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Patients with cystic fibrosis (CF) need costly medical care and adequate therapy with expensive medicinal products. Tigerase® is the first biosimilar of dornase alfa, developed by the lead Russian biotechnology company GENERIUM. The aim of the manuscript to present post hoc sub-analysis of patients' data with cystic fibrosis and severe pulmonary impairment of a larger comparative study (phase III open label, prospective, multi-centre, randomized study (NCT04468100)) of a generic version of recombinant human DNase Tigerase® to the only comparable drug, Pulmozyme®. In the analyses included subgroup of 46 severe pulmonary impairment patients with baseline FEV1 level 40-60% of predicted (23 patients in each treatment group) out of 100 patients registered in the study phase III open label, prospective, multi-center, randomized study (NCT04468100), and compared efficacy endpoints (FEV1, FVC, number and time of exacerbations, body weight, St.George's Respiratory Questionnaire) as well as safety parameters (AEs, SAEs, anti-drug antibody) within 24 treatment weeks. All outcomes were comparable among the studied groups. In the efficacy dataset, the similar mean FEV1 and mean FVC changes for 24 weeks of both treatment groups were observed. The groups were also comparable in safety, all the secondary efficacy parameters and immunogenicity. The findings from this study support the clinical Tigerase® biosimilarity to Pulmozyme® administered in CF patients with severe impairment of pulmonary function.

Microbial nucleic acid recognition serves as the major stimulus to an antiviral response, implying a requirement to limit the misrepresentation of self nucleic acids as non-self and the induction of autoinflammation. By systematic screening using a panel of interferon-stimulated genes we identify two siblings and a singleton variably demonstrating severe neonatal anemia, membranoproliferative glomerulonephritis, liver fibrosis, deforming arthropathy and increased anti-DNA antibodies. In both families we identify biallelic mutations in DNASE2 , associated with a loss of DNase II endonuclease activity. We record increased interferon alpha protein levels using digital ELISA, enhanced interferon signaling by RNA-Seq analysis and constitutive upregulation of phosphorylated STAT1 and STAT3 in patient lymphocytes and monocytes. A hematological disease transcriptomic signature and increased numbers of erythroblasts are recorded in patient peripheral blood, suggesting that interferon might have a particular effect on hematopoiesis. These data define a type I interferonopathy due to DNase II deficiency in humans. Nucleic acid sensing is important to ensure that an innate immune response is only mounted against microbial nucleic acid. Here, the authors identify loss-of-function mutations in the DNASE2 gene that cause type I interferon-mediated autoinflammation due to enhanced systemic interferon signaling.

Slowly clearing infections in the pleural space are a source of substantial morbidity. This study showed that instillation of recombinant DNase and tissue plasminogen activator (t-PA) is more effective than placebo in clearing radiographic pleural effusions. Pleural infection affects more than 65,000 patients each year in the United States and the United Kingdom, 1 and the incidence is increasing in both countries — in both children 2 – 4 and adults. 5 , 6 The mortality rate from pleural infection is between 10% and 20%, 5 , 7 – 9 and drainage through a chest tube and administration of antibiotics fail in approximately one third of patients, who then require surgical drainage. 5 , 9 The median duration of the hospital stay for these patients is 12 to 15 days, 5 , 6 , 8 , 9 with 25% hospitalized for more than a month. Care of each patient costs . . .

Bacterial type VI secretion systems (T6SSs) inject toxic effectors into adjacent eukaryotic and prokaryotic cells. It is generally thought that this process requires physical contact between the two cells. Here, we provide evidence of contact-independent killing by a T6SS-secreted effector. We show that the pathogen Yersinia pseudotuberculosis uses a T6SS (T6SS-3) to secrete a nuclease effector that kills other bacteria in vitro and facilitates gut colonization in mice. The effector (Tce1) is a small protein that acts as a Ca 2+ - and Mg 2+ -dependent DNase, and its toxicity is inhibited by a cognate immunity protein, Tci1. As expected, T6SS-3 mediates canonical, contact-dependent killing by directly injecting Tce1 into adjacent cells. In addition, T6SS-3 also mediates killing of neighboring cells in the absence of cell-to-cell contact, by secreting Tce1 into the extracellular milieu. Efficient contact-independent entry of Tce1 into target cells requires proteins OmpF and BtuB in the outer membrane of target cells. The discovery of a contact-independent, long-range T6SS toxin delivery provides a new perspective for understanding the physiological roles of T6SS in competition. However, the mechanisms mediating contact-independent uptake of Tce1 by target cells remain unclear. Bacteria can use type VI secretion systems (T6SSs) to inject toxic effector proteins into adjacent cells, in a contact-dependent manner. Here, the authors provide evidence of contact-independent killing by a T6SS effector that is secreted into the extracellular milieu and then taken up by other bacterial cells.

