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Principal Investigator: Joshua A Kritzer
Organization: TUFTS UNIVERSITY MEDFORD
Fiscal Year: 2019
Award: $286,228
Funding agency: National Institute of General Medical Sciences
Cell Penetration Profiling for Biotherapeutics
Biotechnology promises the ability to control biology and disease with laser-like precision. Hundreds of
peptides, proteins and nucleic acids are being developed as diagnostics and therapies, but nearly all of these
have poor cell penetration and unpredictable subcellular localization. Currently, there are no quantitative, high-
throughput tools to measure how much of a biomolecule enters a cell and where it distributes within the cell.
The imprecision and low throughput of current methods for measuring intracellular delivery is a major
barrier for the development of biomolecule therapies.
The Kritzer lab has devised a new method for quantitating cell penetration, called the ChloroAlkane
Penetration Assay (CAPA). CAPA measures the degree to which a molecule penetrates the cytosol,
independent of the molecule's target or biological function. Our published and unpublished data show that
CAPA is quantitative, inexpensive and high-throughput, and that it exclusively measures penetration to single
cellular compartment. Based on these results, we envision that CAPA could be used for comprehensive cell
penetration profiling for a large variety of biomolecules and drug delivery systems. This will provide detailed
data on dose dependence, time course, penetration to different subcellular compartments, and cell type
specificity. It will also allow independent profiling of endocytic uptake, endosomal escape and nuclear import.
We propose applying cell penetration profiling to uncover structure-activity relationships for cell
penetration for several classes of bioactive peptides, cell-penetrant proteins, and antisense oligonucleotides.
Finally, we use cell penetration profiling to understand and improve the delivery of Cas9-sgRNA complexes by
lipid nanoparticles.
Our understanding of how all these classes of biotherapeutics reach the cell interior is severely limited by
current methods. Cell penetration profiling provides a rapid means of measuring endocytic uptake, endosomal
escape, and nuclear import, allowing critical drug delivery problems to be directly assessed and
avoided. Since intra-cellular delivery is the major hurdle for most biotherapeutics, cell penetration profiling has
the potential to become a routine and valuable part of the development process for peptide, protein and nucleic
acid therapies.
Terms: <Address><Adoption><Anti-Sense Oligonucleotides><Antisense Agent><Antisense Oligonucleotides><Area><Assay><Bioassay><Biologic Assays><Biologic Therapy><Biological Assay><Biological Function><Biological Process><Biological Response Modifier Therapy><Biological Therapy><Biology><Biophysics><Biotech><Biotechnology><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cas nuclease technology><Cell Body><Cell Nucleus><Cells><Cellular biology><Charge><Complex><Cytosol><Data><Data Set><Dataset><Dependence><Development><Diagnostic><Disease><Disorder><Dose><Drug Delivery><Drug Delivery Systems><Drugs><Endosomes><FDA approved><Formulation><Foundations><Funding><Genes><Genetic><Hydrophobicity><Kinetics><Laser Electromagnetic><Laser Radiation><Lasers><Libraries><Measurement><Measures><Mediating><Medication><Methods><Modification><Nuclear Import><Nucleic Acids><Nucleus><Organic Synthesis><Penetration><Peptides><Pharmaceutic Preparations><Pharmaceutical Preparations><Process><Property><Proteins><Publishing><Receptosomes><Reporting><Research><Resolution><Series><Specificity><Structure><Structure-Activity Relationship><Technology><Therapeutic><Time><analog><anti-sense agent><anti-sense oligo><antisense oligo><base><biological therapeutic><biological treatment><biophysical foundation><biophysical principles><biophysical sciences><biotherapeutics><biotherapy><case-by-case basis><cell biology><cell type><chemical structure function><combinatorial><design><designing><developmental><drug development><drug/agent><experiment><experimental research><experimental study><improved><insight><lipid nanoparticle><nano particle delivery><nanoparticle delivered><nanoparticle delivery><new approaches><novel approaches><novel strategies><novel strategy><nucleic acid-based therapeutics><peptide drug><phosphorothioate><structure function relationship><technological research and development><technology research and development><therapeutic candidate><therapeutic nanoparticles><therapeutic nucleic acids><therapeutic peptide><tool><uptake>