Annely Lorents

The therapeutic potential of mRNA is vast, and yet translating this potential into effective treatments requires overcoming significant challenges of achieving safe and efficient delivery. This process is hampered by biological barriers that limit cellular uptake, degrade exposed mRNA, and thus necessitate effective endosomal escape to reach the cytoplasm. To address these challenges, we developed a hPep peptide-based nanoparticle (PNP) system that encapsulates mRNA, forming stable and biocompatible particles, which are rapidly taken up by the cells and enable efficient mRNA delivery across various cell culture models. Following systemic administration in mice, lead hPep3/mRNA PNPs achieve broad mRNA expression across multiple tissues, including the lungs, liver, and spleen, and enable effective mRNA delivery to the central nervous system upon local administration. Furthermore, we established a high-yield (≥70%) microfluidics-based protocol to scale up the production of well-defined, sterile hPep3/mRNA PNP formulations (approximately 70 nm, PDI around 0.170). Most importantly, in a proof-of-concept study in nonhuman primates (NHPs), we demonstrate that hPep PNPs loaded with human erythropoietin (hEPO) mRNA induce dose-dependent expression of hEPO protein in monkey serum, reaching up to 10 ng/ml at 1.0 mg/kg dose, following both single and repeated administration, while remaining systemically well-tolerated. These findings underscore the potential of hPep peptide-based nanoparticles as a versatile platform for mRNA delivery across multiple tissues, highlighting their promise in advancing the development of mRNA therapeutics.

The development of therapeutic small interfering RNAs (siRNAs) has lately gained significant momentum due to their ability to silence genes in a highly specific manner. The main obstacle withholding the wider translation of siRNA‐based drug modalities is their limited half‐life and poor bioavailability, especially in extra‐hepatic tissues. Consequently, various drug delivery systems (DDSs) have been developed to improve the delivery of siRNAs, including short delivery peptides called cell‐penetrating peptides (CPPs). In this study, we explore the potential of using alkenyl‐alanine modifications to enhance the siRNA delivery efficacy with CPPs. We demonstrate on hPep peptides that incorporation of alkenyl‐alanines enhances the encapsulation of siRNAs into stable nanoparticles and contributes to increased cellular uptake. Furthermore, we demonstrate that the lead peptide, hPep3, induces effective RNAi‐mediated gene silencing in a reporter cell model as well as on the disease‐implicated endogenous CD45 gene target. The biodistribution studies in mice show that the alkenyl‐alanines are systemically well tolerated, and employing such modifications in the peptide backbone improves siRNA delivery in several tissues, including extra‐hepatic sites. As demonstrated on hPep peptides, alkenyl‐alanines offer a simple yet robust way to enhance the delivery efficacy of CPPs and have the potential to advance siRNA therapeutics beyond the liver targets.

Nucleic acid molecules can be transferred into cells to alter gene expression and, thus, alleviate certain pathological conditions. Cell-penetrating peptides (CPPs) are vectors that can be used for transfecting nucleic acids as well as many other compounds. CPPs associate nucleic acids non-covalently, forming stable nanoparticles and providing efficient transfection of cells in vitro. However, in vivo, expected efficiency is achieved only in rare cases. One of the reasons for this discrepancy is the formation of protein corona around nanoparticles, once they are exposed to a biological environment, e.g., blood stream. In this study, we compared protein corona of CPP-nucleic acid nanoparticles formed in the presence of bovine, murine and human serum. We used Western blot and mass-spectrometry to identify the major constituents of protein corona forming around nanoparticles, showing that proteins involved in transport, haemostasis and complement system are its major components. We investigated physical features of nanoparticles and measured their biological efficiency in splice-correction assay. We showed that protein corona constituents might alter the fate of nanoparticles in vivo, e.g., by subjecting them to phagocytosis. We demonstrated that composition of protein corona of nanoparticles is species-specific that leads to dissimilar transfection efficiency and should be considered while developing delivery systems for nucleic acids.

Proficient transport vectors called cell-penetrating peptides (CPPs) internalize into eukaryotic cells mostly via endocytic pathways and facilitate the uptake of various cargo molecules attached to them. However, some CPPs are able to induce disturbances in the plasma membrane and translocate through it seemingly in energy-independent manner. For understanding this phenomenon, giant plasma membrane vesicles (GPMVs) derived from the cells is a beneficial model system, since GPMVs have a complex membrane composition comparable to the cells, yet lack cellular energy-dependent mechanisms. We investigated the translocation of arginine-rich CPPs into GPMVs with different membrane composition. Our results demonstrate that lower cholesterol content favors accumulation of nona-arginine and, additionally, sequestration of cholesterol increases the uptake of the CPPs in vesicles with higher cholesterol packing density. Furthermore, the proteins on the surface of vesicles are essential for the uptake of arginine-rich CPPs: downregulation of nucleolin decreases the accumulation and digestion of proteins on the membrane suppresses translocation even more efficiently.