Cell Penetration Profiling for Biotherapeutics

对于许多种类的肽、蛋白质和核酸药物，细胞内递送仍然是主要障碍 在药物开发中。这一障碍具有挑战性，因为许多用于测量的方法 “细胞穿透”实际上测量总细胞摄取，包括捕获在细胞表面或细胞内的物质。 核内体很长一段时间以来，测量胞质渗透的方法是不可用的，这使得 大大减缓了几类生物治疗药物的发展。此外，如果没有基本的理解， 细胞溶质渗透的时间分辨，细胞类型特异性的方式，我们永远不会超越试验- 作为开发用于特定组织中的特定疾病靶点的生物治疗剂的手段。 在以前的工作中，我们开发了氯代烷烃渗透试验（CAPA），以解决一些问题， 以前的方法。CAPA已被大量的学术和工业实验室采用， 成为测量胞质渗透的新“金标准”。在本更新建议中，我们描述了 解决生物治疗发展中具有挑战性的问题的新机遇。首先是如何 测量不同细胞类型中的胞质渗透。在目标1中，我们描述了使用腺相关病毒载体。 病毒（AAV），以在任何感兴趣的细胞类型中实现通用CAPA测定。这开启了令人兴奋的新 有机会比较几十种细胞系（包括原代细胞）的胞质渗透， 测量对不同亚细胞区室的渗透。第二个重要问题是 了解细胞溶质渗透的动力学。在目标2中，我们通过引入一个新的“开启”来解决这个问题。 CAPA版本，应更敏感并允许实时测量。在目标3中，我们 设想一种可以筛选数千个汇集文库的氯代烷烃渗透筛选（CAPS）， 一次能产生数百万个分子我们将把这种新的筛选应用于大型的环肽文库， 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，和新的屏幕将被纳入一个设计- 测试-学习循环，以产生用于细胞溶质渗透剂分子的数据驱动设计算法。 这个项目非常适合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细胞中的渗透率。 细胞，到任何隔室，并且实时。它们还将允许定制数百万个分子的筛选， 优化胞质渗透，它们将为设计更好的寡核苷酸提供数据驱动规则， 肽类药物像CAPA一样，这些新方法被设计成简单和可访问的，因此它们可以 被从事肽、蛋白质和核酸治疗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.