A major obstacle to understanding and controlling the efficacy of biotherapeutic molecules, such as peptides and antisense oligonucleotides, is the imprecise and qualitative methods currently used to measure cellular penetration. The drawbacks of existing commonly used methods to monitor the cell penetration of biomolecules highlight the importance of a quantitative, high throughput, and minimally perturbing assay to uncover the fate of therapeutically-relevant molecules. To solve this problem, the Kritzer lab has developed the Chloroalkane Penetration Assay (CAPA), which can quantitatively compare the cell penetration and cytosolic access of exogenously added molecules. CAPA uses a<br/>cell line that stably expresses cytosolically localized HaloTag protein. The cells are incubated with chloroalkane-tagged molecules, which react with HaloTag in the cytoplasm, and is then incubated with chloroalkane-tagged fluorescent dye that reacts with free HaloTag active sites. The degree of penetration is quantitated by detecting fluorescence using flow cytometry. Increased penetration of a molecule results in lower fluorescence readout of the dye. To data we have used CAPA to examine the relative cytosolic and nuclear access of peptides, as well as to investigate dependence on dose, endocytosis inhibitors, and time, providing a rich “penetration profile”. CAPA has been used to study<br/>the mechanism of uptake, degree of endosomal escape, and subcellular localization of molecules. We have also used this assay to help explain the differences in autophagy-inducing phenotype observed between similar stapled peptides through analysis of structure-penetration relationships. Enhancing cell penetration would directly improve the therapeutic activity of biotherapeutic molecules. An understanding of the cellular fate of these molecules would be transformative in the design of potent therapeutics.

Chloroalkane penetration assay (CAPA) to monitor the cellular penetration of biomolecules

256th National Meeting (2018)

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