Optimizing mRNA vaccines and virusmediated CAR T-cell therapy for immuno-oncology: Synthetic tools for gene modification

With the success of mRNA vaccines during the COVID-19 pandemic and CAR T-cell therapies in clinical trials, there is growing opportunity for immunotherapies in the treatment of many types of cancers. Lentiviral vectors have proven effective at delivery of genetic material or gene editing technology for ex vivo processing, but the benefits and promise of Adeno-associated virus (AAV) and mRNA tools for in vivo immunotherapy have garnered recent interest. Here we describe complete synthetic solutions for immuno-oncology research programs using either mRNA-vaccines or virus-mediated cell and gene engineering. These solutions optimize workflows to minimize screening time while maximizing successful research results through: (1) Efficiency in lentiviral packaging with versatility in titer options for high-quality particles. (2) A highthroughput viral packaging process to enable rapid downstream screening. (3) Proprietary plasmid synthesis and preparation techniques to maintain ITR integrity through AAV packaging and improve gene delivery. (4) Rapid synthesis, in vitro transcription, and novel sequencing of mRNA constructs for complete characterization of critical components such as the polyA tail. The reported research demonstrates a streamlined approach that improves data quality through innovative synthesis and sequencing methodologies as compared to current standard practices.

SARS-CoV-2 infection disrupts trophoblast function and placental syncytial barrier integrity via its accessory protein, ORF3a

Problem: Although it is rare for a SARS-CoV-2 infection to transmit vertically to the fetus during pregnancy, there is a significantly increased risk of adverse pregnancy outcomes due to maternalCOVID- 19. However, there is a poor understanding of such risks because mechanistic studies on how SARS-CoV-2 infection disrupts placental homeostasis are significantly lacking. The SARS-CoV-2 proteome includes multiple structural and non-structural proteins, including the non-structural accessory proteinORF3a. The roles of these proteins in mediating placental infection remain undefined. We and others have shown that autophagy activity in placental syncytium is essential for barrier function and integrity. Here, we have used clinical samples and cultured trophoblast cells to evaluate syncytial integrity of placenta exposed to SARS-CoV-2. The objective of our study was to investigate potential mechanisms through which SARS-CoV-2 impairs placental homeostasis and causes adverse pregnancy outcomes. We tested the central hypothesis that an essential SARS-CoV-2 non-structural and accessory protein, ORF3a, uniquely (amongst multiple viral proteins tested) with a novel three-dimensional structure andwith no homology to any other proteins is a key modulator of placental trophoblast cell dynamics via autophagy and intracellular trafficking of a tight junction protein (TJP), ZO-1. Method(s): We used clinical samples and cultured trophoblast cells to evaluate syncytial integrity of placentas exposed to SARS-CoV- 2. Autophagic flux was measured in placental villous biopsies from SARS-CoV-2-exposed and unexposed pregnant women by quantifying the expression of autophagy markers, LC3 and P62. Trophoblast cells (JEG-3, Forskolin-treated JEG-3, HTR8/SVneo, or primary human trophoblasts (PHTs)) were transfected with expression plasmids encoding SARS-CoV-2 proteins including ORF3a. Using western blotting, multi-label immunofluorescence, and confocal imaging, we analyzed the effect of ORF3a on the autophagy, differentiation, invasion, and intracellular trafficking of ZO-1 in trophoblasts. Using coimmunoprecipitation assays, we tested ORF3a interactions with host proteins. t-tests and one-way analyses of variance (ANOVAs) with post hoc tests were used to assess the data, with significance set at P < .05. Result(s): We discovered :1) increased activation of autophagy, but incomplete processing of autophagosome-lysosomal degradation;2) accumulation of protein aggregates in placentas exposed to SARS-CoV- 2. Mechanistically, we showed that the SARS-CoV-2 ORF3a protein, uniquely 3) blocks the autophagy-lysosomal degradation process;4) inhibits maturation of cytotrophoblasts into syncytiotrophoblasts (STBs);5) reduces production ofHCG-beta, a key pregnancy hormone that is also essential for STB maturation;and 6) inhibits trophoblast invasive capacity. Furthermore, ORF3a harbors an intrinsically disordered C-terminus withPDZ-bindingmotifs.We show for the first time that, 7) ORF3a binds to and co-localizes with the PDZ domain of ZO-1, a junctional protein that is essential for STB maturation and the integrity of the placental barrier. Conclusion(s): Our work outlines a new molecular and cellular mechanism involving the SARS-CoV-2 accessory protein ORF3a that may drive the virus's ability to infect the placenta and damage placental syncytial integrity. This implies that the mechanisms facilitating viral maturation, such as the interaction of ORF3a with host factors, can be investigated for additional functionality and even targeted for developing new intervention strategies for treatment or prevention of SARS-CoV-2 infection at the maternal-fetal interface.