Acute respiratory distress syndrome (ARDS) caused by SARS-CoV-2 infection is associated with high mortality rates and respiratory compromise in which excessive neutrophil extracellular trap (NET) production may amplify alveolar inflammation and injury. Dornase alfa, a recombinant DNAse 1, has been proposed to attenuate these effects by degrading extracellular DNA and enhancing alveolar clearance of NETs. In this multicenter, open-label, randomized in two parallel arms (1:1) controlled trial, intubated COVID-19 ARDS patients received either standard-of-care (SOC) alone or SOC plus aerosolized dornase alfa (2500 IU twice daily for 7 days). The primary endpoint was the proportion of patients with ARDS severity improvement at Day 7, defined by at least one-grade improvement on the Berlin criteria scale. Secondary outcomes included 28-day mortality, ventilator-free days, ICU-free days, and changes in key ventilatory parameters. Biological samples were analyzed to assess NET related markers, DNAse drug activity and indicate possible bioavailability issues associated with aerosolization of dornase alfa. Seventy-seven patients were enrolled (dornase alfa group, n  = 39; SOC group, n  = 38). At Day 7, ARDS severity improved in 18% of patients receiving dornase alfa compared with 29% in the SOC group (adjusted OR: 0.33; 95% CI 0.09–1.14; p  = 0.11). Secondary endpoints, including 28-day mortality, ventilator-free days, and ICU-free days, showed no significant differences between groups. Adverse events occurred in 38.5% of patients in the dornase alfa arm versus 31.6% in the SOC arm, indicating comparable safety profiles. Despite early increases in NET plasmatic levels observed in both groups and successful ex vivo NET degradation, aerosolized dornase alfa failed to significantly enhance DNAse activity or reduce NET-related markers in patients’ plasma and mucus, suggesting potential bioavailability limitations with this delivery method. In patients with COVID-19-related ARDS, dornase alfa did neither significantly reduce ARDS severity nor improve clinical outcomes over SOC. Although well tolerated, analysis of biological samples suggests that aerosol administration may have compromised drug bioavailability. Further trials are needed to determine whether specific patient subgroups could benefit more from dornase alfa or if alternative drug delivery methods might enhance treatment efficacy. ClinicalTrials.gov, NCT04355364. Registered on 21/04/2020.

In the type III CRISPR–Cas immune response of prokaryotes, infection triggers the production of cyclic oligoadenylates that bind and activate proteins that contain a CARF domain 1 , 2 . Many type III loci are associated with proteins in which the CRISPR-associated Rossman fold (CARF) domain is fused to a restriction  endonuclease-like domain 3 , 4 . However, with the exception of the well-characterized Csm6 and Csx1 ribonucleases 5 , 6 , whether and how these inducible effectors provide defence is not known. Here we investigated a type III CRISPR accessory protein, which we name cyclic-oligoadenylate-activated single-stranded ribonuclease and single-stranded deoxyribonuclease 1 (Card1). Card1 forms a symmetrical dimer that has a large central cavity between its CRISPR-associated Rossmann fold and restriction endonuclease domains that binds cyclic tetra-adenylate. The binding of ligand results in a conformational change comprising the rotation of individual monomers relative to each other to form a more compact dimeric scaffold, in which a manganese cation coordinates the catalytic residues and activates the cleavage of single-stranded—but not double-stranded—nucleic acids (both DNA and RNA). In vivo, activation of Card1 induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used in CRISPR systems to provide immunity. Structural analyses of the type III CRISPR accessory protein Card1, which induces dormancy in infected hosts to provide immunity against phage infection, reveal the mechanisms by which it cleaves single-stranded RNA and DNA.

Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation 1 – 5 . Gabija is one of the most prevalent anti-phage defence systems, occurring in more than 15% of all sequenced bacterial and archaeal genomes 1 , 6 , 7 , but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-electron microscopy (cryo-EM) to define how Gabija proteins assemble into a supramolecular complex of around 500 kDa that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defence complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajA–GajB assembly (hereafter, GajAB) that is essential for phage resistance in vivo. We show that a phage-encoded protein, Gabija anti-defence 1 (Gad1), directly binds to the Gabija GajAB complex and inactivates defence. A cryo-EM structure of the virally inhibited state shows that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defence complex and define a unique mechanism of viral immune evasion. X-ray crystallography, cryo-EM and biochemical analysis provide insight into the assembly of the bacterial Gabija complex, an anti-phage system, and reveal how viruses can evade this defence mechanism.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.

Platelet and fibrin clots occlude blood vessels in hemostasis and thrombosis. Here we report a noncanonical mechanism for vascular occlusion based on neutrophil extracellular traps (NETs), DNA fibers released by neutrophils during inflammation. We investigated which host factors control NETs in vivo and found that two deoxyribonucleases (DNases), DNase1 and DNase1-like 3, degraded NETs in circulation during sterile neutrophilia and septicemia. In the absence of both DNases, intravascular NETs formed clots that obstructed blood vessels and caused organ damage. Vascular occlusions in patients with severe bacterial infections were associated with a defect to degrade NETs ex vivo and the formation of intravascular NET clots. DNase1 and DNase1-like 3 are independently expressed and thus provide dual host protection against deleterious effects of intravascular NETs.

Transcription activator-like effector nucleases (TALENs) comprise a nonspecific nuclease fused to a sequence-specific DNA-binding domain. This domain can be engineered so that TALENs can target virtually any sequence. TALENs are an efficient tool to modify genes in a wide range of cell types and organisms. Engineered nucleases enable the targeted alteration of nearly any gene in a wide range of cell types and organisms. The newly-developed transcription activator-like effector nucleases (TALENs) comprise a nonspecific DNA-cleaving nuclease fused to a DNA-binding domain that can be easily engineered so that TALENs can target essentially any sequence. The capability to quickly and efficiently alter genes using TALENs promises to have profound impacts on biological research and to yield potential therapeutic strategies for genetic diseases.