A Kit to Quantify Cell Penetration
Tufts investigator, Joshua Kritzer, has developed a novel high throughput assay, Chloroalkane Penetration Assay (CAPA), to quantitatively measure the compartment-specific cell penetration of any externally applied agent. CAPA uses a cell line expressing a HaloTag fusion protein that is exclusively localized to the cytosol, nucleus, or other cellular compartment. Cells are incubated with a molecule of interest that is conjugated to the chloroalkane. The extent of penetration is then quantitated by reading out the amount of free HaloTag (Fig. A). Using small molecule controls and known cell-penetrating peptides, Dr. Kritzer has confirmed that readouts from CAPA correlate directly with the amount of molecule that can access the cytosol (Fig. B).
Current methods used to judge cell penetration of an external agent have difficulty distinguishing between material that is trapped in endosomes from material in the cytosol, and they are further prone to artifacts including peptide aggregation. These methods also require large tags or dyes, are qualitative, and/or have low throughput.
To address these shortcomings, the CAPA assay uses a small chloroalkane tag (Fig. A), is quantitative (graph in Fig. B), and has exceptional throughput.
PATH TO COMMERCIALIZATION
Catering to academic and commercial research labs, the assay could be offered as a kit comprising multiple cell lines expressing a HaloTag fusion protein localized to the appropriate cytosolic organelle and a HaloTag labeling reagent.
Extensive profiles of cell penetration can be obtained inexpensively and with high throughput, including dose dependence, time dependence, and sensitivity to inhibitors of different active transport pathways. Thus, detailed mechanistic data can be obtained for each molecule of interest. Additionally, Dr. Kritzer has shown that CAPA can be used to report on cytosolic delivery of lipid nanoparticles and their protein cargoes in a highly sensitive and quantitative manner.
US Publication No. 2018-0188260 (July 5, 2018)