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This article presents long-term data on the efficacy of gene replacement therapy (GRT) for spinal muscular atrophy (SMA) using onasemnogene abeparvovec (OA). The study includes patients who were older, heavier, or more severely affected than those in previous clinical trials, as well as a large cohort of presymptomatic SMA patients identified through newborn screening. The results show that younger patients treated with OA had better motor function outcomes compared to those treated at a later age. The study provides valuable evidence for future treatment decisions, particularly for patients with limited trial data available. [Extracted from the article]

For drugs to be active they have to reach their targets. Within cells this requires crossing the cell membrane, and then free diffusion, distribution, and availability. Here, we explored the in-cell diffusion rates and distribution of a series of small molecular fluorescent drugs, in comparison to proteins, by microscopy and fluorescence recovery after photobleaching (FRAP). While all proteins diffused freely, we found a strong correlation between p K a and the intracellular diffusion and distribution of small molecule drugs. Weakly basic, small-molecule drugs displayed lower fractional recovery after photobleaching and 10- to-20-fold slower diffusion rates in cells than in aqueous solutions. As, more than half of pharmaceutical drugs are weakly basic, they, are protonated in the cell cytoplasm. Protonation, facilitates the formation of membrane impermeable ionic form of the weak base small molecules. This results in ion trapping, further reducing diffusion rates of weakly basic small molecule drugs under macromolecular crowding conditions where other nonspecific interactions become more relevant and dominant. Our imaging studies showed that acidic organelles, particularly the lysosome, captured these molecules. Surprisingly, blocking lysosomal import only slightly increased diffusion rates and fractional recovery. Conversely, blocking protonation by N- acetylated analogues, greatly enhanced their diffusion and fractional recovery after FRAP. Based on these results, N -acetylation of small molecule drugs may improve the intracellular availability and distribution of weakly basic, small molecule drugs within cells., Competing Interests: DD, SM, AP, OG, LW, TS, GS No competing interests declared, (© 2024, Dey et al.)

Janus kinases (JAKs) bind to class I and II cytokine receptors, activating signaling and regulating gene transcription through signal transducer and activator of transcription (STAT) proteins. Type I interferons (IFNs) require the JAK members TYK2 and JAK1, which bind to the receptor subunits IFNAR1 and IFNAR2, respectively. We investigated the role of JAKs in regulating IFNAR signaling activity. Synthetic IFNARs in which the extracellular domains of IFNAR1 and IFNAR2 are replaced with nanobodies had near-native type I IFN signaling, whereas the homomeric variant of IFNAR2 initiated much weaker signaling, despite harboring docking sites for JAKs and STATs. Cells with JAK1 and TYK2 knockout (KO) showed residual signaling, suggesting partial complementation by the remaining JAKs, particularly when they were overexpressed. Live-cell micropatterning experiments confirmed the promiscuous binding of JAK1, JAK2, and TYK2 to IFNAR1 and IFNAR2, and their recruitment correlated with their relative cellular abundances. However, each JAK had a different efficacy in inducing cross-phosphorylation and downstream signaling. JAK binding was also promiscuous for other cytokine receptors, including IFN-L1, IL-10Rβ, TPOR, and GHR, but not for EPOR, which activated different downstream signaling pathways. These findings suggest that competitive binding of JAKs to cytokine receptors together with the varying absolute and relative abundances of the JAKs in different cell types can account for the cell type-dependent signaling pleiotropy of cytokine receptors.