Gain and loss of function CFTR alleles modulate COVID-19 clinical outcome

Background/Objectives: We previously demonstrated that carrying a single pathogenic CFTR allele increases the risk for COVID-19 severity and mortality rate. We now aim to clarify the role of several uncharacterized rare alleles, including complex (cis) alleles, and in trans combinations. Method(s): LASSO logistic regression was used for the association of sets of variants, stratified by MAF, with severity. Immortalized cystic fibrosis bronchial epithelial cell lines and Fischer Rat Thyroid cells were transfected by plasmid carrying specific CFTR mutations. YFP-based assays were used to measure CFTR activity. Result(s): Here we functionally demonstrate that the rare (MAF=0.007) complex G576V/R668C allelemitigates the disease by a gain of function mechanism. Several novel CFTR ultra-rare (MAF <0.001) alleles were proved to have a reduced function;they are associated with disease severity either alone (single or complex alleles) or with another hypomorphic allele in the second chromosome, with a global reduction of CFTR activity between 40 to 72%. Conclusion(s): CFTR is a bidirectional modulator of COVID-19 outcome. At-risk subjects do not have open cystic fibrosis before viral infection and therefore are not easily recognisable in the general population unless a genetic analysis is performed. As the CFTR activity is partially retained, CFTR potentiator drugs could be an option as add-on therapy for at-risk patients.

Gene Editing/Gene Therapies: BIOREACTOR RECOMBINANT PRODUCTION OF SARS-COV-2 VIRUS ANTIGENS IN CHO CELL CLONES BY USING NEW UCOE LENTIVIRAL VECTOR SYSTEM

Background & Aim: The new UCOE models we have recently developed, tested on many cell groups (including mouse ES and human iPS cells) and human mAb recombinant production studies as well, shows a powerful resistance to DNA methylation- mediated silencing and provides a higher and stable transfection profile. By the urgent need of vaccine development for COVID-19 during the pandemic, in this study we aimed to produce a potential recombinant vaccine by using the new generation UCOEs models of our own design. Methods, Results & Conclusion(s): Existing new-generation UCOE models and standard plasmid vectors to be used as control group were provided. Then, the sequences related to the PCR method were amplified for sufficient stock generation and cloning experiments. Verification in the plasmid vector was carried out in gel electrophoresis. Transfection of 293T cells was performed with clone plasmids carrying antigen genes and plasmids carrying genetic information of lentivirus units for the production of lentiviral vectors. Afterwards, 293T cells produced lentiviral vectors carrying antigen genes. Harvesting of these vectors was carried out during 48th and 72nd hours. Afterwards, CHO cells were transduced with appropriate quantity of lentiviral vectors. Isolation and purification of targeted proteins from the relevant medium were performed by HPLC and Q-TOF methods. A part of the spike and nucleocapsid gene sequences of COVID-19 were firstly cloned into our UCOE models. These UCOEs plasmids were then transferred into 293T cells along with plasmids carrying the genes that will form the lentivirus vectors (LVs). After harvesting and calculation of LV vector titers, the cloned vectors were then transfected into the CHO cells which the targeted recombinant production of the antigen proteins will be carried out. Antigenic structures were then isolated from the culture medium of CHO cells in following days for confirmation. Using HPLC and qTOF mass spectrometer methods, these structures in the medium were confirmed to be the units of spike and nucleocapsid proteins of the COVID-19 virus. In order to produce large amount of the recombinant antigens, the culture was then carried out with bioreactors in liters. At the final stage, these recombinantly produced antigen proteins were tested on rats to measure their immunogenic responses, and the study recently been completed successfully as a potential recombinant vaccine against COVID-19.Copyright © 2023 International Society for Cell & Gene Therapy

Characterization of blaKPC-2 and blaNDM-1 Plasmids of a K. pneumoniae ST11 Outbreak Clone.

The most common resistance mechanism to carbapenems is the production of carbapenemases. In 2021, the Pan American Health Organization warned of the emergence and increase in new carbapenemase combinations in Enterobacterales in Latin America. In this study, we characterized four Klebsiella pneumoniae isolates harboring blaKPC and blaNDM from an outbreak during the COVID-19 pandemic in a Brazilian hospital. We assessed their plasmids' transference ability, fitness effects, and relative copy number in different hosts. The K. pneumoniae BHKPC93 and BHKPC104 strains were selected for whole genome sequencing (WGS) based on their pulsed-field gel electrophoresis profile. The WGS revealed that both isolates belong to ST11, and 20 resistance genes were identified in each isolate, including blaKPC-2 and blaNDM-1. The blaKPC gene was present on a ~56 Kbp IncN plasmid and the blaNDM-1 gene on a ~102 Kbp IncC plasmid, along with five other resistance genes. Although the blaNDM plasmid contained genes for conjugational transfer, only the blaKPC plasmid conjugated to E. coli J53, without apparent fitness effects. The minimum inhibitory concentrations (MICs) of meropenem/imipenem against BHKPC93 and BHKPC104 were 128/64 and 256/128 mg/L, respectively. Although the meropenem and imipenem MICs against E. coli J53 transconjugants carrying the blaKPC gene were 2 mg/L, this was a substantial increment in the MIC relative to the original J53 strain. The blaKPC plasmid copy number was higher in K. pneumoniae BHKPC93 and BHKPC104 than in E. coli and higher than that of the blaNDM plasmids. In conclusion, two ST11 K. pneumoniae isolates that were part of a hospital outbreak co-harbored blaKPC-2 and blaNDM-1. The blaKPC-harboring IncN plasmid has been circulating in this hospital since at least 2015, and its high copy number might have contributed to the conjugative transfer of this particular plasmid to an E. coli host. The observation that the blaKPC-containing plasmid had a lower copy number in this E. coli strain may explain why this plasmid did not confer phenotypic resistance against meropenem and imipenem.

