PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation

Abstract

Numerous human genetic diseases are caused by mutations that give rise to aberrant alternative splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides (SCOs) that modify splicing patterns and restore the phenotype in experimental models. However, translational approaches are required to transform SCOs into usable drug products. In this study, we present a new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers SCOs to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne’s muscular dystrophy (DMD). Non-covalent PF14-SCO nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector Lipofectamine™ 2000 (LF2000) and remain active in the presence of serum. Furthermore, we demonstrate the feasibility of incorporating this delivery system into solid formulations that could be suitable for several therapeutic applications. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to same procedure. This shows that using PF14 is a very promising translational approach for the delivery of SCOs in different pharmaceutical forms.

Exon Skipping and Gene Transfer Restore Dystrophin Expression in Human Induced Pluripotent Stem Cells-Cardiomyocytes Harboring DMD Mutations

“…Delivery of AONs expressed from viral vectors (e.g., adeno-associated virus [27] or lentivirus [28]) has been achieved but this strategy is costly and it is time consuming to screen large numbers of target sequences. Similarly, the system could be used to test the efficiency of uptake of new AON chemistries or methods of uptake, such as the use of cell-penetrating peptides [29,30]. It is notable that even when methods deliver limited quantities of skipping agents, dystrophin expression can be restored.…”

Peptides for nucleic acid delivery

“…PepFect peptides are another intriguing group of amphipathic CPPs that have successfully deployed stearic acid modification and shown delivery potential for a wide variety of nucleic acid-based cargoes, including SSOs, siRNAs, and plasmids [78][79][80][81][82][83]. In the first generation, TP10 peptide was modified with stearic acid in its N-terminus, generating the PepFect3 (stearyl-TP10) peptide [78].…”

“…In PepFect14, changes in the amino acid sequence of TP10 were carried out, most prominently introducing ornithines instead of lysines as a source of positive charge. Interestingly, the PepFect14 peptide proved to be a very effective vector for many nucleic acid-and ON-based molecules, including SSOs, siRNAs and pDNA [81][82][83]. An important finding using PF14 was that it could be formulated with SSO in presence of different pharmaceutical excipients and dried as solid formulations and be re-used several months later with maintained activity [81].…”

Membrane Active Peptides and Their Biophysical Characterization

“…Most of the currently available peptide based drugs target extracellular regions of receptors [345] while the number of AMPs and other peptide pharmaceuticals in clinical trials is increasing [346,347]. Modification with PEG [348,349] or nanoparticles [350], residue modifications such as mutation of lysine residues to ornithine [96], cyclization of the peptides using internal linkages [351] are some of the approaches that have been used to enhance the stability and bioavailability of peptides. Targeted methodologies that pack the peptide in nanoparticles have been used to target only the cancer cells and hence reduce their toxicity related to their low selectivity [352,353].…”