Background: Real-world data on gene addition therapy (GAT) with onasemnogene abeparvovec (OA), including all age groups and with or without symptoms of the disease before treatment are needed to provide families with evidence-based advice and realistic therapeutic goals. Aim of this study is therefore a population-based analysis of all patients with SMA treated with OA across Germany, Austria and Switzerland (D-A-CH)., Methods: This observational study included individuals with Spinal Muscular Atrophy (SMA) treated with OA in 29 specialized neuromuscular centers in the D-A-CH-region. A standardized data set including WHO gross motor milestones, SMA validated motor assessments, need for nutritional and respiratory support, and adverse events was collected using the SMArtCARE registry and the Swiss-Reg-NMD. Outcome data were analyzed using a prespecified statistical analysis plan including potential predictors such as age at GAT, SMN2 copy number, past treatment, and symptom status., Findings: 343 individuals with SMA (46% male, 54% female) with a mean age at OA of 14.0 months (range 0-90, IQR 20.0 months) were included in the analysis. 79 (23%) patients were clinically presymptomatic at the time of treatment. 172 (50%) patients received SMN2 splice-modifying drugs prior to GAT (risdiplam: n = 16, nusinersen: n = 154, both: n = 2). Functional motor improvement correlated with lower age at GAT, with the best motor outcome in those younger than 6 weeks, carrying 3 SMN2 copies, and being clinically presymptomatic at time of treatment. The likelihood of requiring ventilation or nutritional support showed a significantly increase with older age at the time of GAT and remained stable thereafter. Pre-treatment had no effect on disease trajectories. Liver-related adverse events occurred significantly less frequently up to 8 months of age. All other adverse events showed an even distribution across all age and weight groups., Interpretation: Overall, motor, respiratory, and nutritional outcome were dependent on timing of GAT and initial symptom status. It was best in presymptomatic children treated within the first six weeks of life, but functional motor scores also increased significantly after treatment in all age groups up to 24 months. Additionally, OA was best tolerated when administered at a young age. Our study therefore highlights the need for SMA newborn screening and immediate treatment to achieve the best possible benefit-risk ratio., Funding: The SMArtCARE and Swiss-Reg-NMD registries are funded by different sources (see acknowledgements)., Competing Interests: No author received financial support for the present manuscript. CW received honoraria for presentations and/or travel support from Novartis, Roche and Biogen and participated on advisory boards for Novartis, Roche and Biogen. JF received honoraria from Novartis for presentations. AB received honoraria for presentations from Roche and Pfizer and attend advisory boards for Roche and Pfizer. SI received honoraria for presentations and advisory board meetings from Novartis and Roche. OS received honoraria from Biogen, support for attending meetings from Novartis and participated on an advisory board for Novartis. GB received honoraria for advisory boards and/or presentations from Novartis, Roche and Biogen and support for attending meetings from Novartis. MvdH received grants from Deutsche Gesellschaft für Muskelkranke and Innovationsfond (INTEGRATE ATMP and KoCoN), honoraria from Pfizer, Biogen and PTC Therapeutics, and participated on advisory boards for Roche, Sarepta, Novartis, and Pfizer. RAH received consulting fees from Biogen and honoraria for presentations from Novartis, and participated on advisory boards by Novartis and Roche. KG participated on an industry symposium and an advisory board by Novartis. JK received funding for clinical research from Biogen, Novartis, Roche, ScholarRock and Biohaven, consulting fees from Biogen, Novartis and Roche, payment for educational activities from Biogen, Novartis and Roche and attended a data safety monitoring board for Biogen. AP received research funding form Roche, Novartis and Biogen. MFB received consulting fees for advisory boards from Roche, Novartis and Biogen and payment for presentations from Novartis and Biogen. GS received honoraria for a case report and participated on an advisory board for Novartis. US received honoraria for presentations from Biogen, Novartis and Roche and support for attending meetings from Roche and Novartis. BP received honoraria for a presentation from Biogen and participated on advisory boards for Novartis and Biogen. RT received honoraria for presentations from Desitin, PTC and Roche and participated on advisory boards for PTC, Roche and Santhera. VH attended an adivisory board for Biogen. MB received compensation for advisory boards and speakers honoraria from Novartis, Biogen and Roche and compensation for travel costs to meetings from Roche. AK is clinical lead of the Swiss Reg NMD, attended advisory board meetings of Novartis and received honorarium for a presentation by Novartis. AE received honoraria and payment for expert testimony from Biogen, Roche and Novartis, support for attending meetings from Biogen and Roche and participated on an advisory board for Biogen, Roche and Novartis. GMS participated on advisory boards from Biogen, Novartis and Roche and is member of the steering board for Swiss-Reg-NMD. SC received payment for a presentation. JJ received compensation for advisory boards and funding for travel or speaker honoraria from Avexis/Novartis, Biogen, IFT, Roche, PTC, Pfizer and Sarepta Therapeutics. AZ received compensation for advisory boards and funding for travel or speaker honoraria from Avexis/Novartis, Biogen, ITF, Roche, Pfizer and Sarepta Therapeutics. LLB, AH, EW, CK, OH, and SFG indicated no potential conflicts of interest to disclose., (© 2024 The Author(s).)

