Understanding the impact of in vitro transcription byproducts and contaminants.

The success of messenger (m)RNA-based vaccines against SARS-CoV-2 during the COVID-19 pandemic has led to rapid growth and innovation in the field of mRNA-based therapeutics. However, mRNA production, whether in small amounts for research or large-scale GMP-grade for biopharmaceutics, is still based on the In Vitro Transcription (IVT) reaction developed in the early 1980s. The IVT reaction exploits phage RNA polymerase to catalyze the formation of an engineered mRNA that depends on a linearized DNA template, nucleotide building blocks, as well as pH, temperature, and reaction time. But depending on the IVT conditions and subsequent purification steps, diverse byproducts such as dsRNA, abortive RNAs and RNA:DNA hybrids might form. Unwanted byproducts, if not removed, could be formulated together with the full-length mRNA and cause an immune response in cells by activating host pattern recognition receptors. In this review, we summarize the potential types of IVT byproducts, their known biological activity, and how they can impact the efficacy and safety of mRNA therapeutics. In addition, we briefly overview non-nucleotide-based contaminants such as RNases, endotoxin and metal ions that, when present in the IVT reaction, can also influence the activity of mRNA-based drugs. We further discuss current approaches aimed at adjusting the IVT reaction conditions or improving mRNA purification to achieve optimal performance for medical applications.

Dental aerosol-producing treatments: Comparison of contamination patterns of face shields and surgical masks.

During the COVID-19 pandemic, dental face shields were recommended to protect the eyes. This study aimed to examine to what extent face shield and mask contamination differ when a pre-procedural mouth rinsing with Chlorhexidine (CHX) is conducted before treatment. In this prospective, randomized study, three groups of subjects were formed (rinsing with 0.1% CHX, water, or no rinsing (control) before aerosol-producing treatments). After each of the 301 treatments, the practitioner's face shield was swabbed with eSwab and the mask was brought into contact with agar plates. Sampling was done from the exterior surface only. Samples were cultured for 48 h at 35 °C under aerobic and anaerobic conditions. Bacteria were classified by phenotypic characteristics, biochemical test methods, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Colony-forming units were counted and mean values were compared (WSR, H-test, U-test, p < 0.05). Within each subject group, face shields showed significantly more contamination than surgical masks (control group: 350 CFU, 50 CFU; intervention water: 270 CFU, 40 CFU; intervention CHX: 250 CFU, 30 CFU). Comparison of face shields of the different subject groups did not reveal any statistically significant differences. However, CHX resulted in a statistically significant bacterial reduction on surgical masks compared to the water and control group (control: 50 CFU, intervention water: 40 CFU, intervention CHX: 30 CFU). Contamination of face shields and surgical masks was highest in the control group, followed by the water group, and lowest in the intervention group with CHX. Streptococcus spp. and Staphylococcus spp. dominated, representing the oral and cutaneous flora. Contamination of masks worn with or without face shields did not differ. Presumably, face shields intercept first splashes and droplets, while the masks were mainly exposed to bioaerosol mist. Consequently, face shields protect the facial region and surroundings from splashes and droplets, but not the mask itself. A pre-procedural mouth rinse with CHX had no statistically significant reducing effect on contamination of the face shield, but a statistically significant reducing effect was observed on contamination of the mask.

Detection of viable oral bacteria of the patient on the surgical mask of dentists.

INTRODUCTION AND AIM: Bioaerosols contaminate the personal protective equipment (PPE), especially masks. The PPE harbors microorganisms from various sources. However, no previous studies have investigated the specific sources of bacteria found on used masks and their correlation with those from the treated patient. SETTING, DESIGN, MATERIAL AND METHODS: Intraoral samples from the patient were collected prior to dental aerosol-producing treatments using a nylon flock fiber swab. After treatment, the practitioner's mask was imprinted onto agar plates. MAIN OUTCOME METHODS: Following cultivation, colony forming units were counted and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). After the samples were analyzed, the intraoral samples as well as the mask samples were assessed for the presence of identical species, which were subsequently quantified. RESULTS: 126 treatments were included. One species match occurred most frequently (26.2%), followed by two (11.9%%) and three or more (3.97%). In the intraoral samples, Neisseria subflava occurred most often, within mask samples Staphylococcus epidermidis were detected most. Staphylococcus aureus could be cultivated three times more often in intraoral samples than on the mask. DISCUSSION AND CONCLUSION: Oral microorganisms originating from the patient's oral cavity can be found on the outside of masks. When using PPE during treatments, it should therefore always be in mind that potentially pathogenic microorganisms may land on the mask becoming a source of for itself.

