Cell Penetration Profiling for Biotherapeutics

对于许多种类的多肽、蛋白质和核酸药物来说，细胞内给药仍然是主要的障碍 在药物开发方面。这一障碍一直具有挑战性，因为许多用于测量的方法 “细胞渗透率”实际上是测量细胞的总摄取量，包括细胞表面或细胞内的物质。 内噬菌体。在很长一段时间里，测量细胞质渗透的方法是不可获得的，这已经 极大地减缓了几类生物疗法的发展。此外，在没有基本了解的情况下 以一种时间分辨的、特定细胞类型的方式进行胞浆渗透，我们将永远不会超越试验- 以及-错误，作为开发针对特定组织中特定疾病靶点的生物疗法的手段。 在以前的工作中，我们开发了氯烷烃渗透分析(CAPA)来解决一些问题 以前的方法。CAPA已经被大量的学术和工业实验室采用，并且它是 成为衡量胞质渗透率的新“黄金标准”。在这份续签建议书中，我们描述了 解决生物疗法发展中具有挑战性的问题的新机遇。第一个是如何 测量不同类型细胞的胞浆渗透率。在目标1中，我们描述了使用腺相关 病毒(AAVs)在任何感兴趣的细胞类型中启用多功能CAPA检测。这开启了令人兴奋的新 有机会比较数十种细胞系(包括原代细胞)的胞浆渗透性，并允许 对不同亚细胞隔室的渗透率测量。第二个重要问题是 了解细胞质穿透的动力学。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，它应该更敏感，并允许实时测量。最后，在目标3中，我们 设想一种氯烷渗透屏幕(CAPS)，它可以筛选数千到 一次可以产生数百万个分子。我们将把这一新的筛选应用于环肽的大型文库，并 反义寡核苷酸。这些数据将代表着我们对结构理解的巨大飞跃-- 这些分子的穿透关系，新的屏幕将被纳入设计- 测试-学习循环，以产生数据驱动的细胞质渗透分子的设计算法。 该项目非常适合PAR-19-253，重点技术研究和开发，因为它是 着力创新方法。这些新方法将允许测量分子在任何 牢房，到任何隔间，并实时。他们还将允许定制数百万分子的屏幕，以 优化胞质渗透，他们将提供数据驱动的规则，设计更好的寡核苷酸和 多肽类药物。与CAPA一样，这些新方法被设计为简单和可访问的，因此它们可以 被多肽、蛋白质和核酸疗法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.