SARS-CoV-2 has the capacity to evolve mutations that escape vaccine- and infection-acquired immunity and antiviral drugs. A variant-agnostic therapeutic agent that protects against severe disease without putting selective pressure on the virus would thus be a valuable biomedical tool that would maintain its efficacy despite the ongoing emergence of new variants. Here, we challenge male rhesus macaques with SARS-CoV-2 Delta-the most pathogenic variant in a highly susceptible animal model. At the time of challenge, we also treat the macaques with aerosolized RBD-62, a protein developed through multiple rounds of in vitro evolution of SARS-CoV-2 RBD to acquire 1000-fold enhanced ACE2 binding affinity. RBD-62 treatment equivalently suppresses virus replication in both upper and lower airways, a phenomenon not previously observed with clinically approved vaccines. Importantly, RBD-62 does not block the development of virus-specific T- and B-cell responses and does not elicit anti-drug immunity. These data provide proof-of-concept that RBD-62 can prevent severe disease from a highly virulent variant., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Importance: There is increasing evidence that early diagnosis and treatment are key for outcomes in infants with spinal muscular atrophy (SMA), and newborn screening programs have been implemented to detect the disease before onset of symptoms. However, data from controlled studies that reliably confirm the benefits of newborn screening are lacking., Objective: To compare data obtained on patients with SMA diagnosed through newborn screening and those diagnosed after clinical symptom onset., Design, Setting, and Participants: This nonrandomized controlled trial used data from the SMARTCARE registry to evaluate all children born between January 2018 and September 2021 with genetically confirmed SMA and up to 3 SMN2 copies. The registry includes data from 70 participating centers in Germany, Austria, and Switzerland. Data analysis was performed in February 2023 so that all patients had a minimal follow-up of 18 months., Exposure: Patients born in 2 federal states in Germany underwent screening in a newborn screening pilot project. All other patients were diagnosed after clinical symptom onset. All patients received standard care within the same health care system., Main Outcomes: The primary end point was the achievement of motor milestones., Results: A total of 234 children (123 [52.6%] female) were identified who met inclusion criteria and were included in the analysis: 44 (18.8%) in the newborn screening cohort and 190 children (81.2%) in the clinical symptom onset cohort. The mean (SD) age at start of treatment with 1 of the approved disease-modifying drugs was 1.3 (2.2) months in the newborn screening cohort and 10.7 (9.1) months in the clinical symptom onset cohort. In the newborn screening cohort, 40 of 44 children (90.9%) gained the ability to sit independently vs 141 of 190 (74.2%) in the clinical symptom onset cohort. For independent ambulation, the ratio was 28 of 40 (63.6%) vs 28 of 190 (14.7%)., Conclusions and Relevance: This nonrandomized controlled trial demonstrated effectiveness of newborn screening for infants with SMA in the real-world setting. Functional outcomes and thus the response to treatment were significantly better in the newborn screening cohort compared to the unscreened clinical symptom onset group., Trial Registration: German Clinical Trials Register: DRKS00012699.

Interferons (IFNs) are essential for defense against viral infections but also drive recruitment of inflammatory cells to sites of infection, a key feature of severe COVID-19. Here, we explore the complexity of the IFN response in COVID-19, examine the effects of manipulating IFN on SARS-CoV-2 viral replication and pathogenesis, and highlight pre-clinical and clinical studies evaluating the therapeutic efficacy of IFN in limiting COVID-19 severity., Competing Interests: The authors declare no conflict of interest.

Newborn screening for 5qSMA offers the potential for early, ideally pre-symptomatic, therapeutic intervention. However, limited data exist on the outcomes of individuals with 4 copies of SMN2, and there is no consensus within the SMA treatment community regarding early treatment initiation in this subgroup. To provide evidence-based insights into disease progression, we performed a retrospective analysis of 268 patients with 4 copies of SMN2 from the SMArtCARE registry in Germany, Austria and Switzerland. Inclusion criteria required comprehensive baseline data and diagnosis outside of newborn screening. Only data prior to initiation of disease-modifying treatment were included. The median age at disease onset was 3.0 years, with a mean of 6.4 years. Significantly, 55% of patients experienced symptoms before the age of 36 months. 3% never learned to sit unaided, a further 13% never gained the ability to walk independently and 33% of ambulatory patients lost this ability during the course of the disease. 43% developed scoliosis, 6.3% required non-invasive ventilation and 1.1% required tube feeding. In conclusion, our study, in line with previous observations, highlights the substantial phenotypic heterogeneity in SMA. Importantly, this study provides novel insights: the median age of disease onset in patients with 4 SMN2 copies typically occurs before school age, and in half of the patients even before the age of three years. These findings support a proactive approach, particularly early treatment initiation, in this subset of SMA patients diagnosed pre-symptomatically. However, it is important to recognize that the register will not include asymptomatic individuals., (© 2024. The Author(s).)

