# Cell Penetration Profiling for Biotherapeutics

> **NIH NIH R01** · TUFTS UNIVERSITY MEDFORD · 2022 · $360,446

## Abstract

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.

## Key facts

- **NIH application ID:** 10364261
- **Project number:** 2R01GM127585-05
- **Recipient organization:** TUFTS UNIVERSITY MEDFORD
- **Principal Investigator:** Joshua A Kritzer
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $360,446
- **Award type:** 2
- **Project period:** 2018-04-10 → 2023-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10364261

## Citation

> US National Institutes of Health, RePORTER application 10364261, Cell Penetration Profiling for Biotherapeutics (2R01GM127585-05). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10364261. Licensed CC0.

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