Design and evaluation of bioinspired lipid nanoparticles for optimized gene therapy

Gene therapy offers the possibility to prevent and treat numerous diseases, thereby improving the well-being of millions of individuals. The success of such treatments requires efficient delivery of nucleic acids to cells. For this, various gene delivery strategies, including modified oligonucleotides, viruses, and lipid nanoparticles (LNPs), have been widely explored and used in clinical practice. LNPs have demonstrated their effectiveness with the success of the messenger ribonucleic acid vaccines from Pfizer-BioNTech and Moderna against the coronavirus disease 2019. However, despite this monumental achievement, the LNP technology still faces challenges, including limited efficacy and potency, safety concerns, and regulatory considerations.
In the first part of this thesis, the aim was to optimize the performance of LNPs in terms of efficacy and potency. To achieve this, bioinspired and ionizable cationic lipid modifications were explored. Two bioinspired formulations were subsequently developed: phosphatidylserine-LNPs and hybrid LNPs. The first formulation incorporates phosphatidylserine, a lipid typically found in viruses, while the second results from the fusion of LNPs with extracellular vesicles, which are a naturally occurring gene delivery system. Impressively, these formulations surpassed the performance of standard LNPs, exhibiting up to 14-fold higher reporter gene expression in vitro and in vivo in zebrafish larvae and mice. Further investigations highlighted the potential of ionizable cationic lipid modifications to boost the performance of LNPs. The improvements of these formulations were primarily linked to enhancements in cellular uptake and intracellular processing, especially endosomal escape.
In the second part, aiming to bridge the gap between in vitro and in vivo, an endosomal escape reporter cell line, zebrafish larvae, and intravital imaging were employed to assess the behavior and performance of various nanomedicines. The insights gained from these experiments were invaluable, helping to determine certain in vitro to in vivo prediction aspects. Notably, the in vitro endosomal escape capability of LNPs and their biodistribution and circulation behavior in zebrafish larvae have emerged as important indicators of their in vivo behavior and performance in rodents.
In conclusion, modifying the lipid composition of LNPs significantly improves their performance with respect to efficacy and potency. However, persisting challenges related to cell specificity and cellular uptake emphasize the potential of active targeting strategies, as indicated by preliminary studies. Ultimately, the combination of lipid composition modifications, active targeting strategies, and both in vitro and in vivo models, will serve as a firm foundation for the design of efficacious and potent LNP-based gene therapies.