Introduction: In this review, recent developments and applications with cell-penetrating peptides (CPP) are discussed. CPPs are widely used tools for the delivery of various macromolecular therapeutics, such as proteins and nucleic acids. Areas covered: The current review focuses on recent important advances and reports that demonstrate high clinical and translational potential. Most important clinical developments have occurred with the CPP-drug conjugate approaches that target various protein-protein interactions, and these have been highlighted subsequently. Most of the applications are targeting cancer, but recently, noteworthy advances have taken place in the field of antisense oligonucleotides and muscular dystrophies, lung targeting, and trans-BBB targeting. Expert opinion: Successful applications and clinical development with the drug conjugate approaches are discussed. On the other hand, the reasons of why the nanoparticle approaches are not as far in development are analyzed.
Therapeutic proteins are currently at the apex of innovation in pharmaceutical medicine. However, their industrial production is technically challenging and improved methods for transient transfection of mammalian cell cultures are necessary. We aimed to find a fast, microliter-scale transfection assay that allows the prediction of protein expression in the transient production settings. We used an array of lipid, polymeric and cell-penetrating peptide transfection reagents, and compared their performance in various high throughput transfection assays to their performance in protein (antibody) expression in professional protein-producer cell lines. First, we show that some of the most frequently used microliter-scale transfection efficacy assays fail to predict performance in the protein production in milliliter and liter scale settings. We found that CHO suspension culture post-transfection EGFP(+) population and SEAP quantitation correlate with large-scale protein production, whereas the adhesion culture assays and transfection of pLuc are non-predictive. Second, we demonstrated that cell-penetrating peptide-based transfection achieves significantly higher protein yields compared to PEI and lipoplex methods in both CHO and HEK293 producer cell lines. In this work we demonstrate a CPP-based transient protein expression approach that significantly outperformed the current industry standard workhorse method of PEI.
Therapeutic proteins are currently at the hotspot of innovation in the pharmaceutical medicine. However, their industrial production is technically challenging and improved methods for transcriptional manipulation of mammalian industrial cell cultures are needed. In this work we show that some of the most frequently used lab scale transfection efficacy assays fail to predict performance in the protein production settings. We compare the efficacies of a number of transfection reagents using adherent and suspension mammalian cell cultures and assessment based on several assays that utilize reporter protein quantitation, transfected cell population and post-transfection viability of cells. We validate reporter assays for assessing transfection methods in the lab that predict protein production in industrial settings. We also demonstrate that cell penetrating peptide-based transfection achieve significantly higher protein yields compared to PEI and lipoplex methods in both CHO and HEK293 producer cell lines. Availability of fast lab scale screening methods allows future development of improved transfection methods for protein production. One such potentially effective transient transfection method is the CPP-based approach presented currently.
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