Journal: Advanced Drug Delivery Reviews

Article Title: Messenger RNA-based vaccines: Past, present, and future directions in the context of the COVID-19 pandemic

doi: 10.1016/j.addr.2021.114000

Figure Lengend Snippet: A summary of the various mRNA vaccine candidates for the prophylactic treatment of SARS-CoV-2

Article Snippet: Pfizer/BioNTech , mRNA , LNP-encapsulated modified mRNA encoding for COVID-19 spike protein delivered intramuscularly over two doses with a potential third dose booster , Emergency use authorization in several countries around the world.

Techniques: Modification, Plasmid Preparation, Injection, Virus, Clinical Proteomics, Formulation

Journal: Infection and Drug Resistance

Article Title: The Development of mRNA Vaccines for Infectious Diseases: Recent Updates

doi: 10.2147/IDR.S341694

Figure Lengend Snippet: mRNA Vaccines Against Infectious Diseases

Article Snippet: SARS-CoV-2 , CV2nCoV/CVnCoV, CureVac AG , Two-dose regimen, 21 days apart, delivered intramuscularly , Preclinical , N/A , LNP- encapsulated sequence optimized spike mRNA , [ ] .

Techniques: Sequencing, Neutralization, Expressing

Journal: Cell

Article Title: A Unified Model for the Function of YTHDF Proteins in Regulating m 6 A-Modified mRNA

doi: 10.1016/j.cell.2020.05.012

Figure Lengend Snippet: (A-D) m6A-modified mRNAs show substantially higher increase in expression upon simultaneous silencing of DF1, DF2, and DF3 in HeLa cells. The abundance of each mRNA (based on RNA-seq counts) was compared between the control and the conditions of DF paralog(s) silencing, as indicated. mRNAs were binned based on the number of m6A sites. The expression distribution of each of m6A mRNA subgroup is quantified in the boxplots. The center of each box represents the median fold change (log2), the boundaries contain genes within a quartile of the median, whiskers represent 1.5 × interquartile ranges. The width of the boxplot is proportional to the number of genes in each category. m6A mRNAs do not change in expression upon silencing of DF1 or DF3 compared to non-methylated mRNAs. However, m6A mRNAs show a small increase in their expression upon silencing of DF2. m6A-modified mRNAs show substantially higher increase in expression upon simultaneous silencing of DF1, DF2, and DF3. Only significant p-values are shown in each graph, two-tailed Mann–Whitney test. n=3 replicates. n=2425 mRNAs for 0, n=894 mRNAs for 1, n=1521 mRNAs for 2–4, n=2022 mRNAs for 5+ m6A sites.

Article Snippet: To account for differences in extraction efficiency, 2 ng of a spike-in luciferase mRNA (TriLink Biotechnologies) was added to each fraction before RNA extraction.

Techniques: Modification, Expressing, RNA Sequencing, Control, Methylation, Two Tailed Test, MANN-WHITNEY

Journal: Cell

Article Title: A Unified Model for the Function of YTHDF Proteins in Regulating m 6 A-Modified mRNA

doi: 10.1016/j.cell.2020.05.012

Figure Lengend Snippet: (A) DF paralogs are not highly associated with actively translated mRNAs. DF1, DF2, and DF3 were not enriched in polysomal fractions. Instead, they were predominantly associated with the mRNA ribonucleoprotein complex fraction (mRNPs). The ribosomal protein RPS6 was used as a marker for ribosome-enriched fractions.

Article Snippet: To account for differences in extraction efficiency, 2 ng of a spike-in luciferase mRNA (TriLink Biotechnologies) was added to each fraction before RNA extraction.

Techniques: Marker

Journal: Cell

Article Title: A Unified Model for the Function of YTHDF Proteins in Regulating m 6 A-Modified mRNA

doi: 10.1016/j.cell.2020.05.012

Figure Lengend Snippet: (A) TNFRSF1B mRNA expression is increased upon depletion of all DF paralogs. TNFRSF1B mRNA levels were measured by qRT-PCR in MOLM-13 cells after knockdown of each paralog. DF2 silencing lead to increased levels of TNFRSF1B, consistent with previous results (Paris et al., 2019). However, a larger and statistically significant increase in its expression level was seen upon silencing all three DF paralogs, suggesting that the combined activity of the DF paralogs mediates the destabilization of TNFRSF1B. TNFRSF1B levels were normalized to RPS28 expression levels, a non-methylated mRNA (Vu et al., 2017). Non-parametric ANOVA test, ****p ≤ 0.0001, n=3 mean± SEM.

Article Snippet: To account for differences in extraction efficiency, 2 ng of a spike-in luciferase mRNA (TriLink Biotechnologies) was added to each fraction before RNA extraction.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Activity Assay, Methylation

Journal: Vaccines

Article Title: Nanomaterial Delivery Systems for mRNA Vaccines

doi: 10.3390/vaccines9010065

Figure Lengend Snippet: mRNA lipid nanoparticle structure. Recent studies using cryoelectron microscopy , small-angle neutron scattering and small-angle X-ray scattering have shown that the mRNA Lipid nanoparticle includes low copy numbers of mRNA (1–10) and that the mRNA is bound by the ionizable lipid that occupies the central core of the LNP. The polyethylene glycol (PEG) lipid forms the surface of the lipid nanoparticle (LNP), along with DSPC, which is bilayer forming. Cholesterol and the ionizable lipid in charged and uncharged forms can be distributed throughout the LNP. Structural schematics of other delivery systems are available in a recent review .

Article Snippet: BioNTech Pfizer , nucleoside modified mRNA , membrane bound prefusion stabilized spike , 30 , Acuitas ALC-0315 [ ] confirmed in [ ] , [ , , , ] .

Techniques: Cryo-Electron Microscopy

Journal: Vaccines

Article Title: Nanomaterial Delivery Systems for mRNA Vaccines

doi: 10.3390/vaccines9010065

Figure Lengend Snippet: mRNA lipid nanoparticle assembly is achieved by ( A ) rapid mixing in a microfluidic or T-junction mixer of four lipids (ionizable lipid, DSPC, cholesterol, PEG–lipid) in ethanol with mRNA in an aqueous buffer near pH4. ( B ) When the ionizable lipid meets the aqueous phase, it becomes protonated at a pH ~5.5, which is intermediate between the pKa of the buffer and that of the ionizable lipid. ( C ) The ionizable lipid then electrostatically binds the anionic phosphate backbone of the mRNA while it experiences hydrophobicity in the aqueous phase, driving vesicle formation and mRNA encapsulation. ( D ) After initial vesicle formation, the pH is raised by dilution, dialysis or filtration, which results in the neutralization of the ionizable lipid, rendering it more hydrophobic and thereby driving vesicles to fuse and causing the further sequestration of the ionizable lipid with mRNA into the interior of the solid lipid nanoparticles. The PEG–lipid content stops the fusion process by providing the LNP with a hydrophilic exterior, determining its thermodynamically stable size, and the bilayer forming DSPC is present just underneath this PEG–lipid layer.

Article Snippet: BioNTech Pfizer , nucleoside modified mRNA , membrane bound prefusion stabilized spike , 30 , Acuitas ALC-0315 [ ] confirmed in [ ] , [ , , , ] .

Techniques: Encapsulation, Filtration, Neutralization