Dnazymes as a Method for Nipah Henipavirus Detection

Intro: Deoxyribozymes (Dz) are short synthetic DNA oligonucleotides that catalyze the cleavage of a phosphodiester bond between nucleotides in the presence of divalent metal ions. The use of DNAzymes in the in vitro diagnostics increases the specificity and versatility of the analysis. Method(s): We took the well-studied Dz 10-23 with high catalytic activity as the basis of our system. The biosensor is divided into two fragments according to the binary probe principle (Dz1 and Dz2), which consist of target RNA binding sites, a fluorescent substrate (Fsub), and half of the Dz 10-23 catalytic center sequence. Assembly of the Dz 10-23 active center with subsequent Fsub cleavage and registration of a fluorescent signal is possible only if the target RNA is present in the sample. Finding(s): To assess the diagnostic potential of the biosensor, we measured FAM fluorescence in a solution containing synthetic RNA 35 nucleotides long (nip35) corresponding to the NiV target sequence, Fsub labeled with the FAM-BHQ1 and Dz_NiV pair. A mixture of Dz_NiV and Fsub was used as a control. The detection limit of the target RNA reached 5 nM, the signal development time was 30 minutes at a temperature of 37 C . Discussion(s): The specificity of Dz_NiV was evaluated in the presence of synthetic RNAs from six other RNA viruses of similar length: Hendra, Machupo, Sabia, Junin, Guanarito, and SARS-CoV. A fluorescent signal was recorded only in the presence of nip35 in the reaction mixture. The efficiency of Dz_NiV on a long fragment was tested using a plasmid with a cloned target sequence. The site is about 700 b.p. was amplified by PCR, followed by transcription. Conclusion(s): It was developed the highly specific biosensor Dz_NiV for the detection of Nipah virus RNA with a sensitivity limit of 5 nM at 37 C .Copyright © 2023

Cloning, expression and identification of truncated spike gene of IBV (Sf200) and chicken granulocyte- macrophage colony stimulating factor (GM-CSF)

The aim of this study was to clone, express and identify the truncated S1 gene of nephrotropic infectious bronchitis virus (IBV) and granulocyte-monocyte colony stimulating factor (GM-CSF) of chicken. Firstly, two genes were amplified by polymerase chain reaction (PCR) and cloned into pMD18-T vector. The truncated S1 gene designated as Sf200 containing five antigenic sites of S1 glycoprotein on amino acid residues (aa) 24-61, (aa) 291-398 and (aa) 497-543 and GM-CSF were then amplified from the respective recombinant pMD18-T plasmids and cloned into pET-32a (+) vector resulting pET-Sf200, pET-GM which were identified by restriction enzyme digestion and sequencing analysis. The in vitro expression of truncated Sf200 and GM-CSF constructs were later expressed in E. coli BL21 with a molecular mass of approximately 38 kDa and 29 kDa respectively as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Polyclonal antibodies were developed by injecting E. coli expressed Sf200 and GM-CSF into the SPF mice and were used to identify the recombinant proteins by Western blot analysis. These findings indicated that the polyclonal antibodies produced in mice could be used to detect the recombinant truncated Sf200 and GM-CSF and vice versa.

Antibiotic resistance of Klebsiella pneumoniae against the background of the COVID-19 pandemic: experience of the multidisciplinary hospital