ASL mRNA-LNP Therapeutic for the Treatment of Argininosuccinic Aciduria Enables Survival Benefit in a Mouse Model.

Argininosuccinic aciduria (ASA) is a metabolic disorder caused by a deficiency in argininosuccinate lyase (ASL), which cleaves argininosuccinic acid to arginine and fumarate in the urea cycle. ASL deficiency (ASLD) leads to hepatocyte dysfunction, hyperammonemia, encephalopathy, and respiratory alkalosis. Here we describe a novel therapeutic approach for treating ASA, based on nucleoside-modified messenger RNA (modRNA) formulated in lipid nanoparticles (LNP). To optimize ASL-encoding mRNA, we modified its cap, 5' and 3' untranslated regions, coding sequence, and the poly(A) tail. We tested multiple optimizations of the formulated mRNA in human cells and wild-type C57BL/6 mice. The ASL protein showed robust expression in vitro and in vivo and a favorable safety profile, with low cytokine and chemokine secretion even upon administration of increasing doses of ASL mRNA-LNP. In the ASLNeo/Neo mouse model of ASLD, intravenous administration of the lead therapeutic candidate LNP-ASL CDS2 drastically improved the survival of the mice. When administered twice a week lower doses partially protected and 3 mg/kg LNP-ASL CDS2 fully protected the mice. These results demonstrate the considerable potential of LNP-formulated, modified ASL-encoding mRNA as an effective alternative to AAV-based approaches for the treatment of ASA.

CHX and a Face Shield Cannot Prevent Contamination of Surgical Masks.

Background: Bacterial contamination on surgical masks puts a threat to medical staff and patients. The aim of the study was to investigate its contamination during dental treatments, wearing a face shield and performing a pre-procedural mouth rinsing with chlorhexidine (CHX). Methods: In this prospective, randomized study, 306 treatments were included, 141 single-tooth (restorations) and 165 total dentition treatments (preventive or periodontal supportive ultrasonic application). A total of three groups (each: n = 102) were formed: participants rinsed for 60 s with 0.1 % CHX or with water before treatment, and, for control, a non-rinsing group was included. In view of the COVID-19 pandemic, a face shield covering the surgical mask enhanced personal protective equipment. After treatment, masks were imprinted on agar plates and incubated at 35°C for 48 h. Bacteria were classified by phenotypic characteristics, biochemical assay methods, and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Colonies (CFU) were counted and mean values were compared (Kruskal-Wallis-, U test, p < 0.05). Results: Chlorhexidine led to a statistically significant reduction of bacterial contamination of the surgical mask (mean: 24 CFU) in comparison with water (mean: 47 CFU) and non-rinsing (mean: 80 CFU). Furthermore, rinsing with water reduced CFU significantly in comparison with the non-rinsing group. There were no significant differences between single or total dentition treatments. Streptococcus spp., Staphylococcus spp., Micrococcus spp., and Bacillus spp. dominated, representing the oral and cutaneous flora. Conclusion: A pre-procedural mouth rinse is useful to reduce the bacterial load of the surgical mask. However, contamination cannot be prevented completely, even by applying a face shield. In particular, during pandemic, it is important to consider that these additional protective measures are not able to completely avoid the transmission of pathogens bearing aerosols to the facial region. If antiseptic rinsing solutions are not available, rinsing with water is also useful.

Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA.

The presence of the cap structure on the 5'-end of in vitro-transcribed (IVT) mRNA determines its translation and stability, underpinning its use in therapeutics. Both enzymatic and co-transcriptional capping may lead to incomplete positioning of the cap on newly synthesized RNA molecules. IVT mRNAs are rapidly emerging as novel biologics, including recent vaccines against COVID-19 and vaccine candidates against other infectious diseases, as well as for cancer immunotherapies and protein replacement therapies. Quality control methods necessary for the preclinical and clinical stages of development of these therapeutics are under ongoing development. Here, we described a method to assess the presence of the cap structure of IVT mRNAs. We designed a set of ribozyme assays to specifically cleave IVT mRNAs at a unique position and release 5'-end capped or uncapped cleavage products up to 30 nt long. We purified these products using silica-based columns and visualized/quantified them using denaturing polyacrylamide gel electrophoresis (PAGE) or liquid chromatography and mass spectrometry (LC-MS). Using this technology, we determined the capping efficiencies of IVT mRNAs with different features, which include: Different cap structures, diverse 5' untranslated regions, different nucleoside modifications, and diverse lengths. Taken together, the ribozyme cleavage assays we developed are fast and reliable for the analysis of capping efficiency for research and development purposes, as well as a general quality control for mRNA-based therapeutics.

Bacterial contamination of forehead skin and surgical mask in aerosol-producing dental treatment.

BACKGROUND: Bacterial contamination of dental professionals' facial skin and protective equipment from treatment-related aerosols and droplets are poorly studied. METHODS: This prospective study analyzed samples from 67 consecutive aerosol-producing dental treatments. Sterile nylon swabs served to collect samples from dental professionals' foreheads before and after exposure. Contact samples were obtained from used surgical masks. Samples were incubated on agar under aerobic and anaerobic conditions. Bacteria were classified by MALDI-TOF mass spectrometry. We determined the frequencies of obligate and facultative oral bacteria and scored bacterial growth (0: none; 1: < 100 colonies; 2: >100 colonies; 3: dense). RESULTS: Bacteria were detected in 95% of skin-swab and 76% of mask samples. Median bacterial scores were 2 for forehead samples before and after treatment, and 1 for masks. Obligate and facultative oral bacteria were more frequent (6% and 30%) in samples from exposed forehead skin, which also showed increased bacterial scores (28%). 5% of samples contained methicillin-sensitive Staphylococcus aureus; 3% contained obligate anaerobes. CONCLUSION: Exposed forehead skin was significantly less contaminated with obligate oral bacteria than expected based on surgical mask findings. Exposed forehead skin showed increased contamination attributable to aerosol-producing procedures. The forehead's physiological skin microbiota may offer some protection against bacterial contamination.

BNT162b2 vaccine induces neutralizing antibodies and poly-specific T cells in humans.

BNT162b2, a nucleoside-modified mRNA formulated in lipid nanoparticles that encodes the SARS-CoV-2 spike glycoprotein (S) stabilized in its prefusion conformation, has demonstrated 95% efficacy in preventing COVID-191. Here we extend a previous phase-I/II trial report2 by presenting data on the immune response induced by BNT162b2 prime-boost vaccination from an additional phase-I/II trial in healthy adults (18-55 years old). BNT162b2 elicited strong antibody responses: at one week after the boost, SARS-CoV-2 serum geometric mean 50% neutralizing titres were up to 3.3-fold above those observed in samples from individuals who had recovered from COVID-19. Sera elicited by BNT162b2 neutralized 22 pseudoviruses bearing the S of different SARS-CoV-2 variants. Most participants had a strong response of IFNγ+ or IL-2+ CD8+ and CD4+ T helper type 1 cells, which was detectable throughout the full observation period of nine weeks following the boost. Using peptide-MHC multimer technology, we identified several BNT162b2-induced epitopes that were presented by frequent MHC alleles and conserved in mutant strains. One week after the boost, epitope-specific CD8+ T cells of the early-differentiated effector-memory phenotype comprised 0.02-2.92% of total circulating CD8+ T cells and were detectable (0.01-0.28%) eight weeks later. In summary, BNT162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes that are conserved in a broad range of variants, at well-tolerated doses.

