Showing papers by "Howard Hughes Medical Institute published in 2020"

Autoantibodies against type I IFNs in patients with life-threatening COVID-19.

Abstract: Interindividual clinical variability in the course of SARS-CoV-2 infection is immense. We report that at least 101 of 987 patients with life-threatening COVID-19 pneumonia had neutralizing IgG auto-Abs against IFN-ω (13 patients), the 13 types of IFN-α (36), or both (52), at the onset of critical disease; a few also had auto-Abs against the other three type I IFNs. The auto-Abs neutralize the ability of the corresponding type I IFNs to block SARS-CoV-2 infection in vitro. These auto-Abs were not found in 663 individuals with asymptomatic or mild SARS-CoV-2 infection and were present in only 4 of 1,227 healthy individuals. Patients with auto-Abs were aged 25 to 87 years and 95 were men. A B cell auto-immune phenocopy of inborn errors of type I IFN immunity underlies life-threatening COVID-19 pneumonia in at least 2.6% of women and 12.5% of men.

Convergent antibody responses to SARS-CoV-2 in convalescent individuals.

Abstract: During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S) Although there is no vaccine, it is likely that antibodies will be essential for protection However, little is known about the human antibody response to SARS-CoV-21-5 Here we report on 149 COVID-19 convalescent individuals Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titres: less than 1:50 in 33% and below 1:1,000 in 79%, while only 1% showed titres above 1:5,000 Antibody sequencing revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals Despite low plasma titres, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC50 values) as low as single digit nanograms per millitre Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective

Inborn errors of type I IFN immunity in patients with life-threatening COVID-19

Abstract: Clinical outcome upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ranges from silent infection to lethal coronavirus disease 2019 (COVID-19). We have found an enrichment in rare variants predicted to be loss-of-function (LOF) at the 13 human loci known to govern Toll-like receptor 3 (TLR3)- and interferon regulatory factor 7 (IRF7)-dependent type I interferon (IFN) immunity to influenza virus in 659 patients with life-threatening COVID-19 pneumonia relative to 534 subjects with asymptomatic or benign infection. By testing these and other rare variants at these 13 loci, we experimentally defined LOF variants underlying autosomal-recessive or autosomal-dominant deficiencies in 23 patients (3.5%) 17 to 77 years of age. We show that human fibroblasts with mutations affecting this circuit are vulnerable to SARS-CoV-2. Inborn errors of TLR3- and IRF7-dependent type I IFN immunity can underlie life-threatening COVID-19 pneumonia in patients with no prior severe infection.

A framework for advancing our understanding of cancer-associated fibroblasts

Abstract: Cancer-associated fibroblasts (CAFs) are a key component of the tumour microenvironment with diverse functions, including matrix deposition and remodelling, extensive reciprocal signalling interactions with cancer cells and crosstalk with infiltrating leukocytes. As such, they are a potential target for optimizing therapeutic strategies against cancer. However, many challenges are present in ongoing attempts to modulate CAFs for therapeutic benefit. These include limitations in our understanding of the origin of CAFs and heterogeneity in CAF function, with it being desirable to retain some antitumorigenic functions. On the basis of a meeting of experts in the field of CAF biology, we summarize in this Consensus Statement our current knowledge and present a framework for advancing our understanding of this critical cell type within the tumour microenvironment.

Longitudinal analyses reveal immunological misfiring in severe COVID-19.

Abstract: Recent studies have provided insights into the pathogenesis of coronavirus disease 2019 (COVID-19)1-4. However, the longitudinal immunological correlates of disease outcome remain unclear. Here we serially analysed immune responses in 113 patients with moderate or severe COVID-19. Immune profiling revealed an overall increase in innate cell lineages, with a concomitant reduction in T cell number. An early elevation in cytokine levels was associated with worse disease outcomes. Following an early increase in cytokines, patients with moderate COVID-19 displayed a progressive reduction in type 1 (antiviral) and type 3 (antifungal) responses. By contrast, patients with severe COVID-19 maintained these elevated responses throughout the course of the disease. Moreover, severe COVID-19 was accompanied by an increase in multiple type 2 (anti-helminths) effectors, including interleukin-5 (IL-5), IL-13, immunoglobulin E and eosinophils. Unsupervised clustering analysis identified four immune signatures, representing growth factors (A), type-2/3 cytokines (B), mixed type-1/2/3 cytokines (C), and chemokines (D) that correlated with three distinct disease trajectories. The immune profiles of patients who recovered from moderate COVID-19 were enriched in tissue reparative growth factor signature A, whereas the profiles of those with who developed severe disease had elevated levels of all four signatures. Thus, we have identified a maladapted immune response profile associated with severe COVID-19 and poor clinical outcome, as well as early immune signatures that correlate with divergent disease trajectories.

SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies.

Abstract: The coronavirus disease 2019 (COVID-19) pandemic presents an urgent health crisis. Human neutralizing antibodies that target the host ACE2 receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein1–5 show promise therapeutically and are being evaluated clinically6–8. Here, to identify the structural correlates of SARS-CoV-2 neutralization, we solved eight new structures of distinct COVID-19 human neutralizing antibodies5 in complex with the SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed us to classify the antibodies into categories: (1) neutralizing antibodies encoded by the VH3-53 gene segment with short CDRH3 loops that block ACE2 and bind only to ‘up’ RBDs; (2) ACE2-blocking neutralizing antibodies that bind both up and ‘down’ RBDs and can contact adjacent RBDs; (3) neutralizing antibodies that bind outside the ACE2 site and recognize both up and down RBDs; and (4) previously described antibodies that do not block ACE2 and bind only to up RBDs9. Class 2 contained four neutralizing antibodies with epitopes that bridged RBDs, including a VH3-53 antibody that used a long CDRH3 with a hydrophobic tip to bridge between adjacent down RBDs, thereby locking the spike into a closed conformation. Epitope and paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 to escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects and suggesting combinations for clinical use, and provide insight into immune responses against SARS-CoV-2. Eight structures of human neutralizing antibodies that target the SARS-CoV-2 spike receptor-binding domain are reported and classified into four categories, suggesting combinations for clinical use.

Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants.

Abstract: Neutralizing antibodies elicited by prior infection or vaccination are likely to be key for future protection of individuals and populations against SARS-CoV-2. Moreover, passively administered antibodies are among the most promising therapeutic and prophylactic anti-SARS-CoV-2 agents. However, the degree to which SARS-CoV-2 will adapt to evade neutralizing antibodies is unclear. Using a recombinant chimeric VSV/SARS-CoV-2 reporter virus, we show that functional SARS-CoV-2 S protein variants with mutations in the receptor-binding domain (RBD) and N-terminal domain that confer resistance to monoclonal antibodies or convalescent plasma can be readily selected. Notably, SARS-CoV-2 S variants that resist commonly elicited neutralizing antibodies are now present at low frequencies in circulating SARS-CoV-2 populations. Finally, the emergence of antibody-resistant SARS-CoV-2 variants that might limit the therapeutic usefulness of monoclonal antibodies can be mitigated by the use of antibody combinations that target distinct neutralizing epitopes.

Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors

Abstract: The development of new CRISPR-Cas genome editing tools continues to drive major advances in the life sciences. Four classes of CRISPR-Cas-derived genome editing agents-nucleases, base editors, transposases/recombinases and prime editors-are currently available for modifying genomes in experimental systems. Some of these agents have also moved rapidly into the clinic. Each tool comes with its own capabilities and limitations, and major efforts have broadened their editing capabilities, expanded their targeting scope and improved editing specificity. We analyze key considerations when choosing genome editing agents and identify opportunities for future improvements and applications in basic research and therapeutics.

Sex differences in immune responses that underlie COVID-19 disease outcomes.

Abstract: There is increasing evidence that coronavirus disease 2019 (COVID-19) produces more severe symptoms and higher mortality among men than among women1-5. However, whether immune responses against severe acute respiratory syndrome coronavirus (SARS-CoV-2) differ between sexes, and whether such differences correlate with the sex difference in the disease course of COVID-19, is currently unknown. Here we examined sex differences in viral loads, SARS-CoV-2-specific antibody titres, plasma cytokines and blood-cell phenotyping in patients with moderate COVID-19 who had not received immunomodulatory medications. Male patients had higher plasma levels of innate immune cytokines such as IL-8 and IL-18 along with more robust induction of non-classical monocytes. By contrast, female patients had more robust T cell activation than male patients during SARS-CoV-2 infection. Notably, we found that a poor T cell response negatively correlated with patients' age and was associated with worse disease outcome in male patients, but not in female patients. By contrast, higher levels of innate immune cytokines were associated with worse disease progression in female patients, but not in male patients. These findings provide a possible explanation for the observed sex biases in COVID-19, and provide an important basis for the development of a sex-based approach to the treatment and care of male and female patients with COVID-19.

Kawasaki-like multisystem inflammatory syndrome in children during the covid-19 pandemic in Paris, France: prospective observational study.

Abstract: Objectives To describe the characteristics of children and adolescents affected by an outbreak of Kawasaki-like multisystem inflammatory syndrome and to evaluate a potential temporal association with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Design Prospective observational study. Setting General paediatric department of a university hospital in Paris, France. Participants 21 children and adolescents (aged ≤18 years) with features of Kawasaki disease who were admitted to hospital between 27 April and 11 May 2020 and followed up until discharge by 15 May 2020. Main outcome measures The primary outcomes were clinical and biological data, imaging and echocardiographic findings, treatment, and outcomes. Nasopharyngeal swabs were prospectively tested for SARS-CoV-2 using reverse transcription-polymerase chain reaction (RT-PCR) and blood samples were tested for IgG antibodies to the virus. Results 21 children and adolescents (median age 7.9 (range 3.7-16.6) years) were admitted with features of Kawasaki disease over a 15 day period, with 12 (57%) of African ancestry. 12 (57%) presented with Kawasaki disease shock syndrome and 16 (76%) with myocarditis. 17 (81%) required intensive care support. All 21 patients had noticeable gastrointestinal symptoms during the early stage of illness and high levels of inflammatory markers. 19 (90%) had evidence of recent SARS-CoV-2 infection (positive RT-PCR result in 8/21, positive IgG antibody detection in 19/21). All 21 patients received intravenous immunoglobulin and 10 (48%) also received corticosteroids. The clinical outcome was favourable in all patients. Moderate coronary artery dilations were detected in 5 (24%) of the patients during hospital stay. By 15 May 2020, after 8 (5-17) days of hospital stay, all patients were discharged home. Conclusions The ongoing outbreak of Kawasaki-like multisystem inflammatory syndrome among children and adolescents in the Paris area might be related to SARS-CoV-2. In this study an unusually high proportion of the affected children and adolescents had gastrointestinal symptoms, Kawasaki disease shock syndrome, and were of African ancestry.

Cells of the adult human heart.

Abstract: Cardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and therapeutic strategies require a deeper understanding of the molecular processes involved in the healthy heart. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavour. Here, using state-of-the-art analyses of large-scale single-cell and single-nucleus transcriptomes, we characterize six anatomical adult heart regions. Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes and fibroblasts, and reveal distinct atrial and ventricular subsets of cells with diverse developmental origins and specialized properties. We define the complexity of the cardiac vasculature and its changes along the arterio-venous axis. In the immune compartment, we identify cardiac-resident macrophages with inflammatory and protective transcriptional signatures. Furthermore, analyses of cell-to-cell interactions highlight different networks of macrophages, fibroblasts and cardiomyocytes between atria and ventricles that are distinct from those of skeletal muscle. Our human cardiac cell atlas improves our understanding of the human heart and provides a valuable reference for future studies.

Seasonality of Respiratory Viral Infections.

Abstract: The seasonal cycle of respiratory viral diseases has been widely recognized for thousands of years, as annual epidemics of the common cold and influenza disease hit the human population like clockwork in the winter season in temperate regions. Moreover, epidemics caused by viruses such as severe acute respiratory syndrome coronavirus (SARS-CoV) and the newly emerging SARS-CoV-2 occur during the winter months. The mechanisms underlying the seasonal nature of respiratory viral infections have been examined and debated for many years. The two major contributing factors are the changes in environmental parameters and human behavior. Studies have revealed the effect of temperature and humidity on respiratory virus stability and transmission rates. More recent research highlights the importance of the environmental factors, especially temperature and humidity, in modulating host intrinsic, innate, and adaptive immune responses to viral infections in the respiratory tract. Here we review evidence of how outdoor and indoor climates are linked to the seasonality of viral respiratory infections. We further discuss determinants of host response in the seasonality of respiratory viruses by highlighting recent studies in the field. Expected final online publication date for the Annual Review of Virology, Volume 7 is September 29, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays.