Frequency of bacterial co-infections among patients with COVID-19 is not high, and over-prescribing of antibiotics may contribute the selection of resistant strains of enterobacteria and gram-negative non-fermenting bacteria. The aim of the study was to assess the local features of antibiotic resistance of K. pneumoniae and its genetic mechanisms against background of the COVID-19 infection pandemic. Material and methods. There was selected 37 carbapenem-resistant K. pneumoniae strains isolated in 2016, 2017 and 2020 from hospitalized patients, including 15 strains, isolated from patients with COVID-19 infection. Minimal inhibitory concentrations (MICs) of meropenem and colistin were determined by broth microdilution method. Determination of MICs of eravacycline, ceftazidime/avibactam, meropenem/vaborbactam, imipenem/relebactam was performed using Sensititre diagnostic system on EUMDROXF plates. Susceptibility to 11 combinations of 2 antibiotics was detected by modified method of multiply combination bactericidal testing. For 4 K. pneumoniae strains high-throughput sequencing was performed, followed with the subsequent search for determinants of antibiotic resistance and virulence, assessment of plasmid profiles. Results. All strains were resistant to meropenem (MIC50 32 mg/l, MIC90 128 mg/l) and produced KPC and OXA-48 carbapenemases. Strains isolated in 2016-2017 were susceptible to colistin (MIC <=2 mg/l), in 2020 only 26.7% of the strains retained their susceptibility (MIC50 64 mg/l, MIC90 256 mg/l). Susceptibility to combinations of two antibiotics with colistin included reduced from 84.6-100% in 2016-2017 till 26.6-66.7% in 2020. The strains isolated in 2020 retained their susceptibility to ceftazidime/avibactam (MIC <=1 mg/l). 5 strains resistant to cefiderocol with a MIC 8 mg/l were identified. Strains 2564 and 3125 isolated in 2020 from sputum of patients with COVID-19 infection belonged to different sequence-types (ST12 and ST23) and contained the blaOXA-48 carbapenemase gene, additionally strain 2564 contained the blaKPC-27carbapenemase gene. Resistance to colistin was caused by inactivation of the mgrB genes due to insertion of IS1 and IS5-like transposons. Conclusion. The performed genetic studies demonstrate a diversity of mechanisms of antibiotic resistance in K. pneumoniae leading to the formation of resistance including to antibiotics that haven't been used in Belarus till now.Copyright © 2021 Geotar Media Publishing Group. All Rights Reserved.

Production of Sars-Cov-2 Membrane Protein as Better Target Protein for Diagnostic Application

Intro: The COVID-19 pandemic is caused by the SARS-CoV-2 virus, an enveloped RNA of the coronavirus family. The advancement in molecular technology and biochemistry has accelerated the development of diagnostic reagents and assays. Much attention has been focused on the S protein, but the high mutation rate in this region could lead to false negative results. Thus, a better target protein for diagnostic application is needed for accurate detection. Method(s): Nucleotide sequences encoded for membrane (M) glycoprotein gene region of SARS-CoV-2 from Malaysian isolates were extracted from GISAID, aligned, and selected accordingly. The DNA plasmid was commercially synthesized with codon optimization for Escherichia coli (E. coli), and the presence of the M gene was confirmed by PCR. The plasmid was then transformed into E. coli. Later, the expression of M glycoprotein was induced, separated on an SDS-PAGE gel, and transferred onto a nitrocellulose membrane, followed by immunostaining. Finding(s): The analysis of the M glycoprotein against the Omicron strains demonstrated that the amino acid is conserved (99.5%). The M glycoprotein was successfully expressed and detected with antibodies from SARS-CoV-2 infected patients at ~26 kDa. The protein is currently upscale for the generation of monoclonal Ab (Mab). Discussion(s): The M protein of SARS-CoV-2 is more conserved among the virus and also has been reported to confer antigenic properties. Selection of M protein perhaps a better option compared to current detection assays that use spike (S) protein, which could lead to false negative results, as this gene region particularly the ribosome-binding domain (RBD) rapidly undergoes mutations. The utilization of M protein potentially improves negative predictive value (NPV) of the diagnostic test. Conclusion(s): Further development of diagnostic reagents is needed to improve the assay's specificity. The newly developed M protein and the MAb can be used to generate a more accurate viral detection assay.Copyright © 2023

The Role of SARS-CoV Non-structural protein 1 in Regulating RNA stability

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected more than 600 million people across 219 countries during the past three years. SARS-CoV-2 consists of a positive-strand RNA genome that encodes structural and nonstructural proteins and shares a 79% sequence homology with severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1). Nonstructural proteins are necessary for viral replication and suppression of the host cell immune response. Nonstructural protein 1 (nsp1), a small protein conserved among most beta-coronaviruses, inhibits host messenger RNA (mRNA) translation by binding to ribosomal mRNA channels. Nsp1 also triggers degradation of host mRNA while viral RNA remains intact. We have previously shown that nsp1 localizes within stress granules (SGs), non-membranous vesicles of stalled mRNA that form in response to viral infection. We also found that upon induction of stress, SGs disperses within 60- 120 minutes in the presence of nsp1. Since SGs are known to store and protect translationally stalled mRNAs that are target of nsp1, we sought to analyze the level of mRNAs accumulation in SGs in the presence of nsp1. The goal of this project is to identify the impact of nsp1 on stress granule formation during SARS-CoV infection. We used human embryonic kidney cells (HEK293) and transfected them with DNA expressing SARSCoV- 1 nsp1 or a control plasmid. Cells were then incubated at 37degreeC under 5% CO2 concentration for 16 hours. Following incubation, cells were subjected to 30 min of oxidative stress using sodium arenite. Cells were collected and lysed using lysis buffer, then centrifuge at 18 000xg to collect SG pellets used for RNA isolation. Isolated mRNAs were quantified using quantitative RT-PCR. We specifically targeted mRNAs that tend to show a preferential accumulation in SGs without any viral infection. When nsp1 was expressed, we found majority of mRNAs have shown a 2-fold decrease in accumulation in SGs. These results suggest there is a direct effect of nsp1 in dispersing of RNA from SGs. We are currently investigating the effect of viral leader sequence in their accumulation in SGs in the presence of nsp1. This project was supported by the DRP award from SC INBRE (NIGMS, P20GM103499).Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Development of PCR diagnostic method for Trichomonas foetus infection based on 18S rRNA.

