A flexible, efficient, and scalable platform to produce circular RNAs as new therapeutics (preprint)

Messenger RNA (mRNA) has recently emerged as a new drug modality with great therapeutic potential. However, linear mRNAs are relatively unstable and also require base modification to reduce their immunogenicity, imposing a limitation to the broad application. With improved stability, the circular RNA (circRNA) presents a better alternative for prolonged expression of the proteins, however the in vitro circularization of RNA at a large scale is technically challenging. Here we developed a new self-catalyzed system to efficiently produce circRNAs in a co-transcriptional fashion. By rational sequence design, we can efficiently produce scarless circRNAs that do not contain foreign sequences. The resulting circRNAs are very stable and have low immunogenicity, enabling prolonged protein translation in different cells without cellular toxicity. The circRNAs generated from this platform can be encapsulated in lipid nanoparticles and efficiently delivered into mice to direct robust protein expression. Finally, the circRNAs encoding RBD of SARS-CoV-2 S protein induced strong antibody productions, with neutralization antibody titers higher than the preclinical data from the linear mRNAs. Collectively, this study provided a general platform for efficient production of circRNAs, demonstrating the potential of circRNAs as the new generation of mRNA therapy.

Differential transcriptomic landscapes of multiple organs from SARS-CoV-2 early infected rhesus macaques.

SARS-CoV-2 infection causes complicated clinical manifestations with variable multi-organ injuries, however, the underlying mechanism, in particular immune responses in different organs, remains elusive. In this study, comprehensive transcriptomic alterations of 14 tissues from rhesus macaque infected with SARS-CoV-2 were analyzed. Compared to normal controls, SARS-CoV-2 infection resulted in dysregulation of genes involving diverse functions in various examined tissues/organs, with drastic transcriptomic changes in cerebral cortex and right ventricle. Intriguingly, cerebral cortex exhibited a hyperinflammatory state evidenced by significant upregulation of inflammation response-related genes. Meanwhile, expressions of coagulation, angiogenesis and fibrosis factors were also up-regulated in cerebral cortex. Based on our findings, neuropilin 1 (NRP1), a receptor of SARS-CoV-2, was significantly elevated in cerebral cortex post infection, accompanied by active immune response releasing inflammatory factors and signal transmission among tissues, which enhanced infection of the central nervous system (CNS) in a positive feedback way, leading to viral encephalitis. Overall, our study depicts a multi-tissue/organ transcriptomic landscapes of rhesus macaque with early infection of SARS-CoV-2, and provides important insights into the mechanistic basis for COVID-19-associated clinical complications.

SARS-CoV-2 treatment effects induced by ACE2-expressing microparticles are explained by the oxidized cholesterol-increased endosomal pH of alveolar macrophages.

Exploring the cross-talk between the immune system and advanced biomaterials to treat SARS-CoV-2 infection is a promising strategy. Here, we show that ACE2-overexpressing A549 cell-derived microparticles (AO-MPs) are a potential therapeutic agent against SARS-CoV-2 infection. Intranasally administered AO-MPs dexterously navigate the anatomical and biological features of the lungs to enter the alveoli and are taken up by alveolar macrophages (AMs). Then, AO-MPs increase the endosomal pH but decrease the lysosomal pH in AMs, thus escorting bound SARS-CoV-2 from phago-endosomes to lysosomes for degradation. This pH regulation is attributable to oxidized cholesterol, which is enriched in AO-MPs and translocated to endosomal membranes, thus interfering with proton pumps and impairing endosomal acidification. In addition to promoting viral degradation, AO-MPs also inhibit the proinflammatory phenotype of AMs, leading to increased treatment efficacy in a SARS-CoV-2-infected mouse model without side effects. These findings highlight the potential use of AO-MPs to treat SARS-CoV-2-infected patients and showcase the feasibility of MP therapies for combatting emerging respiratory viruses in the future.

SARS-CoV-2 crosses the blood-brain barrier accompanied with basement membrane disruption without tight junctions alteration.

SARS-CoV-2 has been reported to show a capacity for invading the brains of humans and model animals. However, it remains unclear whether and how SARS-CoV-2 crosses the blood-brain barrier (BBB). Herein, SARS-CoV-2 RNA was occasionally detected in the vascular wall and perivascular space, as well as in brain microvascular endothelial cells (BMECs) in the infected K18-hACE2 transgenic mice. Moreover, the permeability of the infected vessel was increased. Furthermore, disintegrity of BBB was discovered in the infected hamsters by administration of Evans blue. Interestingly, the expression of claudin5, ZO-1, occludin and the ultrastructure of tight junctions (TJs) showed unchanged, whereas, the basement membrane was disrupted in the infected animals. Using an in vitro BBB model that comprises primary BMECs with astrocytes, SARS-CoV-2 was found to infect and cross through the BMECs. Consistent with in vivo experiments, the expression of MMP9 was increased and collagen IV was decreased while the markers for TJs were not altered in the SARS-CoV-2-infected BMECs. Besides, inflammatory responses including vasculitis, glial activation, and upregulated inflammatory factors occurred after SARS-CoV-2 infection. Overall, our results provide evidence supporting that SARS-CoV-2 can cross the BBB in a transcellular pathway accompanied with basement membrane disrupted without obvious alteration of TJs.