Abstract: SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.

Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets.

Abstract: The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse transcription–PCR (RT–qPCR) assays are being used by clinical, research and public health laboratories. However, it is currently unclear whether results from different tests are comparable. Our goal was to make independent evaluations of primer–probe sets used in four common SARS-CoV-2 diagnostic assays. From our comparisons of RT–qPCR analytical efficiency and sensitivity, we show that all primer–probe sets can be used to detect SARS-CoV-2 at 500 viral RNA copies per reaction. The exception for this is the RdRp-SARSr (Charite) confirmatory primer–probe set which has low sensitivity, probably due to a mismatch to circulating SARS-CoV-2 in the reverse primer. We did not find evidence for background amplification with pre-COVID-19 samples or recent SARS-CoV-2 evolution decreasing sensitivity. Our recommendation for SARS-CoV-2 diagnostic testing is to select an assay with high sensitivity and that is regionally used, to ease comparability between outcomes. This is a comparative analysis of the performance of the primer–probe sets from four open-source molecular diagnostic assays for SARS-CoV-2 recommended by the World Health Organization.

SARS-CoV-2 viral load is associated with increased disease severity and mortality.

Abstract: The relationship between SARS-CoV-2 viral load and risk of disease progression remains largely undefined in coronavirus disease 2019 (COVID-19). Here, we quantify SARS-CoV-2 viral load from participants with a diverse range of COVID-19 disease severity, including those requiring hospitalization, outpatients with mild disease, and individuals with resolved infection. We detected SARS-CoV-2 plasma RNA in 27% of hospitalized participants, and 13% of outpatients diagnosed with COVID-19. Amongst the participants hospitalized with COVID-19, we report that a higher prevalence of detectable SARS-CoV-2 plasma viral load is associated with worse respiratory disease severity, lower absolute lymphocyte counts, and increased markers of inflammation, including C-reactive protein and IL-6. SARS-CoV-2 viral loads, especially plasma viremia, are associated with increased risk of mortality. Our data show that SARS-CoV-2 viral loads may aid in the risk stratification of patients with COVID-19, and therefore its role in disease pathogenesis should be further explored.

A connectome and analysis of the adult Drosophila central brain

Abstract: Animal brains of all sizes, from the smallest to the largest, work in broadly similar ways. Studying the brain of any one animal in depth can thus reveal the general principles behind the workings of all brains. The fruit fly Drosophila is a popular choice for such research. With about 100,000 neurons – compared to some 86 billion in humans – the fly brain is small enough to study at the level of individual cells. But it nevertheless supports a range of complex behaviors, including navigation, courtship and learning. Thanks to decades of research, scientists now have a good understanding of which parts of the fruit fly brain support particular behaviors. But exactly how they do this is often unclear. This is because previous studies showing the connections between cells only covered small areas of the brain. This is like trying to understand a novel when all you can see is a few isolated paragraphs. To solve this problem, Scheffer, Xu, Januszewski, Lu, Takemura, Hayworth, Huang, Shinomiya et al. prepared the first complete map of the entire central region of the fruit fly brain. The central brain consists of approximately 25,000 neurons and around 20 million connections. To prepare the map – or connectome – the brain was cut into very thin 8nm slices and photographed with an electron microscope. A three-dimensional map of the neurons and connections in the brain was then reconstructed from these images using machine learning algorithms. Finally, Scheffer et al. used the new connectome to obtain further insights into the circuits that support specific fruit fly behaviors. The central brain connectome is freely available online for anyone to access. When used in combination with existing methods, the map will make it easier to understand how the fly brain works, and how and why it can fail to work correctly. Many of these findings will likely apply to larger brains, including our own. In the long run, studying the fly connectome may therefore lead to a better understanding of the human brain and its disorders. Performing a similar analysis on the brain of a small mammal, by scaling up the methods here, will be a likely next step along this path.

Neutralizing Antibodies Correlate with Protection from SARS-CoV-2 in Humans during a Fishery Vessel Outbreak with a High Attack Rate.

