Exposure of the Basophilic Cell Line KU812 to Liposomes Reveals Activation Profiles Associated with Potential Anaphylactic Responses Linked to Physico-Chemical Characteristics.

Lipidic nanoparticles (LNP), particularly liposomes, have been proven to be a successful and versatile platform for intracellular drug delivery for decades. Whilst primarily developed for small molecule delivery, liposomes have recently undergone a renaissance due to their success in vaccination strategies, delivering nucleic acids, in the COVID-19 pandemic. As such, liposomes are increasingly being investigated for the delivery of nucleic acids, beyond mRNA, as non-viral gene delivery vectors. Although not generally considered toxic, liposomes are increasingly shown to not be immunologically inert, which may have advantages in vaccine applications but may limit their use in other conditions where immunological responses may lead to adverse events, particularly those associated with complement activation. We sought to assess a small panel of liposomes varying in a number of physico-chemical characteristics associated with complement activation and inflammatory responses, and examine how basophil-like cells may respond to them. Basophils, as well as other cell types, are involved in the anaphylactic responses to liposomes but are difficult to isolate in sufficient numbers to conduct large scale analysis. Here, we report the use of the human KU812 cell line as a surrogate for primary basophils. Multiple phenotypic markers of activation were assessed, as well as the release of histamine and inflammasome activity within the cells. We found that larger liposomes were more likely to result in KU812 activation, and that non-PEGylated liposomes were potent stimulators of inflammasome activity (four-fold greater IL-1ß secretion than untreated controls), and a lower ratio of cholesterol to lipid was also associated with greater IL-1ß secretion ([Cholesterol:DSPC ratio] 1:10; 0.35 pg/mL IL-1ß vs. 5:10; 0.1 pg/mL). Additionally, PEGylation appeared to be associated with direct KU812 activation. These results suggest possible mechanisms related to the consequences of complement activation that may be underpinned by basophilic cells, in addition to other immune cell types. Investigation of the mechanisms behind these responses, and their impact on use in vivo, are now warranted.

Current Status and Future Perspectives on MRNA Drug Manufacturing.

The coronavirus disease of 2019 (COVID-19) pandemic launched an unprecedented global effort to rapidly develop vaccines to stem the spread of the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2). Messenger ribonucleic acid (mRNA) vaccines were developed quickly by companies that were actively developing mRNA therapeutics and vaccines for other indications, leading to two mRNA vaccines being not only the first SARS-CoV-2 vaccines to be approved for emergency use but also the first mRNA drugs to gain emergency use authorization and to eventually gain full approval. This was possible partly because mRNA sequences can be altered to encode nearly any protein without significantly altering its chemical properties, allowing the drug substance to be a modular component of the drug product. Lipid nanoparticle (LNP) technology required to protect the ribonucleic acid (RNA) and mediate delivery into the cytoplasm of cells is likewise modular, as are technologies and infrastructure required to encapsulate the RNA into the LNP. This enabled the rapid adaptation of the technology to a new target. Upon the coattails of the clinical success of mRNA vaccines, this modularity will pave the way for future RNA medicines for cancer, gene therapy, and RNA engineered cell therapies. In this review, trends in the publication records and clinical trial registrations are tallied to show the sharp intensification in preclinical and clinical research for RNA medicines. Demand for the manufacturing of both the RNA drug substance (DS) and the LNP drug product (DP) has already been strained, causing shortages of the vaccine, and the rise in development and translation of other mRNA drugs in the coming years will exacerbate this strain. To estimate demand for DP manufacturing, the dosing requirements for the preclinical and clinical studies of the two approved mRNA vaccines were examined. To understand the current state of mRNA-LNP production, current methods and technologies are reviewed, as are current and announced global capacities for commercial manufacturing. Finally, a vision is rationalized for how emerging technologies such as self-amplifying mRNA, microfluidic production, and trends toward integrated and distributed manufacturing will shape the future of RNA manufacturing and unlock the potential for an RNA medicine revolution.

The role of nanoparticle format and route of administration on self-amplifying mRNA vaccine potency.

The efficacy of RNA-based vaccines has been recently demonstrated, leading to the use of mRNA-based COVID-19 vaccines. The application of self-amplifying mRNA within these formulations may offer further enhancement to these vaccines, as self-amplifying mRNA replicons enable longer expression kinetics and more potent immune responses compared to non-amplifying mRNAs. To investigate the impact of administration route on RNA-vaccine potency, we investigated the immunogenicity of a self-amplifying mRNA encoding the rabies virus glycoprotein encapsulated in different nanoparticle platforms (solid lipid nanoparticles (SLNs), polymeric nanoparticles (PNPs) and lipid nanoparticles (LNPs)). These were administered via three different routes: intramuscular, intradermal and intranasal. Our studies in a mouse model show that the immunogenicity of our 4 different saRNA vaccine formulations after intramuscular or intradermal administration was initially comparable; however, ionizable LNPs gave higher long-term IgG responses. The clearance of all 4 of the nanoparticle formulations from the intramuscular or intradermal administration site was similar. In contrast, immune responses generated after intranasal was low and coupled with rapid clearance for the administration site, irrespective of the formulation. These results demonstrate that both the administration route and delivery system format dictate self-amplifying RNA vaccine efficacy.

Manufacturing Considerations for the Development of Lipid Nanoparticles Using Microfluidics.

In the recent of years, the use of lipid nanoparticles (LNPs) for RNA delivery has gained considerable attention, with a large number in the clinical pipeline as vaccine candidates or to treat a wide range of diseases. Microfluidics offers considerable advantages for their manufacture due to its scalability, reproducibility and fast preparation. Thus, in this study, we have evaluated operating and formulation parameters to be considered when developing LNPs. Among them, the flow rate ratio (FRR) and the total flow rate (TFR) have been shown to significantly influence the physicochemical characteristics of the produced particles. In particular, increasing the TFR or increasing the FRR decreased the particle size. The amino lipid choice (cationic-DOTAP and DDAB; ionisable-MC3), buffer choice (citrate buffer pH 6 or TRIS pH 7.4) and type of nucleic acid payload (PolyA, ssDNA or mRNA) have also been shown to have an impact on the characteristics of these LNPs. LNPs were shown to have a high (>90%) loading in all cases and were below 100 nm with a low polydispersity index (≤0.25). The results within this paper could be used as a guide for the development and scalable manufacture of LNP systems using microfluidics.