Distinct uptake, amplification, and release of SARS-CoV-2 by M1 and M2 alveolar macrophages.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) invades the alveoli, where abundant alveolar macrophages (AMs) reside. How AMs respond to SARS-CoV-2 invasion remains elusive. Here, we show that classically activated M1 AMs facilitate viral spread; however, alternatively activated M2 AMs limit the spread. M1 AMs utilize cellular softness to efficiently take up SARS-CoV-2. Subsequently, the invaded viruses take over the endo-lysosomal system to escape. M1 AMs have a lower endosomal pH, favoring membrane fusion and allowing the entry of viral RNA from the endosomes into the cytoplasm, where the virus achieves replication and is packaged to be released. In contrast, M2 AMs have a higher endosomal pH but a lower lysosomal pH, thus delivering the virus to lysosomes for degradation. In hACE2 transgenic mouse model, M1 AMs are found to facilitate SARS-CoV-2 infection of the lungs. These findings provide insights into the complex roles of AMs during SARS-CoV-2 infection, along with potential therapeutic targets.

Corrigendum: Protecting Healthcare Workers Amid the COVID-19 Crisis: A Safety Protocol in Wuhan.

[This corrects the article DOI: 10.3389/fpubh.2020.577499.].

Application of an optimized pharyngeal swab-assisted device in pharyngeal sampling for COVID-19 patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has triggered a global pandemic. Healthcare workers are placed at an elevated risk of nosocomial cross-infection from clinical exposure. One diagnostic criterion for COVID-19 is a positive result from a real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) assay of pharyngeal swab specimens, which has been a routine procedure for healthcare workers during the outbreak. In the context of a global shortage of personal protective equipment (PPE), we aimed to lower the probability of clinical cross-infection without impacting the results of pharynx sampling through an optimized pharyngeal swab assisted device (OPAD). METHODS: To evaluate the efficacy and feasibility of an OPAD for the detection of SARS-CoV-2, 22 confirmed COVID-19 cases were enrolled in our self-controlled study. The results of two pharyngeal sampling qRT-PCR tests using the OPAD or the traditional method were recorded each. Clinical data including baseline characteristics, laboratory tests, and computed tomography (CT) results were also collected. The procedure duration and levels of pharynx exposure with the OPAD, and the diagnostic consistency between the OPAD and the traditional method for pharyngeal sampling qRT-PCR, were evaluated individually. Additionally, a questionnaire was designed for healthcare workers who had performed the pharyngeal swab to deepen our understanding of their attitude during their service on the frontline. RESULTS: In all 44 samplings (22 samples with each method), the qRT-PCR results of 18 pairs (81.82%) were consistent, while 3 (13.64%) were single positive with the OPAD. The positive rate was slightly higher with the OPAD (54.55%, 12/22) than with the traditional method (45.45%, 10/22). Using the OPAD, the average procedure duration of sampling was 30 s (30±13 s). Pharynx exposure was excellent in 21 subjects (95.45%, 21/22), which meant that the operator could acquire the swabs without difficulty. CONCLUSIONS: As the COVID-19 pandemic escalates, our OPAD has identical efficacy compared to the traditional method for pharyngeal swabs, and it can also contribute to protecting the safety of healthcare workers.

Sec-Eliminating the SARS-CoV-2 by AlGaN Based High Power Deep Ultraviolet Light Source.

The world-wide spreading of coronavirus disease (COVID-19) has greatly shaken human society, thus effective and fast-speed methods of non-daily-life-disturbance sterilization have become extremely significant. In this work, by fully benefitting from high-quality AlN template (with threading dislocation density as low as ≈6×108 cm-2) as well as outstanding deep ultraviolet (UVC-less than 280 nm) light-emitting diodes (LEDs) structure design and epitaxy optimization, high power UVC LEDs and ultra-high-power sterilization irradiation source are achieved. Moreover, for the first time, a result in which a fast and complete elimination of SARS-CoV-2 (the virus causes COVID-19) within only 1 s is achieved by the nearly whole industry-chain-covered product. These results advance the promising potential in UVC-LED disinfection particularly in the shadow of COVID-19.

