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bioRxiv · 10.1101/2022.04.13.488231

In-cell penetration selection-mass spectrometry produces noncanonical peptides for antisense delivery

Schissel, C. K.·Farquhar, C. E.·Loas, A.·Malmberg, A. B.·Pentelute, B. L.
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Abstract

Peptide-mediated delivery of macromolecules in cells has significant potential therapeutic benefits, but no therapy employing cell-penetrating peptides (CPPs) has reached the market after 30 years of investigation due to challenges in the discovery of new, more efficient sequences. We developed a method for in-cell penetration selection-mass spectrometry (in-cell PS-MS) to discover peptides from a synthetic library capable of delivering macromolecule cargo to the cytosol. This method was inspired by recent in vivo selection approaches for cell-surface screening, with an added spatial dimension resulting from subcellular fractionation. A representative peptide discovered in the cytosolic extract, Pep1a, is nearly 100-fold more active toward antisense phosphorodiamidate morpholino oligomer (PMO) delivery compared to a sequence identified from a whole cell extract, which includes endosomes. Pep1a is composed of D-amino acids and two non--amino acids. Pulse-chase and microscopy experiments revealed that while the PMO-Pep1a conjugate is likely taken up by endosomes, it can escape to localize to the nucleus. In-cell PS-MS introduces a means to empirically discover unnatural synthetic peptides for subcellular delivery of therapeutically relevant cargo.

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Schissel, C. K., Farquhar, C. E., Loas, A., Malmberg, A. B., Pentelute, B. L.. 2022-04-14. In-cell penetration selection-mass spectrometry produces noncanonical peptides for antisense delivery. https://doi.org/10.1101/2022.04.13.488231

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