Cell-penetrating peptides (CPPs) can facilitate the intracellular delivery of large therapeutically relevant molecules, including proteins and oligonucleotides. Although hundreds of CPP sequences are described in the literature, predicting efficacious sequences remains difficult. Here, we focus specifically on predicting CPPs for the delivery of phosphorodiamidate morpholino oligonucleotides (PMOs), a compelling type of antisense therapeutic that has recently been FDA approved for the treatment of Duchenne muscular dystrophy. Using literature CPP sequences, 64 covalent PMO–CPP conjugates were synthesized and evaluated in a fluorescence-based reporter assay for PMO activity. Significant discrepancies were observed between the sequences that performed well in this assay and the sequences that performed well when conjugated to only a small-molecule fluorophore. As a result, we envisioned that our PMO–CPP library would be a useful training set for a computational model to predict CPPs for PMO delivery. We used the PMO activity data to fit a random decision forest classifier to predict whether or not covalent attachment of a given peptide would enhance PMO activity at least 3-fold. To validate the model experimentally, seven novel sequences were generated, synthesized, and tested in the fluorescence reporter assay. All computationally predicted positive sequences were positive in the assay, and one sequence performed better than 80% of the tested literature CPPs. These results demonstrate the power of machine learning algorithms to identify peptide sequences with particular functions and illustrate the importance of tailoring a CPP sequence to the cargo of interest.

Exon-skipping antisense oligonucleotides are effective treatments for genetic diseases, yet exon-skipping activity requires that these macromolecules reach the nucleus. While cell-penetrating peptides can improve delivery, proteolytic instability often limits efficacy. It is hypothesized that the bicyclization of arginine-rich peptides would improve their stability and their ability to deliver oligonucleotides into the nucleus. Two methods were introduced for the synthesis of arginine-rich bicyclic peptides using cysteine perfluoroarylation chemistry. Then, the bicyclic peptides were covalently linked to a phosphorodiamidate morpholino oligonucleotide (PMO) and assayed for exon skipping activity. The perfluoroaryl cyclic and bicyclic peptides improved PMO activity roughly 14-fold over the unconjugated PMO. The bicyclic peptides exhibited increased proteolytic stability relative to the monocycle, demonstrating that perfluoroaryl bicyclic peptides are potent and stable delivery agents.

Phosphorodiamidate morpholino oligonucleotides (PMOs) are a promising class of therapeutics for genetic disease. PMOs designed for "exon skipping" must be internalized into cells, reach the nucleus, and act on pre-mRNA to mediate their effects. One tactic for improving PMO delivery and exon skipping is to covalently conjugate PMOs to cell-penetrating peptides (CPPs). Here we report the synthesis of PMOs conjugated to CPP chimeras, constructed by combining multiple CPPs into one sequence. The chimeric CPPs synergistically improve PMO activity up to 70-fold over the PMO alone, beyond the expected effects of each component peptide. By investigating the design space of CPP chimeras, we demonstrate that all components must be covalently attached, that the order of the two sequences matters, and that peptide identity can tune activity.We identified one chimera (pVEC-Bpep) to investigate in more detail and found that it engages different mechanisms of endocytosis than its parent peptides. We also examined the extent to which the beneficial effect comes from improved cellular uptake as opposed to the downstream steps required for exon skipping. Given the complexity of intracellular delivery, we anticipate this work will lead researchers to consider combining molecules with different physicochemical properties in order to aid in the delivery of biologic cargoes. TOC Graphic, For Table of Contents Use Only:we envision that the approach of combining CPPs from different classes can be applied to the intracellular delivery of a variety of macromolecular cargoes. ASSOCIATED CONTENT Supporting Information:Methods for peptide synthesis and conjugation, lactate dehydrogenase assay results, flow cytometry analysis of PMO-chimeras in the presence of endocytosis inhibitors, LC-MS analysis of peptide conjugates, flow cytometry analysis of fluorophore-labeled PMO-chimeras, and colocalization analysis and nuclear fluorescence quantification of confocal images.

Current therapies for Duchenne muscular dystrophy (DMD) use phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping in the dystrophin pre-mRNA, enabling the translation of a shortened but functional dystrophin protein. This strategy has been hampered by insufficient delivery of PMO to cardiac and skeletal muscle. To overcome these limitations, we developed the FORCETM platform consisting of an antigen-binding fragment, which binds the transferrin receptor 1, conjugated to an oligonucleotide. We demonstrate that a single dose of the mouse-specific FORCE–M23D conjugate enhances muscle delivery of exon skipping PMO (M23D) in mdx mice, achieving dose-dependent and robust exon skipping and durable dystrophin restoration. FORCE–M23D-induced dystrophin expression reached peaks of 51%, 72%, 62%, 90% and 77%, of wild-type levels in quadriceps, tibialis anterior, gastrocnemius, diaphragm, and heart, respectively, with a single 30 mg/kg PMO-equivalent dose. The shortened dystrophin localized to the sarcolemma, indicating expression of a functional protein. Conversely, a single 30 mg/kg dose of unconjugated M23D displayed poor muscle delivery resulting in marginal levels of exon skipping and dystrophin expression. Importantly, FORCE–M23D treatment resulted in improved functional outcomes compared with administration of unconjugated M23D. Our results suggest that FORCE conjugates are a potentially effective approach for the treatment of DMD.

Exon-skipping antisense oligonucleotides are effective treatments for genetic diseases,y et exon-skipping activity requires that these macromolecules reacht he nucleus.W hile cell-penetrating peptides can improve delivery,p roteolytic instability often limits efficacy.I ti sh ypothesized that the bicyclization of arginine-richp eptides would improve their stability and their ability to deliver oligonucleotides into the nucleus.T wo methods were introduced for the synthesis of arginine-richb icyclic peptides using cysteine perfluoroarylation chemistry.T hen, the bicyclic peptides were covalently linked to ap hosphorodiamidate morpholino oligonucleotide (PMO) and assayed for exon skipping activity.T he perfluoroaryl cyclic and bicyclic peptides improved PMO activity roughly 14-fold over the unconjugated PMO.T he bicyclic peptides exhibited increased proteolytic stability relative to the monocycle,demonstrating that perfluoroaryl bicyclic peptides are potent and stable delivery agents.