Mucus production stimulated by IFN-AhR signaling triggers hypoxia of COVID-19.

Silent hypoxia has emerged as a unique feature of coronavirus disease 2019 (COVID-19). In this study, we show that mucins are accumulated in the bronchoalveolar lavage fluid (BALF) of COVID-19 patients and are upregulated in the lungs of severe respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected mice and macaques. We find that induction of either interferon (IFN)-ß or IFN-γ upon SARS-CoV-2 infection results in activation of aryl hydrocarbon receptor (AhR) signaling through an IDO-Kyn-dependent pathway, leading to transcriptional upregulation of the expression of mucins, both the secreted and membrane-bound, in alveolar epithelial cells. Consequently, accumulated alveolar mucus affects the blood-gas barrier, thus inducing hypoxia and diminishing lung capacity, which can be reversed by blocking AhR activity. These findings potentially explain the silent hypoxia formation in COVID-19 patients, and suggest a possible intervention strategy by targeting the AhR pathway.

Protecting Healthcare Workers Amid the COVID-19 Crisis: A Safety Protocol in Wuhan.

Coronavirus disease 2019 (COVID-19), which is caused by a distinct coronavirus, is an acute infectious disease that spreads mainly via the respiratory route. During the COVID-19 outbreak, many healthcare systems faced a severe burden when a large number of healthcare workers (HCWs) became infected due to the lack of adequate protection. Consequently, it was apparent that it is important to ensure the health and safety of HCWs in order to control the outbreak throughout society. In this article, we share our successful protocol for protecting the safety of HCWs in the course of their daily work in an orthopedics department with the aim of eventually reducing the risk of nosocomial infection. None of our HCWs or their families contracted the infection during the COVID-19 pandemic.

Analysis of the Risk Factors for Mortality in Adult COVID-19 Patients in Wuhan: A Multicenter Study.

Objectives: An outbreak of coronavirus disease (COVID-19) in 2019 in Wuhan, China, has spread quickly worldwide. However, the risk factors associated with COVID-19-related mortality remain controversial. Methods: A total of 245 adult patients with laboratory-confirmed COVID-19 from two centers were analyzed. Chi-square, Fisher's exact, and the Mann-Whitney U-tests were used to compare the clinical characteristics between the survivors and non-survivors. To explore the risk factors associated with in-hospital death, univariable and multivariable cox regression analyses were used. Results: Of the 245 patients included in this study, 23 (9.4%) died in the hospital. The multivariate regression analysis showed increased odds of in-hospital deaths associated with age, D-dimer levels >1,000 ng/L, platelet count <125, and higher serum creatinine levels. Conclusions: We identified risk factors that show significant association with mortality in adult COVID-19 patients, and our findings provide valuable references for clinicians to identify high-risk patients with COVID-19 at an early stage.

Ischemic Necrosis of Lower Extremity in COVID-19: A Case Report.

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an acute infectious disease that spreads mainly via the respiratory route. Elderly patients or those with underlying diseases are more seriously affected. We report a case of COVID-19 infection in a geriatric patient with arteriovenous thrombosis of the right lower limb. Despite persistent anticoagulant therapy, the patient's arterial thrombosis continued to progress and presented with ischemic necrosis of the lower extremity. After amputation in this case, the levels of D-dimer and inflammatory cytokine increased progressively, and he presented with acute myocardial infarction, which progressed rapidly to multisystem organ failure. However, whether coronavirus can directly cause the damage of the cardiovascular system and thrombosis needs further investigation.

Surgical treatment of thoracolumbar fracture with incomplete lower limb paralysis in a patient with COVID-19.

Since December 2019, COVID-19, an acute infectious disease, has gradually become a global threat. We report a case of thoracolumbar fractures (T12 and L1) and incomplete lower limb paralysis in a patient with COVID-19. After a series of conservative treatment which did not work at all, posterior open reduction and pedicle screw internal fixation of the thoracolumbar fracture were performed in Wuhan Union Hospital. Three weeks later, the patient could stand up and the pneumonia is almost cured. We successfully performed a surgery in a COVID-19 patient, and to our knowledge it is the first operation for a COVID-19 patient ever reported.

Protection of Rhesus Macaque from SARS-Coronavirus challenge by recombinant adenovirus vaccine (preprint)

A recombinant adenovirus vaccine against the SARS Coronavirus (SARS-CoV) was constructed, which contains fragments from the S, N, and Orf8 genes. Rhesus Macaques immunized with the recombinant adenovirus generated antigen-specific humoral and cellular response. Furthermore, the vaccine provided significant protection against subsequent live SARS-CoV challenge. In contrast, three out of four monkeys immunized with placebo suffered severe alveolar damage and pulmonary destruction.