To establish a PCR detection method for Trichomonas foetus, the primers were designed and synthesized according to the 18S rRNA gene sequence of T. foetus published by GenBank. The positive recombinant plasmid pUCm-T-TF18S of T. foetus was used as the template, and the genomic DNA of Giardia felis, Coccidia +e-lis, feline parvovirus and cDNA of feline coronavirus were used as the control for PCR detection to analyze the specificity of this method. The positive T. foetus recombinant plasmid was serial to 8 different concentrations with a gap of 10 folds, and PCR was performed to analyze the sensitivity of this method. The pUCm-T-TF18S plasmids stored at -20 " for 3, 6, 9 and 12 months were detected by PCR to analyze the stability of the method. Twenty cat fecal samples were tested using this established PCR assay and compared with those of microscopic examination. The results showed that the recombinant plasmid pUCm-T- TF18S gave specific bands after PCR amplification. The sequencing results showed that the length of the product sequence was 1 264 bp, and the BLAST sequence comparison analysis showed 99.53% sequence identity, which is consistent with that of T. foetus from cats (GenBank registration number M81842.1). The PCR method for detection of T. foetus had no cross-reactivities with C. felis, G. felis, feline coronavirus and feline parvovirus;the minimum detectable template concentration is 4.52 X 105 copies/xl;The target band of T. foetus DNA can still be detected after being stored in the refrigerator at -20 " for 12 months. This method detected 16 positive samples of T. foetus nucleic acid from 20 cat fecal samples, which is more accurate and sensitive than the results from traditional microscopy (13 samples). It is suggested that the PCR method for the detection of T. foetus is highly specific, sensitive and stable, and can be used for clinical detection and epidemiological investigation of T. foetus.Copyright © 2022, National Institute of Parasitic Diseases. All rights reserved.

Optimized nanoparticle-mediated base editing of cystic fibrosis-causing variants

Background: Next-generation SARS-CoV-2 vaccines demonstrated that nanoparticle messenger ribonucleic acid (mRNA) delivery is effective and safe for in vivo delivery in humans. Current treatments for cystic fibrosis (CF) primarily focus on modulator drug therapies designed to correct malfunctioning CF transmembrane conductance regulator (CFTR) protein, but these modulators are ineffective for the 10% of people with CF with variants that do not allow protein production. Among these is the splice variant 3120 + 1G >A, the most common CF-causing mutation in native Africans. Gene editing would allow production of CFTR protein and enhancement of function using available CFTR modulators. We have demonstrated that electroporation of a modified CRISPR-Cas9 base editor to primary human bronchial epithelial cells carrying 3120 + 1G >A and F508del mutant alleles achieved 75% genome editing of the splice variant, resulting in approximately 40% wild-type (WT) CFTR function [1]. Here,we evaluate the effectiveness of several new nanoparticle formulations at delivering green fluorescent protein (GFP) mRNA to CF bronchial epithelial (CFBE41o-) cells. Using the optimal formulation,we then tested the efficacy correction of the 3120 + 1G >Avariant in a CFTR expression minigene (EMG) integrated into the genome of isogenic CFBE cells using mRNA and plasmid deoxyribonucleic acid (DNA) encoding adenine base editor (ABE) and guide (g)RNA. Method(s): GFP served as a reporter to evaluate transfection efficiency, cell viability, and mean fluorescence intensity (MFI) for three dosages (150, 75, 32.5 ng of mRNA), four polymer-to-mRNA to weight (w/w) ratios (60, 40, 30, 20), and four polymers (R, Y, G, B). 7-AAD served as a live/dead stain to quantify viability, with flow cytometry results analyzed using FlowJo software. CFBE cells stably expressing the 3120 + 1G >A EMG were transfected with the optimized nanoparticle formulation to deliver ABE and gRNA at two dosages (150, 75 ng) of mRNA and DNA. CFTR function in CFBE cellswas measured by short circuit current, forskolin stimulation, and inh-172 inhibition as a measure of editing efficiency. Result(s): Flow cytometry showed that polymer R achieved more than 85% GFP transfection, compared with a maximum of approximately 35% for the other three polymers at the maximum 150-ng dose, with approximately 80% viability normalized to untreated cells. In addition, polymer R achieved GFP MFI more than one order of magnitude as high as other formulations (~30 000 vs 2700 MFI) for the other three polymers at 150-ng dose and 40 w/w ratio. CFBE cells transfected with polymer R nanoparticles containing ABE and guide RNA at 75 ng and 150 ng showed mean CFTR function increase to 10 muA 6 (standard error of the mean [SEM] 1.1 muA) (~10% of WT) and 6.3 muA (SEM 0.9 muA) (~6% of WT), respectively. Greater toxicity at the higher dose could explain the larger increase in CFTR current at the lower dose. DNA-encoded ABE plasmid and gRNA showed a less robust increase in CFTR function (2.9 muA [SEM 0.4 muA] for 75-ng dose;3.0 muA [SEM 0.4 muA] for 150-ng dose), which was probably a result of the nanoparticle formulation being optimized for RNA instead of DNA cargo or the additional intracellular barriers that must be overcome for successful DNA delivery. Conclusion(s): We demonstrated that an optimized nanoparticle formulation containing ABE and gRNA can correct splicing of isogenic cells bearing the 3120 + 1G >A CFTR variant, resulting in recovery of CFTR function. In ongoing work, we are adapting these nanoparticles for RNA- and DNAencoded ABE and gRNA delivery to primary human bronchial epithelial cells.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved

