Enhancing Cellular Uptake of Native Proteins through Bio-Orthogonal Conjugation with Chemically Synthesized Cell-Penetrating Peptides.

The potential for native proteins to serve as a platform for biocompatible, targeted, and personalized therapeutics in the context of genetic and metabolic disorders is vast. Nevertheless, their clinical application encounters challenges, particularly in overcoming biological barriers and addressing the complexities involved in engineering transmembrane permeability. This study is dedicated to the development of a multifunctional nanoentity in which a model therapeutic protein is covalently linked to a cell-penetrating peptide, NickFect 55, with the objective of enhancing its intracellular delivery. Successful binding of the nanoentity fragments was achieved through the utilization of an intein-mediated protein-trans splicing reaction. Our research demonstrates that the fully assembled nanoentity-containing protein was effectively internalized by the cells, underscoring the potential of this approach in overcoming barriers associated with protein-based therapeutics for the treatment of genetic disorders.

A Method for Using Cell-Penetrating Peptides for Loading Plasmid DNA into Secreted Extracellular Vesicles.

The low bioavailability and high toxicity of plasmid DNA (pDNA)-based therapeutics pose challenges for their in vivo application. Extracellular vesicles (EVs) have great potential to overcome these limitations, as they are biocompatible native cargo carriers. Various methods for loading pDNA into EVs, including electroporation, sonication, and co-incubation, have been previously investigated, but their success has been questionable. In this study, we report a unique method for loading EVs with pDNA through transient transfection using cell-penetrating peptides (CPPs). With this method, we found a 104-fold increase in the expression levels of the luciferase reporter protein in recipient cells compared to the untreated cells. These data point to the high transfection efficacy and bioavailability of the delivered encapsulated nucleic acid. Furthermore, the in vivo experimental data indicate that the use of pDNA-loaded EVs as native delivery vehicles reduces the toxic effects associated with traditional nucleic acid (NA) delivery and treatment.

The Development of Cell-Penetrating Peptides for Efficient and Selective In Vivo Expression of mRNA in Spleen Tissue.

mRNA-based therapeutics are presently one of the nucleic acid-based therapeutics with a high potential for extraordinary success as preventive vaccines. Current applications with mRNA therapeutics rely on lipid nanoparticle (LNP) mediated delivery of nucleic acids. In order to achieve the transition from preventive to therapeutic vaccines, there is a challenge of delivering the mRNA into non-hepatic tissues, especially into lymphoid tissues such as the spleen and lymph nodes. In this work, we characterize new cell-penetrating peptides NF424 and NF436 that exhibit preferential delivery of mRNA into the spleen after a single i.v. injection, without the use of any active targeting mechanisms. We show that between the spleen, liver, and the lungs, >95% of mRNA expression arises in the spleen tissue and the majority of expression occurs in the dendritic cells. The cell-penetrating peptides NF424 and NF436 represent promising candidates for cancer immunotherapeutic applications with tumor antigens.

Aggregation Limiting Cell-Penetrating Peptides Derived from Protein Signal Sequences.

Alzheimer's disease (AD) is the most common neurodegenerative disease (ND) and the leading cause of dementia. It is characterized by non-linear, genetic-driven pathophysiological dynamics with high heterogeneity in the biological alterations and the causes of the disease. One of the hallmarks of the AD is the progression of plaques of aggregated amyloid-ß (Aß) or neurofibrillary tangles of Tau. Currently there is no efficient treatment for the AD. Nevertheless, several breakthroughs in revealing the mechanisms behind progression of the AD have led to the discovery of possible therapeutic targets. Some of these include the reduction in inflammation in the brain, and, although highly debated, limiting of the aggregation of the Aß. In this work we show that similarly to the Neural cell adhesion molecule 1 (NCAM1) signal sequence, other Aß interacting protein sequences, especially derived from Transthyretin, can be used successfully to reduce or target the amyloid aggregation/aggregates in vitro. The modified signal peptides with cell-penetrating properties reduce the Aß aggregation and are predicted to have anti-inflammatory properties. Furthermore, we show that by expressing the Aß-EGFP fusion protein, we can efficiently assess the potential for reduction in aggregation, and the CPP properties of peptides in mammalian cells.

Predicting Transiently Expressed Protein Yields: Comparison of Transfection Methods in CHO and HEK293.

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.

Utilization of Cell-Penetrating Peptides for In Vivo Delivery of Bioactive Cargo: The Effect of Nanoparticle Formulation.

The efficacy of nanoparticle drugs necessitates the high bioactivity of constituents, but the distribution of the nanoparticles in organisms is mostly determined by their physical properties. Therefore, generation of stable particles with strictly defined characteristics is highly essential. Here we describe a formulation protocol of stable and homogenous CPP/pDNA nanoparticles for in vivo applications.