Contamination of surgical mask during aerosol-producing dental treatments.

OBJECTIVES: Surgical masks are usually contaminated during dental treatment. So far it has not been investigated whether a surgical mask itself can be a source of microbial transmission. The aim of this study was therefore to investigate the microbiological contamination of surgical masks during dental treatment and the transfer of microorganisms from the mask to the hands. MATERIALS AND METHODS: Five dental treatment modalities were studied: carious cavity preparation (P-caries, n = 10), tooth substance preparation (P-tooth, n = 10), trepanation and root canal treatment (P-endo, n = 10), supragingival ultrasonic application (US-supra, n = 10), and subgingival periodontal ultrasonic instrumentation (US-sub, n = 10). Bacterial contamination of mask and gloves worn during treatment was tested by imprinting on agar plates. Additionally, before masks were tested, their outer surface was touched with a new sterile glove. This glove was also imprinted on agar. Bacteria were identified by MALDI TOF mass spectrometry. Colony-forming units (CFU) were scored: score 0: 0 CFU, score 1: < 102 CFU, score 2: > 102 CFU, score 3: dense microbial growth. RESULTS: All masks and all gloves used during treatment displayed bacterial contamination (sample scores 0/1/2/3: masks 0/46/3/1 and gloves 0/31/10/9). After touching the masks with new sterile gloves, microorganisms were recovered with the following contamination scores: P-caries: 4/6/0/0, P-tooth: 2/8/0/0: P-endo: 7/3/0/0, US-supra: 0/9/1/0, US-sub: 2/8/0/0. No statistically significant differences were detected between the treatment modalities. Streptococci spp. and Staphylococci spp. representing the oral and cutaneous flora dominated. CONCLUSIONS: Surgical masks are contaminated after aerosol-producing dental treatment procedures. Used masks have a potential to be a source of bacterial contamination of the hands. CLINICAL RELEVANCE: Dental staff should avoid touching the outer surface of masks with their hands to prevent transmission of pathogens. It is recommendable to change the mask after each treated patient followed by hand disinfection.

Cyclobutane pyrimidine dimers from UVB exposure induce a hypermetabolic state in keratinocytes via mitochondrial oxidative stress.

Ultraviolet B radiation (UVB) is an environmental complete carcinogen, which induces and promotes keratinocyte carcinomas, the most common human malignancies. UVB induces the formation of cyclobutane pyrimidine dimers (CPDs). Repairing CPDs through nucleotide excision repair is slow and error-prone in placental mammals. In addition to the mutagenic and malignancy-inducing effects, UVB also elicits poorly understood complex metabolic changes in keratinocytes, possibly through CPDs. To determine the effects of CPDs, CPD-photolyase was overexpressed in keratinocytes using an N1-methyl pseudouridine-containing in vitro-transcribed mRNA. CPD-photolyase, which is normally not present in placental mammals, can efficiently and rapidly repair CPDs to block signaling pathways elicited by CPDs. Keratinocytes surviving UVB irradiation turn hypermetabolic. We show that CPD-evoked mitochondrial reactive oxygen species production, followed by the activation of several energy sensor enzymes, including sirtuins, AMPK, mTORC1, mTORC2, p53, and ATM, is responsible for the compensatory metabolic adaptations in keratinocytes surviving UVB irradiation. Compensatory metabolic changes consist of enhanced glycolytic flux, Szent-Györgyi-Krebs cycle, and terminal oxidation. Furthermore, mitochondrial fusion, mitochondrial biogenesis, and lipophagy characterize compensatory hypermetabolism in UVB-exposed keratinocytes. These properties not only support the survival of keratinocytes, but also contribute to UVB-induced differentiation of keratinocytes. Our results indicate that CPD-dependent signaling acutely maintains skin integrity by supporting cellular energy metabolism.