SARS-CoV-2 REPLICON SYSTEM FOR THE PHENOTYPIC EVALUATION OF NSP GENE SUBSTITUTIONS

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the current global pandemic of the COVID-19, which has persisted partly through the emergence of new variants. A non-infectious, convenient, and reproducible in vitro system is needed to assess drug susceptibility of new variants of concern and potential drug resistance mutations. Method(s): The SARS-CoV-2 replicon protocol was adapted and optimized based on {Zhang 2021}. The replicon RNA was produced by in vitro transcription of full-length replicon DNA assembled by ligation of plasmid fragments encoding for the SARS-CoV-2 non-structural proteins (Nsps), nucleoprotein and gaussia luciferase reporter protein. Wild-type and mutant replicon RNAs were transfected into Huh7-1CN cells by electroporation and treated with remdesivir (RDV). To determine EC50 values, luciferase activity was determined at 48 hours post transfection. A recombinant SARS-CoV-2 virus rescue system {Xie 2020} was used to generate matching Nsp mutants for comparison with the replicon system. Result(s): The selected substitutions reflective of Omicron BA.5 sub-lineage BF.7 variant: the triple mutants (Nsp12 (P323L) +Nsp13 (R392C) + Nsp14 (I42V), and a single Nsp12 L247F mutant as well as several specific Nsp12 mutations identified by in vitro resistance selection with RDV or RDV parent nucleoside analog GS-441524 were cloned into the replicon and tested for susceptibility to RDV. RDV inhibited the SARS-CoV-2 wild-type replicon with a mean EC50 value of 14.7 +/- 3.5 nM (N=9). The Nsp12 P323L substitution, a common polymorphism in all major variants of concern including Omicron, was fully susceptible to RDV with a 0.6-fold change in EC50 from the wild-type. The Omicron BF.7 triple mutants and L247F were also fully susceptible to RDV with 0.5- and 0.4-fold changes, respectively. Nsp12 substitutions F480L, V557L, V792I, S759A+V792I, and C799F resulting from in vitro resistance selections with RDV showed minimal to moderate levels of reduced susceptibility to RDV (1.8 to 18.3-fold change) (Table 1). The RDV EC50 fold changes correlated between the noninfectious replicon and recombinant infection virus system (Table 1). Conclusion(s): The replicon system is a convenient and reproducible model to test the susceptibility of SARS-CoV-2 mutant variants to RDV and potentially other antivirals. The common Nsp12 polymorphisms in all variants including the highly transmissible Omicron variant were fully susceptible to RDV.

Extracellular vesicles as delivery vectors to enhance killing of bacteria and viruses by airway-recruited human neutrophils

