Branching Ionizable Lipids Can Enhance the Stability, Fusogenicity, and Functional Delivery of mRNA

Ionizable lipids with branched tails have been used in lipid nanoparticles (LNPs)-based messenger RNA (mRNA) therapeutics like COVID-19 vaccines. However, due to the limited commercial availability of branched ingredients, a systematic analysis of how the branched tails affect LNP quality has been lacking to date. Herein, a-branched tail lipids are focused, as they can be synthesized from simple commercially available chemicals, and the length of each chain can be independently controlled. Furthermore, symmetry and total carbon number can be used to describe a-branched tails, facilitating the design of a systematic lipid library to elucidate "structure-property-function" relationships. Consequently, a lipid library is developed containing 32 different types of a-branched tails. This library is used to demonstrate that branched chains increase LNP microviscosity and headgroup ionization ability in an acidic environment, which in turn enhances the stability and in vivo efficacy of mRNA-LNPs. Of the branched lipids, CL4F 8-6 LNPs carrying Cas9 mRNA and sgRNA could achieve 54% genome editing and 77% protein reduction with a single dose of 2.5 mg kg(-1). This mechanism-based data on branched lipids is expected to provide insights into rational lipid design and effective gene therapy in the future.

Investigation of Treatment-Emergent Amino Acid Substitutions in Nonstructural Protein 5 from Ph2a Study of Ensitrelvir, a Novel Oral SARS-CoV-2 3C-Like Protease Inhibitor

Background. Ensitrelvir is a novel oral SARS-CoV-2 3C-like protease inhibitor, and under late clinical development stage for COVID-19 disease. In Ph2a and Ph2b studies, ensitrelvir demonstrated rapid decline of viral titer and viral RNA compared with placebo, and tolerability. To investigate treatment-emergent amino acid substitutions (TEAASs), we analyzed viral RNA sequences of nonstructural protein 5 (NSP5), target of ensitrelvir from Ph2a study in Japan. Methods. In Ph2a study, 69 patients with confirmed SARS-CoV-2 infection were randomized 1:1:1 to ensitrelvir with the loading dose on Day 1/maintenance dose on Day 2-5 (375/125 mg or 750/250 mg), or placebo. Intent-to-treat (ITT) population was defined as participants confirmed with positivity by qualitative RT-PCR at baseline. TEAASs were defined as novel amino acid substitutions identified after treatment with ensitrelvir. NSP5 sequence analysis was performed with sanger sequencing using nasopharyngeal swab samples at Day 1, 6, 9, 14, and 21 with allowances from ITT population with ensitrelvir. Infectious viral titer was measured by virus-induced cytopathic effects in VeroE6/TMPRSS2 cells. Viral RNA was quantified by RT-PCR. Results. By NSP5 sequence analysis, TEAASs were observed in 1 patient from 375/125 mg group and 2 patients from 750/250 mg group. H246Y in 375/125 mg was detected from specimen on Day 8, and A234S and T198I in 750/250 mg were detected from specimens on Day 8 and Day 14, respectively. Structural analysis revealed that these mutations are located outside of the active center of 3C-like protease which is the binding site of ensitrelvir. Viral titer and viral RNA in each specimen, in which TEAASs were observed, were below lower limit of detection and lower limit of quantification, respectively. Furthermore, H246Y, A234S and T198I are rare substitution (< 0.05%) among SARS-CoV-2 variants according to Global Initiative on Sharing Avian Influenza Data. Conclusion. In Ph2a study, H246Y, A234S, and T198I in NSP5 were detected as TEAASs. However, results of viral titer and viral RNA, and structural information suggest that these mutations do not have the impact on antiviral efficacy of ensitrelvir.

Sustainable, collaborative EEG learning with a nationwide webinar

Purpose: To under stand the needs and issues of electr oencephalogr aphy (EEG) education following the onset of novel coronavirus disease 2019 (COVID-19) pandemic, and report our current EEG webinar management. Methods: We conducted a weekly online EEG lectur e at Hir oshima Univer sity Hospital in a hybrid format from July 2020 to December 2021. We recruited trainees using multiple types of social network services (SNS). We assessed the background of trainees in terms of postgraduate year, hospital size and location, area of expertise, entry route for registration, and clinical interests. We also investigated the current EEG educational needs and other relevant issues. Results: A total of 468 tr ainees (419 clinicians, 49 technicians) r egister ed fr om var ious insti-tutes nationwide (229 hospitals and 9 clinics). As more than 60 webinars were presented over a year, a cumulative total of more than 3,000 trainees joined the webinars. Trainees’ background was variable. SNS were helpful for recruitment. Major reasons for participation were to learn the minimum essentials of reading an EEG (70.1%) and to be able to read a digital EEG (56.5%). Privacy policies and web communication failures were major challenges. Discussion: The study identified r obust and nationwide needs for lear ning EEG r egar dless of area of expertise. Reduced face-to-face chance due to the pandemic greatly affected EEG educa-tion, as the education conventionally took a form of multidisciplinary collaboration in person. Our experience suggests that in addition to face-to-face education, information and communication technology can provide a sustainable learning platform.