Abstract: The development of vaccines against SARS-CoV-2 would be greatly facilitated by the identification of immunological correlates of protection in humans. However, to date, studies on protective immunity have been performed only in animal models and correlates of protection have not been established in humans. Here, we describe an outbreak of SARS-CoV-2 on a fishing vessel associated with a high attack rate. Predeparture serological and viral reverse transcription-PCR (RT-PCR) testing along with repeat testing after return to shore was available for 120 of the 122 persons on board over a median follow-up of 32.5 days (range, 18.8 to 50.5 days). A total of 104 individuals had an RT-PCR-positive viral test with a cycle threshold (CT ) of <35 or seroconverted during the follow-up period, yielding an attack rate on board of 85.2% (104/122 individuals). Metagenomic sequencing of 39 viral genomes suggested that the outbreak originated largely from a single viral clade. Only three crew members tested seropositive prior to the boat's departure in initial serological screening and also had neutralizing and spike-reactive antibodies in follow-up assays. None of the crew members with neutralizing antibody titers showed evidence of bona fide viral infection or experienced any symptoms during the viral outbreak. Therefore, the presence of neutralizing antibodies from prior infection was significantly associated with protection against reinfection (Fisher's exact test, P = 0.002).

Prime genome editing in rice and wheat

Abstract: Prime editors, which are CRISPR-Cas9 nickase (H840A)-reverse transcriptase fusions programmed with prime editing guide RNAs (pegRNAs), can edit bases in mammalian cells without donor DNA or double-strand breaks. We adapted prime editors for use in plants through codon, promoter, and editing-condition optimization. The resulting suite of plant prime editors enable point mutations, insertions and deletions in rice and wheat protoplasts. Regenerated prime-edited rice plants were obtained at frequencies of up to 21.8%.

Systematic comparison of single-cell and single-nucleus RNA-sequencing methods.

Abstract: The scale and capabilities of single-cell RNA-sequencing methods have expanded rapidly in recent years, enabling major discoveries and large-scale cell mapping efforts. However, these methods have not been systematically and comprehensively benchmarked. Here, we directly compare seven methods for single-cell and/or single-nucleus profiling-selecting representative methods based on their usage and our expertise and resources to prepare libraries-including two low-throughput and five high-throughput methods. We tested the methods on three types of samples: cell lines, peripheral blood mononuclear cells and brain tissue, generating 36 libraries in six separate experiments in a single center. To directly compare the methods and avoid processing differences introduced by the existing pipelines, we developed scumi, a flexible computational pipeline that can be used with any single-cell RNA-sequencing method. We evaluated the methods for both basic performance, such as the structure and alignment of reads, sensitivity and extent of multiplets, and for their ability to recover known biological information in the samples.

Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses.

Abstract: The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID-19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARS-CoV-2-specific antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1), and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies.

Lipid-droplet-accumulating microglia represent a dysfunctional and proinflammatory state in the aging brain

Abstract: Microglia become progressively activated and seemingly dysfunctional with age, and genetic studies have linked these cells to the pathogenesis of a growing number of neurodegenerative diseases. Here we report a striking buildup of lipid droplets in microglia with aging in mouse and human brains. These cells, which we call 'lipid-droplet-accumulating microglia' (LDAM), are defective in phagocytosis, produce high levels of reactive oxygen species and secrete proinflammatory cytokines. RNA-sequencing analysis of LDAM revealed a transcriptional profile driven by innate inflammation that is distinct from previously reported microglial states. An unbiased CRISPR-Cas9 screen identified genetic modifiers of lipid droplet formation; surprisingly, variants of several of these genes, including progranulin (GRN), are causes of autosomal-dominant forms of human neurodegenerative diseases. We therefore propose that LDAM contribute to age-related and genetic forms of neurodegeneration.

Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity

Abstract: Applications of adenine base editors (ABEs) have been constrained by the limited compatibility of the deoxyadenosine deaminase component with Cas homologs other than SpCas9. We evolved the deaminase component of ABE7.10 using phage-assisted non-continuous and continuous evolution (PANCE and PACE), which resulted in ABE8e. ABE8e contains eight additional mutations that increase activity (kapp) 590-fold compared with that of ABE7.10. ABE8e offers substantially improved editing efficiencies when paired with a variety of Cas9 or Cas12 homologs. ABE8e is more processive than ABE7.10, which could benefit screening, disruption of regulatory regions and multiplex base editing applications. A modest increase in Cas9-dependent and -independent DNA off-target editing, and in transcriptome-wide RNA off-target editing can be ameliorated by the introduction of an additional mutation in the TadA-8e domain. Finally, we show that ABE8e can efficiently install natural mutations that upregulate fetal hemoglobin expression in the BCL11A enhancer or in the the HBG promoter in human cells, targets that were poorly edited with ABE7.10. ABE8e augments the effectiveness and applicability of adenine base editing.

A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing

Abstract: Author(s): Gordon, David E; Jang, Gwendolyn M; Bouhaddou, Mehdi; Xu, Jiewei; Obernier, Kirsten; O'Meara, Matthew J; Guo, Jeffrey Z; Swaney, Danielle L; Tummino, Tia A; Huttenhain, Ruth; Kaake, Robyn M; Richards, Alicia L; Tutuncuoglu, Beril; Foussard, Helene; Batra, Jyoti; Haas, Kelsey; Modak, Maya; Kim, Minkyu; Haas, Paige; Polacco, Benjamin J; Braberg, Hannes; Fabius, Jacqueline M; Eckhardt, Manon; Soucheray, Margaret; Bennett, Melanie J; Cakir, Merve; McGregor, Michael J; Li, Qiongyu; Naing, Zun Zar Chi; Zhou, Yuan; Peng, Shiming; Kirby, Ilsa T; Melnyk, James E; Chorba, John S; Lou, Kevin; Dai, Shizhong A; Shen, Wenqi; Shi, Ying; Zhang, Ziyang; Barrio-Hernandez, Inigo; Memon, Danish; Hernandez-Armenta, Claudia; Mathy, Christopher JP; Perica, Tina; Pilla, Kala B; Ganesan, Sai J; Saltzberg, Daniel J; Ramachandran, Rakesh; Liu, Xi; Rosenthal, Sara B; Calviello, Lorenzo; Venkataramanan, Srivats; Lin, Yizhu; Wankowicz, Stephanie A; Bohn, Markus; Trenker, Raphael; Young, Janet M; Cavero, Devin; Hiatt, Joe; Roth, Theo; Rathore, Ujjwal; Subramanian, Advait; Noack, Julia; Hubert, Mathieu; Roesch, Ferdinand; Vallet, Thomas; Meyer, Bjorn; White, Kris M; Miorin, Lisa; Agard, David; Emerman, Michael; Ruggero, Davide; Garcia-Sastre, Adolfo; Jura, Natalia; von Zastrow, Mark; Taunton, Jack; Schwartz, Olivier; Vignuzzi, Marco; d'Enfert, Christophe; Mukherjee, Shaeri; Jacobson, Matt; Malik, Harmit S; Fujimori, Danica G; Ideker, Trey; Craik, Charles S | Abstract: An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity- purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.

Senolytic CAR T cells reverse senescence-associated pathologies.

Abstract: Cellular senescence is characterized by stable cell-cycle arrest and a secretory program that modulates the tissue microenvironment1,2 Physiologically, senescence serves as a tumour-suppressive mechanism that prevents the expansion of premalignant cells3,4 and has a beneficial role in wound-healing responses5,6 Pathologically, the aberrant accumulation of senescent cells generates an inflammatory milieu that leads to chronic tissue damage and contributes to diseases such as liver and lung fibrosis, atherosclerosis, diabetes and osteoarthritis1,7 Accordingly, eliminating senescent cells from damaged tissues in mice ameliorates the symptoms of these pathologies and even promotes longevity1,2,8-10 Here we test the therapeutic concept that chimeric antigen receptor (CAR) T cells that target senescent cells can be effective senolytic agents We identify the urokinase-type plasminogen activator receptor (uPAR)11 as a cell-surface protein that is broadly induced during senescence and show that uPAR-specific CAR T cells efficiently ablate senescent cells in vitro and in vivo CAR T cells that target uPAR extend the survival of mice with lung adenocarcinoma that are treated with a senescence-inducing combination of drugs, and restore tissue homeostasis in mice in which liver fibrosis is induced chemically or by diet These results establish the therapeutic potential of senolytic CAR T cells for senescence-associated diseases