Background: Polymorphonuclear neutrophils (PMNs) recruited to the airway lumen in cystic fibrosis (CF) undergo a rapid transcriptional program, resulting in exocytosis of granules and inhibition of bacterial killing. As a result, chronic infection, feed-forward inflammation, and structural tissue damage occur. Because CF airway PMNs are also highly pinocytic, we hypothesized that we could deliver protein- and ribonucleic acid (RNA)-based therapies to modulate their function to benefit patients. We elected to use extracellular vesicles (EVs) as a delivery vector because they are highly customizable, and airway PMNs have previously been shown by our group to process and use their cargo efficiently [1]. Furthermore, our prior work on CF airway PMNs [2] led to identification of the long noncoding RNA MALAT1, the transcription factor Ehf, and the histone deacetylase/long-chain fatty deacylase HDAC11 as potential targets to modulate CF airway PMN dysfunction. Method(s): H441 human club epithelial cells were chosen for EV production because they efficiently communicate with lung-recruited primary human PMNs [1]. Relevant constructs were cloned into an expression plasmid downstream of a constitutive cytomegalovirus or U6 promoter with an additional puromycin selection cassette. EVs were generated in serumdepleted media and purified by differential centrifugation. Quality and concentration of EVs was determined by electron microscopy and nanoparticle tracking analysis and cargo content by western blot (protein) or qualitative reverse transcription polymerase chain reaction (RNA). Enhanced green fluorescent protein and messenger ribonucleic acid (mRNA) were used as controls. To test delivery to primary human PMNs, generated EVs were applied in the apical fluid of an airway transmigration model [2]. PMN activation was assessed by flow cytometry, and bacterial (PA01 and Staphylococcus aureus 8325-4) killing and viral (influenza Avirus [IAV] H1N1/PR/8/34;SARS-CoV-2/Washington) clearance assays were conducted. Result(s): To package protein, we used EV-loading motifs such as the tetraspanin CD63, Basp1 amino acids 1-9, and the palmitoylation signal of Lyn kinase. To load mRNA, a C'D box motif recognized by the RNA-binding protein L7Ae was included in the 3' untranslated region of the expressed RNA, and CD63-L7Ae was co-expressed. Airway-recruited PMNs treated with EVs containing small interfering RNAs against MALAT1 or HDAC11 showed greater ability to clear bacteria. Conversely, PMNs treated with constructs encasing MALAT1 or HDAC11 efficiently cleared IAV and SARSCoV- 2. PMNs expressing Ehf showed greater clearance of bacteria and viruses. Conclusion(s): Our findings suggest mutually exclusive roles of MALAT-1 and HDAC11 in regulating bacterial and viral clearance by airway-recruited PMNs. Expression of Ehf in airway PMNs may be a pathogen-agnostic approach to enhancing clearance by airway-recruited PMNs. Overall, our study brings proof-of-concept data for therapeutic RNA/protein transfer to airway-recruited PMNs in CF and other lung diseases and for use of EVs as a promising method for cargo delivery to these cells. It is our expectation that, by treating the immune compartment of CF airway disease, pathogentherapies, such as antibiotics will be more effective, and epithelial-targeted therapies, such as CFTR modulators, will have greater penetrance into the cell types of interest.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved

Low concentration quaternary ammonium compounds promoted antibiotic resistance gene transfer via plasmid conjugation.

During the pandemic of COVID-19, the amounts of quaternary ammonium compounds (QACs) used to inactivate the virus in public facilities, hospitals and households increased, which raised concerns about the evolution and transmission of antimicrobial resistance (AMR). Although QACs may play an important role in the propagation of antibiotic resistance gene (ARGs), the potential contribution and mechanism remains unclear. Here, the results showed that benzyl dodecyl dimethyl ammonium chloride (DDBAC) and didecyl dimethyl ammonium chloride (DDAC) significantly promoted plasmid RP4-mediated ARGs transfer within and across genera at environmental relevant concentrations (0.0004-0.4 mg/L). Low concentrations of QACs did not contribute to the permeability of the cell plasma membrane, but significantly increased the permeability of the cell outer membrane due to the decrease in content of lipopolysaccharides. QACs altered the composition and content of extracellular polymeric substances (EPS) and were positively correlated with the conjugation frequency. Furthermore, transcriptional expression levels of genes encode for mating pairing formation (trbB), DNA replication and translocation (trfA), and global regulators (korA, korB, trbA) are regulated by QACs. And we demonstrate for the first time that QACs decreased the concentration of extracellular AI-2 signals, which was verified to be involved in regulating conjugative transfer genes (trbB, trfA). Collectively, our findings underscore the risk of increased disinfectant concentrations of QACs on the ARGs transfer and provide new mechanisms of plasmid conjugation.

Molecular detection of plasmid-mediated quinolone resistance genes among clinical isolates of Klebsiella pneumoniae during Covid-19 pandemic

Plasmid-mediated quinolone resistance (PMQR) genes confer low resistance to Fluoroquinolones (FQs). This study aims to detect five PMQR genes among FQs-resistant Klebsiella pneumoniae isolated from various clinical specimens. Out of 120 K. pneumoniae isolates, 68 FQs-resistance K. pneumoniae were included in a molecular study. Standard microbiological tests were used for identification and antimicrobial susceptibility. For the detection of PMQR genes, conventional polymerase chain reaction was used. A molecular study revealed that (73.5%) of samples harbored PMQR genes, and among them, 58% were co-carriages of PMQR gene variants. Aac (6')-Ib-cr gene was predominant (47.1%) among samples, and qepA had the lowest percentage (11.8%), qnr genes were (32.4%) (29.4%) (20.6%) qnrS, qnrB, and qnrA respectively. Overall, high percentages of PMQR genes were detected, and almost all of samples were phenotypically resistant to ciprofloxacin. As well, there was a significant statistical relationship between phenotypically ESBL-producers and qnrB and qepA genes.Copyright © Abdulkareem MM et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Novel pro-and eukaryotic expression plasmid expressing omicron antigens delivered via Salmonella elicited MHC class I and II based protective immunity.

