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.

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

Robust, efficient workflow to establish, culture, and functionally assess primary-isolated airway epithelial cells

Background: Air-liquid interface (ALI) and organoid culture are key techniques for differentiating human airway epithelial cells (HAECs). The efficiency and robustness of these assays often depends on the quality of primary-isolated cells, but primary cell isolation workflows, with which the user controls the choice of isolation method, cell culture medium, and culture format, may reduce reproducibility. Therefore, an optimized, standardized workflow can enhance and support isolation of epithelial cells from diseased donors with potentially rare cystic fibrosis (CF) mutations or particularly sensitive cell populations. We have developed a standardized workflow for isolation and culture of freshly derived airway epithelial cells. Method(s): Briefly, HAECs isolated from primary tissue were expanded in PneumaCult-Ex Plus Medium for 1 week and then seeded into Corning Transwell inserts and expanded until confluency. The cells were then differentiated in PneumaCult-ALI Medium for at least 4 weeks. To assess differentiation efficiency in ALI culture, the cells were immunostained to detect Muc5AC, acetylated tubulin, and ZO-1 to identify goblet cells, ciliated cells, and apical tight junctions, respectively, aswell as SARS-CoV-2 cell entry targets angiotensin-converting enzyme 2 and transmembrane serine protease 2. Ion transport and barrier function of the ALI culturesand response to CF transmembrane conductance regulator (CFTR) correctors were also measured. In addition, freshly derived HAECs were seeded into Corning Matrigel domes in the presence of PneumaCult Airway Organoid Seeding Medium. Oneweek later, the mediumwas changed to PneumaCult Airway Organoid Differentiation Medium and maintained for an additional 3 weeks to promote cell differentiation. These airway organoids were then treated with CFTR corrector VX-809 for 24 hours, followed by 6-hour treatment with amiloride, forskolin, and genistein to induce organoid swelling. Result(s): Our results demonstrate that ALI cultures derived from CF donors displayed partial rescue of CFTR across multiple passages after treatment with VX-809. Airway organoids were found to express functional CFTR, as evidenced by forskolin treatment, which induced a 64 +/- 14% (n = 1 donor) greater organoid area than in vehicle control-treated airway organoids. Airway organoids derived from CF donors displayed a loss of forskolininduced swelling, which could be partially re-established with VX-809 treatment (29 +/- 9%, n = 3). Conclusion(s): In summary, the PneumaCult workflow supports robust, efficient culture of primary-airway epithelial cells that can be used as physiologically relevant models suitable for CF research, CFTR corrector screening, and studying airway biology.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved