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Gene therapy, which involves the introduction, alteration, or deletion of geneticmaterial within cells to treat or prevent disease, holds great promise for addressingvarious genetic disorders and other medical conditions. However, several challengesremain in the field, particularly concerning nucleic acid delivery into target cells. Otherkey 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 theprocess of alternative splicing within cells. SSOs are designed to specifically targetpre-mRNA, influencing the splicing machinery to alter the inclusion or exclusion ofspecific exons during mRNA maturation. However, various obstacles need to beaddressed, particularly pertaining to their inability to efficiently enter cells for theirsuccessful use and widespread application. Cell-penetrating peptides (CPPs) havegained interest in nucleic acid delivery due to their ability to traverse cellularmembranes, aiding the intracellular delivery of nucleic acids such as SSOs. To improvethe membrane-penetrating abilities of CPPs and further facilitate the delivery of theaccompanied therapeutic cargo across cell membranes, lipidation of CPPs is oneestablished strategy but it is typically limited to N- or C-terminal conjugation to thepeptides. Hence, we here utilized hydrocarbon modified amino acids which are usedin peptide stapling for orthogonal introduction of hydrophobic modified amino acidsinto 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 structuresthat have a propensity to form an alpha helix in solution. Upon mixing these novel CPPswith SSOs, nanoparticles are formed that facilitate effective and well-tolerateddelivery both in vitro and in vivo. In conclusion, our discoveries introduce a new andversatile approach to augmenting the delivery properties of CPPs, enabling theintroduction of hydrophobic features into their structures with sequence-specificprecision.In paper II, we broadened our investigation to include other peptide-based vectors,namely dendrimers, for delivery of SSOs. Dendrimers are highly branched, tree-likemacromolecules with well-defined structures that have found extensive applicationsin various fields, including drug delivery, imaging, and materials science. Peptidedendrimersare a class of dendritic polymers constructed using amino acids asbuilding blocks. In our study, we employed lipophilic peptide dendrimers in whichlipophilic components, such as fatty acids or hydrophobic amino acids are conjugatedto the dendrimer core structure. We evaluated the transfection efficiency of differentlipophilic dendrimers and investigated several factors including composition,stereochemistry, and formulation buffer, in reporter cells for SSOs delivery. The impactof stereochemistry was more notable in third-generation peptide dendrimers favoringD-amino acids over L-amino acids. The potential of lipophilic peptide dendrimerspresents promising opportunities for enhancing and evaluating diverse cargos andconjugates in forthcoming investigations.In paper III, our focus shifted towards enhancing the efficacy of viral vectors for genedelivery. 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 amongthe polycationic additives that enhance transduction efficiency, pyran and heparin areexamples of anionic compounds that suppress transduction. In this study, wesurprisingly discovered a substantial improvement in the transduction efficiency oflentiviruses and Respiratory Syncytial Virus (RSV) when employing low concentrationsof heparin and analogs thereof. Moreover, we show that by using lactoferrin, a specificinhibitor of heparan-sulfate proteoglycans (HSPGs), heparin-induced transductionenhancement is suppressed, suggesting that low concentrations of heparin and itsanalogues facilitate transduction by bridging between the virus and HSPGs. Incontrast, heparin and its analogues compete with viral binding to HSPGs at highconcentrations. Our findings indicate that polyanionic compounds display aconcentration-dependent impact on viral uptake enhancing viral transduction atlower concentrations. Thus, these compounds hold promise for potential applicationsin ex vivo gene therapy.In paper IV, we wanted to expand on the concept of bridging observed in paper III byinvestigating the protein corona components associated with CPP-basednanoparticles utilized in paper I. The formation of the protein corona occurs whennanoparticles interact with biological fluids, creating a complex coating ofbiomolecules. The composition of this corona is influenced by factors such as thephysical properties of nanoparticles and physiological conditions, playing a vital rolein determining the fate of nanocarriers during therapeutic delivery. Lipidnanoparticles (LNPs) and CPPs represent two effective types of nanoparticle vectorsin gene delivery. Early research indicated that the protein corona surrounding LNPs,tends to contain a significant amount of apolipoprotein E (apoE) that plays animportant role in hepatic uptake. In this study, we assessed the protein coronasurrounding CPP/mRNA nanoparticle complexes and compared it with the proteincorona of mRNA-containing LNPs that are used clinically. Our results demonstrated agreater enrichment of apoE in the protein corona of CPP/mRNA complexes comparedto LNPs. Moreover, in our comparison of CPP formulations decorated with apoE andtransfected into the HepG2 cell line with undecorated formulations, we observed adose-dependent increase in the transfection efficiency of the decoratedformulations. The outcomes from this study will serve as the foundation for developingsafer and more efficient gene delivery vehicles utilizing nanoparticles, advancing ourunderstanding of their interaction with biological fluids. |
