Enhancing the efficiency of viral and non-viral gene delivery vectors

Sammanfattning: Gene therapy, which involves the introduction, alteration, or deletion of genetic material within cells to treat or prevent disease, holds great promise for addressing various genetic disorders and other medical conditions. However, several challenges remain in the field, particularly concerning nucleic acid delivery into target cells. Other key challenges than efficacious nucleic acid delivery include immunogenicity, toxicity, stability and payload capacity. In paper I, we used splice-switching oligonucleotides (SSOs), which is a class of synthetic nucleic acid molecules designed to modulate the process of alternative splicing within cells. SSOs are designed to specifically target pre-mRNA, influencing the splicing machinery to alter the inclusion or exclusion of specific exons during mRNA maturation. However, various obstacles need to be addressed, particularly pertaining to their inability to efficiently enter cells for their successful use and widespread application. Cell-penetrating peptides (CPPs) have gained interest in nucleic acid delivery due to their ability to traverse cellular membranes, aiding the intracellular delivery of nucleic acids such as SSOs. To improve the membrane-penetrating abilities of CPPs and further facilitate the delivery of the accompanied therapeutic cargo across cell membranes, lipidation of CPPs is one established strategy but it is typically limited to N- or C-terminal conjugation to the peptides. Hence, we here utilized hydrocarbon modified amino acids which are used in peptide stapling for orthogonal introduction of hydrophobic modified amino acids into the CPPs. Our data showed that incorporating α,α-disubstituted alkenyl-alanine (ie. Pentenyl- or octenyl alanine) was successful to impart hydrophobicity to the CPPs. Furthermore, this newly designed peptides have secondary amphipathic structures that have a propensity to form an alpha helix in solution. Upon mixing these novel CPPs with SSOs, nanoparticles are formed that facilitate effective and well-tolerated delivery both in vitro and in vivo. In conclusion, our discoveries introduce a new and versatile approach to augmenting the delivery properties of CPPs, enabling the introduction of hydrophobic features into their structures with sequence-specific precision. In paper II, we broadened our investigation to include other peptide-based vectors, namely dendrimers, for delivery of SSOs. Dendrimers are highly branched, tree-like macromolecules with well-defined structures that have found extensive applications in various fields, including drug delivery, imaging, and materials science. Peptidedendrimers are a class of dendritic polymers constructed using amino acids as building blocks. In our study, we employed lipophilic peptide dendrimers in which lipophilic components, such as fatty acids or hydrophobic amino acids are conjugated to the dendrimer core structure. We evaluated the transfection efficiency of different lipophilic dendrimers and investigated several factors including composition, stereochemistry, and formulation buffer, in reporter cells for SSOs delivery. The impact of stereochemistry was more notable in third-generation peptide dendrimers favoring D-amino acids over L-amino acids. The potential of lipophilic peptide dendrimers presents promising opportunities for enhancing and evaluating diverse cargos and conjugates in forthcoming investigations. In paper III, our focus shifted towards enhancing the efficacy of viral vectors for gene delivery. The utilization of viral vectors in gene therapy has gained significant traction. However, in certain cell types, the efficiency of viral transduction remains insufficient. To overcome this, different additives such as lipids, peptides, polycationic chemicals, and polymers have been employed. While protamine sulfate and polybrene are among the polycationic additives that enhance transduction efficiency, pyran and heparin are examples of anionic compounds that suppress transduction. In this study, we surprisingly discovered a substantial improvement in the transduction efficiency of lentiviruses and Respiratory Syncytial Virus (RSV) when employing low concentrations of heparin and analogs thereof. Moreover, we show that by using lactoferrin, a specific inhibitor of heparan-sulfate proteoglycans (HSPGs), heparin-induced transduction enhancement is suppressed, suggesting that low concentrations of heparin and its analogues facilitate transduction by bridging between the virus and HSPGs. In contrast, heparin and its analogues compete with viral binding to HSPGs at high concentrations. Our findings indicate that polyanionic compounds display a concentration-dependent impact on viral uptake enhancing viral transduction at lower concentrations. Thus, these compounds hold promise for potential applications in ex vivo gene therapy. In paper IV, we wanted to expand on the concept of bridging observed in paper III by investigating the protein corona components associated with CPP-based nanoparticles utilized in paper I. The formation of the protein corona occurs when nanoparticles interact with biological fluids, creating a complex coating of biomolecules. The composition of this corona is influenced by factors such as the physical properties of nanoparticles and physiological conditions, playing a vital role in determining the fate of nanocarriers during therapeutic delivery. Lipid nanoparticles (LNPs) and CPPs represent two effective types of nanoparticle vectors in gene delivery. Early research indicated that the protein corona surrounding LNPs, tends to contain a significant amount of apolipoprotein E (apoE) that plays an important role in hepatic uptake. In this study, we assessed the protein corona surrounding CPP/mRNA nanoparticle complexes and compared it with the protein corona of mRNA-containing LNPs that are used clinically. Our results demonstrated a greater enrichment of apoE in the protein corona of CPP/mRNA complexes compared to LNPs. Moreover, in our comparison of CPP formulations decorated with apoE and transfected into the HepG2 cell line with undecorated formulations, we observed a dose-dependent increase in the transfection efficiency of the decorated formulations. The outcomes from this study will serve as the foundation for developing safer and more efficient gene delivery vehicles utilizing nanoparticles, advancing our understanding of their interaction with biological fluids.