Organic Seminar - Joshua Kritzer

Large-molecule therapeutics including peptides, oligonucleotides, and proteins make up a large and growing portion of the drug development pipeline.  One of the greatest barriers to developing these drugs is cell penetration. Most enter the cell through a complex pathway involving endocytosis followed by endosomal escape. This process which is poorly understood and difficult to study – it is challenging simply to measure how much compound has actually accessed the cytosol.  The Kritzer Lab has developed new tools for making these and related measurements.  The Chloroalkane Penetration Assay (CAPA) is a versatile assay that measures cell penetration using cellularly expressed HaloTag protein and a small chloroalkane tag on the molecule-of-interest.  CAPA has been used by the Kritzer group to measure cell penetration for diverse classes of peptide and oligonucleotide therapeutics, to measure penetration to different subcellular compartments, and to measure relative penetration in different cell types.  CAPA is now used by academic and industrial groups all over the world to investigate cell penetration.  The Kritzer group has also developed new HaloTag variants which work optimally with a fluorogenic benzothiadiazole dye.  The resulting “BenzoTag” system allows for turn-on, no-wash cell labeling in seconds. BenzoTag is currently being applied to produce a “turn-on” version of the CAPA assay for continued investigation of drug delivery and mechanisms of endosomal escape.