mRNA Vaccine Manufacturing: Responding Effectively to a Global Pandemic

We’ve learned a few important lessons from the COVID-19 pandemic.

Perhaps the most significant one is the importance of an early and rapid global response to the initial outbreak. A coordinated response—including widespread use of masks and other personal protective equipment (PPE), travel restrictions, lockdowns and social distancing—could save lives and reduce long-term health effects (1). Widespread availability of effective vaccines goes hand in hand with these measures.

New Boosters to Fight Omicron

Last month, Pfizer/BioNTech announced the US Food and Drug Administration (FDA) had granted emergency use authorization (EUA) for a new adapted-bivalent COVID-19 booster vaccine for individuals 12 years and older. This vaccine combines mRNA encoding the wild-type Spike protein from the original vaccine with another mRNA encoding the Spike protein of the Omicron BA.4/BA.5 subvariants. Moderna also announced FDA EUA for its new Omicron-targeting COVID-19 booster vaccine. The Omicron variant of SARS-CoV-2 shows multiple mutations across its subvariants, and it is currently the dominant SARS-CoV-2 variant of concern across the world.

Genomic epidemiology of SARS-CoV-2 with subsampling focused globally over the past 6 months. This phylogenetic tree shows evolutionary relationships of SARS-CoV-2 viruses from the ongoing COVID-19 pandemic. Image from Nextstrain.org; generated September 20, 2022

Booster doses of vaccines have become a way of life, both due to declining effectiveness of the original vaccines especially in older adults (2), and the rapid mutation rate of SARS-CoV-2 (3). Clinical data for the new Pfizer/BioNTech booster vaccine showed superior effectiveness in eliciting an immune response against Omicron BA.1 compared to the original vaccine. Previously, Moderna published interim results from an ongoing phase 2-3 clinical trial, showing that the new bivalent booster vaccine elicited a superior neutralizing antibody response against Omicron, compared to its original COVID-19 vaccine (4).

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How Does DNA Template Length Influence Gene Expression in Cell Free Systems?

3D sketch of coupled transcription/translation in a cell free system. A recent paper looks at the effect of DNA Template Length  on gene expression.

Cell-free gene expression systems are a staple tool for the researcher seeking to understand the regulation of transcription and translation. Many factors can affect the efficiency of cell-free gene expression including vector sequence, reaction components and the template DNA concentration. One factor that has not been extensively studied is how DNA template length influences gene expression.

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Studying Autophagy in Flies Using CRISPR

 

Transcribed RNA can be used to study RNA structure and how it relates to function or how proteins and RNA interact. It can also be used for gene silencing using RNAi (studied more often as a possible therapeutic option) or simply serve as a molecular standard in Real-time RT-PCR. Transcribed RNA is also used in Class 2 Clustered Regularly Interspaced Short Palindromic Repeat systems, or CRISPR.

The CRISPR system, which is naturally occurring in bacteria, has been manipulated to perform gene editing in a laboratory environment. To perform CRISPR in the laboratory environment, you need two main reagents:

  1. The Brains: Guide RNA (gRNA or sgRNA) – Small piece of RNA containing a nucleotide sequence that is capable of binding the chosen Cas Protein, and contains a portion of the sequence that can bind the DNA the researcher intends to modify – the target DNA.
  2. The Brawn: CRISPR-associated endonuclease (Cas Protein) – The protein that cleaves the target DNA; the most popular Cas protein is called Cas9. The Cas protein is guided by the (gRNA).

Recently, Guo et al. used Promega’s RiboMAX™ Large-Scale RNA Production System to produce gRNA to be used in CRISPR for their study to determine the effects of the loss of, or mutations in, a specific gene in fruit flies (1).  Atg101 is a gene that plays an important role in autophagy, an intracellular pathway for removing toxins or damaged parts of cells. Continue reading “Studying Autophagy in Flies Using CRISPR”

In Vitro Transcription: Common Causes of Reaction Failure

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A widely used molecular biology technique, in vitro transcription uses bacteriophage DNA-dependent RNA polymerases to synthesize template-directed RNA molecules. Enzymes like bacteriophage SP6, T3 and T7 RNA polymerases are used to produce synthetic RNA transcripts, which can be used as hybridization probes, as templates for in vitro translation applications, or in structural studies (X-ray crystallography and NMR). Synthesized RNA transcripts are also used for studying cellular RNA functionality in processes such as splicing, RNA processing, intracellular transport, viral infectivity and translation.

Problems in the transcription reaction can result in complete failure (i.e., no transcript generated) or in transcripts that are the incorrect size (i.e., shorter or longer than expected). Below is a discussion of the most common causes of in vitro transcription problems.

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Optimized Wheat Germ Extract for High-Yield Protein Expression of Functional, Soluble Protein

proteinexpressionCell-free protein synthesis has emerged as powerful alternative to cell based protein expression for functional and structural proteomics. The TNT® SP6 High-Yield Protein Expression System uses a high-yield wheat germ extract supplemented with SP6 RNA polymerase and other components. Coupling transcriptionaland translational activities eliminates the inconvenience of separate in vitro transcription and purification steps for the mRNA, while maintaining the high levels of protein expression (1). Continue reading “Optimized Wheat Germ Extract for High-Yield Protein Expression of Functional, Soluble Protein”