关系，新的屏幕将被纳入设计中- 测试学习循环，为细胞质渗透分子生成数据驱动的设计算法。 该项目非常适合 PAR-19-253（重点技术研究和开发），因为它是 注重创新方法。这些新方法将允许测量分子在任何环境中的渗透 细胞，到任何隔间，并且是实时的。它们还将允许对数百万个分子进行定制筛选 优化细胞质渗透，他们将提供数据驱动的规则来设计更好的寡核苷酸和 肽类药物。与 CAPA 一样，这些新方法的设计简单且易于使用，因此可以 被从事肽、蛋白质和核酸治疗研究的研究人员广泛采用。

For many classes of peptide, protein and nucleic acid drugs, intracellular delivery remains the primary obstacle in drug development. This obstacle has been challenging because many methods that are used to measure “cell penetration” actually measure total cellular uptake, including material trapped at the cell surface or in endosomes. For a long time, methods for measuring cytosolic penetration were not accessible, and this has greatly slowed the development of several classes of biotherapeutics. Further, without basic understanding of cytosolic penetration in a time-resolved, cell-type-specific manner, we will never move beyond trial- and-error as a means for developing biotherapeutics for specific disease targets in specific tissues. In previous work, we developed the ChloroAlkane Penetration Assay (CAPA) to solve some of the problems of previous methods. CAPA has been adopted by a large number of academic and industrial labs, and it is becoming a new “gold standard” for measuring cytosolic penetration. In this renewal proposal, we describe new opportunities to address challenging problems in biotherapeutics development. The first is how to measure cytosolic penetration in different cell types. In Aim 1, we describe using adeno-associated viruses (AAVs) to enable the versatile CAPA assay in any cell type of interest. This unlocks exciting new opportunities to compare cytosolic penetration across dozens of cell lines, including primary cells, and to allow measurement of penetration to different subcellular compartments. A second important problem is to understand the kinetics of cytosolic penetration. In Aim 2, we solve this problem by introducing a new “turn-on” version of CAPA, which should be more sensitive and allow real-time measurements. Finally, in Aim 3, we envision a ChloroAlkane Penetration Screen (CAPS) which can screen pooled libraries of thousands to millions of molecules at a time. We will apply this new screen to large libraries of cyclic peptides and antisense oligonucleotides. These data will represent a huge leap in our understanding of structure- penetration relationships for these classes of molecules, and the new screen will be incorporated into a design- test-learn cycle to produce data-driven design algorithms for cytosol-penetrant molecules. This project is well-suited for PAR-19-253, Focused Technology Research and Development, because it is focused on innovative methods. These new methods will allow for measuring a molecule’s penetration in any cell, to any compartment, and in real-time. They will also allow for custom screens of millions of molecules to optimize cytosolic penetration, and they will provide data-driven rules for designing better oligonucleotide and peptide drugs. Like CAPA, these new methods are designed to be simple and accessible, so that they can be widely adopted by researchers working on peptide, protein, and nucleic acid therapeutics.