Cell-Penetrating D-Peptides Retain Antisense Morpholino Oligomer Delivery Activity

Abstract

Cell-penetrating peptides (CPPs) can cross the cell membrane to enter the cytosol and deliver otherwise non-penetrant macromolecules such as proteins and oligonucleotides. For example, recent clinical trials have shown that a CPP attached to phosphorodiamidate morpholino oligomers (PMO) resulted in higher muscle concentration, increased exon-skipping and dystrophin production relative to another study of the PMO alone in patients of Duchenne muscular dystrophy. Therefore, effective design and study of CPPs could help enhance therapies for difficult-to-treat diseases. So far, the study of CPPs for PMO delivery has been restricted to predominantly canonical L-peptides. We hypothesized that mirror-image D-peptides could have similar PMO delivery activity as well as enhanced proteolytic stability, facilitating their characterization and quantification from biological milieu. We found that several enantiomeric peptide sequences could deliver a PMO-biotin cargo with similar activities, while remaining stable against serum proteolysis. The biotin label allowed for affinity capture of fully intact PMO-peptide conjugates from whole cell and cytosolic lysates. By profiling a mixture of these constructs in cells, we determined their relative intracellular concentrations. When combined with PMO activity, these concentrations provide a new metric for delivery efficiency which may be useful for determining which peptide sequence to pursue in further pre-clinical studies.

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

“…D-Bpep was again used as a control here as it is a known highly active sequence able to escape the endosome and localize to the nucleus. 48 Indeed, at both concentrations diffuse fluorescence was observed in the cytosol and nucleus of PMO-Bpep-treated cells, in addition to punctate fluorescence that co-localized with LysoTracker, suggesting accumulation in endosomes (Fig. 8).…”

“…The protocol for extracting biotinylated sequences was adapted from our recent method of profiling mixtures of PMO-D-CPPs from the cytosol and whole cell using MALDI-ToF. 48,49 Confluent HeLa cells in a 12-well plate were treated with 20 µM of biotin-library or PMO-biotin-library (∼10 3 members, 3.5 nmol individual peptide per bead) for 1 h, before being washed with PBS and heparin to dissociate membrane-bound conjugates. Cells were then lifted and extracellular conjugates digested with Trypsin, pelleted, and washed with PBS.…”

“…We further investigated the differences in activity between the sequences found in the cytosol versus the whole cell lysate and compared them to a benchmark compound, PMO-D-Bpep, using flow cytometry. 48 For this purpose, several SulfoCy5-labeled PMO-CPPs were generated and tested to ensure the fluorophore did not impact PMO delivery activity (Fig. 7A).…”

“…Cells were gently lysed using either RIPA buffer (for whole cell extraction) or digitonin buffer (for cytosolic extraction). 48,50 Digitonin extraction is selective to cholesterol-rich membranes, leaving endosomes and some organelles intact while permeabilizing the cell membrane and semi-permeabilizing the nucleus. 51 Exclusion of endosomes in the cytosolic fraction was confirmed by Western blot, in which the endosomal marker, Rab5, is absent ( Fig.…”

“…5D), demonstrating their potential utility as PMO delivery vehicles. We also compared Pep1a and Pep1c to the known endosomal escape peptide Bpep 48 , composed of eight Arg residues interspaced with non-α-backbone residues β-alanine and 6-aminohexanoic acid, in its D-form which shows an EC50 closer to 3 µM (Fig. 5E).…”