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While lipid nanoparticles (LNPs) are a key enabling technology for RNA-based therapeutics, some outstanding challenges hinder their wider clinical translation and use, particularly in terms of RNA stability and limited shelf life. In response to these limitations, we developed silicon-stabilized hybrid lipid nanoparticles (sshLNPs) as a next-generation nanocarrier with improved physical and temperature stability, as well as the highly advantageous capacity for "post-hoc loading" of RNA. Nevertheless, previously reported sshLNP formulations were produced using lipid thin film hydration, making scale-up impractical. To realize the potential of this emerging delivery platform, a manufacturing process enabling multikilogram batch sizes was required for successful clinical translation and deployment at scale. This was achieved by developing a revised protocol based on solvent injection mixing and incorporating other process adjustments to enable in-flow extrusion of multiliter volumes, while ensuring sshLNPs with the desired characteristics. Optimized procedures for nanoparticle formation, extrusion, and tangential flow filtration (to remove residual organic solvent) currently enable production of 2 kg finished batches. Importantly, sshLNPs produced via the modified large-scale workflow show equivalent physical and functional properties to those derived from the earlier small-scale methods, paving the way for GMP manufacturing protocols to enable vital translational clinical studies., Competing Interests: All authors are employees of SiSaf Ltd. S.S.-S. is CEO, board member, and shareholder of SiSaf Ltd. S.S.-S., N.T.-P., and A.D. are named inventors in a patent application filed by SiSaf Ltd. on the manufacturing process for silicon-stabilized lipid nanoparticles., (Crown Copyright © 2024 Published by Elsevier Inc. on behalf of The American Society of Gene and Cell Therapy.)

Rare skeletal diseases are still in need of proper clinically available transfection agents as the major challenge for first-in-human translation relates to intrinsic difficulty in targeting bone without exacerbating any inherent toxicity due to used vector. SiSaf's silicon stabilized hybrid lipid nanoparticles (sshLNPs) constitute next-generation non-viral vectors able to retain the integrity and stability of constructs and to accommodate considerable payloads of biologicals, without requiring cold-chain storage. sshLNP was complexed with a small interfering RNA (siRNA) specifically designed against the human CLCN7 G215R mRNA. When tested via single intraperitoneal injection in pre-puberal autosomal dominant osteopetrosis type 2 (ADO2) mice, carrying a heterozygous mutation of the Clcn7 gene ( Clcn7 G213R ), sshLNP, this significantly downregulated the Clcn7 G213R related mRNA levels in femurs at 48 h. Confirmatory results were observed at 2 weeks and 4 weeks after treatments (3 intraperitoneal injections/week), with rescue of the bone phenotype and demonstrating safety. The pre-clinical results will enable advanced preclinical development of RNA-based therapy for orphan and genetic skeletal disorders by safely and effectively delivering biologicals of interest to cure human systemic conditions., Competing Interests: A.T. and A.M. served as consultant, P.P had a service agreement, and F.M.S., N.T.-P., P.B.R, C.B., U.N., M.W., and A.D. are scientists working for SiSaf Ltd, a commercial stage biopharmaceutical company. S.S.-S. is founder and CEO of SiSaf Ltd., (© 2023 The Author(s).)