The latest omicron variants are emerging with mutations in the receptor binding domain (RBD) that confer immune evasion and resistance against current vaccines. Such variants have raised the peril of future vaccine effectiveness, as leading vaccines target the spike protein. Type-IV hypersensitivity, and other ailments due to the dominant Th1 response by leading vaccines, is also to be resolved. Therefore, vaccine that target diverse SARS-CoV-2 proteins and provide broad-spectrum protection and a balanced Th1 and Th2 response is an indispensable armament against the pandemic. In that prospect, a novel dual expression plasmid pJHL270 was developed and demonstrated the expression of omicron antigens exogenously from Salmonella and endogenously in the host cells. The simultaneous activation of MHC class I and II molecules culminated in a balanced Th1 and Th2 response, which was evident through the upsurge of IgG, IgA antibodies, IgG2a/IgG1 ratio, cytokine responses and CD4+, CD8+ T-lymphocytes. The level of CD44+ cells showed the trigger for Th1 and Th2 balance and memory-cell activation for long-lasting immunity. The level of IFN-γ + cells and neutralizing antibodies signifies the anti-viral response. The vaccine protected the hamsters from BA.5 and BA.2.75 omicron viral-challenge, exhibited a significant reduction in lung viral-load and histopathological lesions. In addition to two-way antigen expression and bilateral immune elicitation, this Salmonella-based vaccine delivery system can be prospectively applied to humans and a broad range of animals as a convenient alternative to viral and chemical vaccine delivery approaches.

Effects of Various Mrna-Lnp Vaccine Doses on Neuroinflammation in Balb/C Mice

It has been proven that mRNA vaccines are highly effective against the COVID-19 outbreak, and low prevalence of side effects has been shown. However, there are still many gaps in our understanding of the biology and biosafety of nucleic acids as components of lipid nanoparticles (LNPs) most often used as a system for inctracellular delivery of mRNA-based vaccines. It is known that LNPs cause severe injection site inflammation, have broad biodistribution profiles, and are found in multiple tissues of the body, including the brain, after administration. The role of new medications with such pharmacokinetics in inflammation developing in inaccessible organs is poorly understood. The study was aimed to assess the effects of various doses of mRNA-LNP expressing the reporter protein (0, 5, 10, and 20 microg of mRNA encoding the firefly luciferase) on the expression of neuroinflammation markers (Tnfalpha, Il1beta, Gfap, Aif1) in the prefrontal cortex and hypothalamus of laboratory animals 4, 8, and 30 h after the intramuscular injection of LNP nanoemulsion. It was shown that mRNA-LNP vaccines in a dose of 10-20 microg of mRNA could enhance Aif1 expression in the hypothalamus 8 h after vaccination, however, no such differences were observed after 30 h. It was found that the Gfap, l11beta, Tnfalpha expression levels in the hypothalamus observed at different times in the experimental groups were different. According to the results, mRNA-LNPs administered by the parenteral route can stimulate temporary activation of microglia in certain time intervals in the dose-dependent and site specific manner.Copyright © 2022 Pirogov Russian National Research Medical University. All rights reserved.

Genome editing technology and applications with the type I CRISPR system

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, which are representative genome editing technologies, are classified into class 1 and class 2 in terms of evolutionary biology and are further classified into several subtypes. Class 2 CRISPR systems, including type II Cas9 and type V Cas12a, are the most commonly used for genome editing in eukaryotic cells, while type I CRISPR systems within Class 1 are also becoming available. Type I CRISPR recognizes longer target sequences than CRISPR-Cas9 and can induce large deletion mutations of several kilobases. These features demonstrate its potential as a novel and unique genome editing tool that can induce genetic disruption safely and reliably. Thus, it is expected to be utilized for gene therapy and industrial applications. Recently, the DNA cleavage mechanism of type I CRISPR has also revealed details from protein-complex analyses with X-ray crystallography, cryo-electron microscopy, and high-speed atomic force microscopy. The single-strand DNA trans-cleavage activity of type I CRISPR, called collateral activity, has broadened the potential application for CRISPR diagnostics, especially in the development of point-of-care testing methods for COVID-19. In this review, we present an overview of the type I CRISPR system, its application to genome editing, and genetic diagnosis using CRISPR-Cas3.Copyright © 2022