Optimization of Lipid Nanoparticles for saRNA Expression and Cellular Activation Using a Design-of-Experiment Approach

Abstract

Lipid nanoparticles (LNPs) are the leading technology for RNA delivery, given the success of the Pfizer/BioNTech and Moderna COVID-19 mRNA (mRNA) vaccines, and small interfering RNA (siRNA) therapies (patisiran). However, optimization of LNP process parameters and compositions for larger RNA payloads such as self-amplifying RNA (saRNA), which can have complex secondary structures, have not been carried out. Furthermore, the interactions between process parameters, critical quality attributes (CQAs), and function, such as protein expression and cellular activation, are not well understood. Here, we used two iterations of design of experiments (DoE) (definitive screening design and Box−Behnken design) to optimize saRNA formulations using the leading, FDA-approved ionizable lipids (MC3, ALC-0315, and SM-102). We observed that PEG is required to preserve the CQAs and that saRNA is more challenging to encapsulate and preserve than mRNA. We identified three formulations to minimize cellular activation, maximize cellular activation, or meet a CQA profile while maximizing protein expression. The significant parameters and design of the response surface modeling and multiple response optimization may be useful for designing formulations for a range of applications, such as vaccines or protein replacement therapies, for larger RNA cargoes.

Effective Perturbations by Small-Molecule Modulators on Voltage-Dependent Hysteresis of Transmembrane Ionic Currents

“…SM-102 is a synthetic and ionizable amino lipid that has been widely used in combination with other lipids in the formation of lipid nanoparticles [ 50 , 51 , 52 ]. Formulations containing SM-102 have been noticeably used in the development of lipid nanoparticles for the delivery of mRNA-based vaccines.…”

“…Formulations containing SM-102 have been noticeably used in the development of lipid nanoparticles for the delivery of mRNA-based vaccines. For example, SM-102 is known to be one of the ingredients in the Moderna TM COVID-19 vaccine [ 52 ]. Recent investigations have also disclosed that the strength of Hys (V) of I K(erg) elicited by the upright isosceles-triangular V ramp was profoundly decreased as cells were exposed to SM-102 or TurboFectin TM [ 53 ].…”

Self-Amplifying RNA Approach for Protein Replacement Therapy

“…Nevertheless, there are substantially more data on saRNA itself compared to taRNA till now; thus, the latter approach requires more studies for further development. Fortunately, along with developing RNA technologies, carrier platforms also continue to advance, which results in the successful delivery of molecules as large as saRNA [ 129 ]. Evidently, the saRNA-based vaccine for COVID-19 (NCT05012943) is in clinical study Phase 3, and, therefore, it is close to getting approval and becoming the first approved saRNA-based vaccine.…”

How far are the new wave of mRNA drugs from us? mRNA product current perspective and future development

“…Polyethyleneimine (PEI) was a common polymer used for gene delivery, but the high molecular weight of PEI resulted in high cytotoxicity, making its successful use for vaccines in a limited number of animal studies, and chemical modifications of PEI were continually being explored ( 77 ). Liposomes began to be used as a carrier-optimized route of administration in 1978, and cationic lipids were first proposed to mediate mRNA transcription in 1989, until the successful launch of mRNA COVID-19 vaccines in 2020 ( 78 – 80 ). Lipids are currently the most widely used class of delivery systems for nucleic acid drugs, including liposomes and lipid nanoparticles, the most commonly used being LNPs containing ionizable lipids ( Figure 5 ).…”