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In-Cell Penetration Selection-Mass Spectrometry Produces Noncanonical Peptides for Antisense Delivery
Carly K Schissel1, Charlotte E Farquhar1, Andrei Loas1
1Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
ACS Chemical Biology
|March 1, 2023
Summary
Researchers developed a new method to discover cell-penetrating peptides (CPPs) for delivering cargo to the cell cytosol. This technique identified a potent peptide, Cyto1a, enhancing therapeutic macromolecule delivery.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Delivery Systems
Background:
- Cell-penetrating peptides (CPPs) show therapeutic promise but their clinical translation is hindered by the lack of efficient peptide discovery methods.
- Existing CPPs face challenges in delivering macromolecules effectively to the cell cytosol, limiting their therapeutic applications.
Purpose of the Study:
- To develop and validate a novel method, in-cell penetration selection-mass spectrometry (in-cell PS-MS), for discovering peptides capable of delivering cargo to the cell cytosol.
- To identify and characterize novel CPPs with enhanced efficiency, stability, and reduced toxicity for therapeutic applications.
Main Methods:
- Developed in-cell penetration selection-mass spectrometry (in-cell PS-MS) by integrating subcellular fractionation with selection approaches.
- Screened a synthetic peptide library using in-cell PS-MS to identify peptides that penetrate the cell and reach the cytosol.
- Synthesized and tested a lead peptide, Cyto1a, for its efficacy in delivering antisense phosphorodiamidate morpholino oligomers (PMOs).
Main Results:
- The in-cell PS-MS method successfully identified a novel peptide, Cyto1a, with superior cytosolic delivery capabilities compared to peptides from whole-cell extracts.
- Cyto1a demonstrated nearly 100-fold higher activity in delivering PMOs compared to a control peptide.
- Cyto1a, composed of d-amino acids and non-α-amino acids, exhibited enhanced stability, reduced toxicity, and efficient endosomal escape for nuclear localization.
Conclusions:
- In-cell PS-MS is an effective empirical method for discovering unnatural synthetic peptides for targeted subcellular delivery.
- The identified peptide Cyto1a represents a promising candidate for advancing therapeutic macromolecule delivery, particularly for nucleic acid-based therapies.
- This approach overcomes limitations of previous CPP discovery methods and opens new avenues for developing efficient and safe drug delivery systems.