Type I and type III interferons (IFNs) are major components in activating the innate immune response. Common to both are two distinct receptor chains (IFNAR1/IFNAR2 and IFNLR1/IL10R2), which form ternary complexes upon binding their respective ligands. This results in close proximity of the intracellularly associated kinases JAK1 and TYK2, which cross phosphorylate each other, the associated receptor chains, and signal transducer and activator of transcriptions, with the latter activating IFN-stimulated genes. While there are clear similarities in the biological responses toward type I and type III IFNs, differences have been found in their tropism, tuning of activity, and induction of the immune response. Here, we focus on how these differences are embedded in the structure/function relations of these two systems in light of the recent progress that provides in-depth information on the structural assembly of these receptors and their functional implications and how these differ between the mouse and human systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Importance: Most studies investigating the characteristics of emerging SARS-CoV-2 variants have been focusing on mutations in the spike proteins that affect viral infectivity, fusogenicity, and pathogenicity. However, few studies have addressed how naturally occurring mutations in the non- spike regions of the SARS-CoV-2 genome impact virological properties. In this study, we proved that multiple SARS-CoV-2 Omicron BA.2 mutations, one in the spike protein and another downstream of the spike gene, orchestrally characterize this variant, shedding light on the importance of Omicron BA.2 mutations out of the spike protein., Competing Interests: The authors declare no conflict of interest.

Type I interferons (IFN-I) are critical mediators of innate control of viral infections but also drive the recruitment of inflammatory cells to sites of infection, a key feature of severe coronavirus disease 2019. Here, IFN-I signaling was modulated in rhesus macaques (RMs) before and during acute SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection using a mutated IFN-α2 (IFN-modulator; IFNmod), which has previously been shown to reduce the binding and signaling of endogenous IFN-I. IFNmod treatment in uninfected RMs was observed to induce a modest up-regulation of only antiviral IFN-stimulated genes (ISGs); however, in SARS-CoV-2-infected RMs, IFNmod reduced both antiviral and inflammatory ISGs. IFNmod treatment resulted in a potent reduction in SARS-CoV-2 viral loads both in vitro in Calu-3 cells and in vivo in bronchoalveolar lavage (BAL), upper airways, lung, and hilar lymph nodes of RMs. Furthermore, in SARS-CoV-2-infected RMs, IFNmod treatment potently reduced inflammatory cytokines, chemokines, and CD163 + MRC1 - inflammatory macrophages in BAL and expression of Siglec-1 on circulating monocytes. In the lung, IFNmod also reduced pathogenesis and attenuated pathways of inflammasome activation and stress response during acute SARS-CoV-2 infection. Using an intervention targeting both IFN-α and IFN-β pathways, this study shows that, whereas early IFN-I restrains SARS-CoV-2 replication, uncontrolled IFN-I signaling critically contributes to SARS-CoV-2 inflammation and pathogenesis in the moderate disease model of RMs.

Collagen XII, belonging to the fibril-associated collagens, is a homotrimeric secreted extracellular matrix (ECM) protein encoded by the COL12A1 gene. Mutations in the human COL12A1 gene cause an Ehlers-Danlos/myopathy overlap syndrome leading to skeletal abnormalities and muscle weakness. Here, we studied the role of collagen XII in joint pathophysiology by analyzing collagen XII deficient mice and human patients. We found that collagen XII is widely expressed across multiple connective tissue of the developing joint. Lack of collagen XII in mice destabilizes tendons and the femoral trochlear groove to induce patellar subluxation in the patellofemoral joint. These changes are associated with an ECM damage response in tendon and secondary quadriceps muscle degeneration. Moreover, patellar subluxation was also identified as a clinical feature of human patients with collagen XII deficiency. The results provide an explanation for joint hyperlaxity in mice and human patients with collagen XII deficiency., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Spinal muscular atrophy (SMA) is a rare genetic disorder, with the most common form being 5q SMA. Survival of children with severe SMA is poor, yet major advances have been made in recent years in pharmaceutical treatment, such as gene-therapy, which has improved patient survival. Therefore, clinical problems, such as the development of spinal deformities in these genetically treated SMA children represent an unknown challenge in clinical work. In a retrospective case series, the development of spinal deformities was analyzed in 16 SMA children (9 male, 7 female) treated with onasemnogene abeparvovec in two institutions during the years 2020 to 2022. Ten out of sixteen patients had a significant kyphosis, and nine out of sixteen patients had significant scoliosis, with the mean curvature angles of 24 ± 27° for scoliosis, and 69 ± 15° for kyphosis. Based on these preliminary data, it can be assumed that early-onset kyphosis presents a clinical challenge in gene-therapy-treated SMA children. Larger datasets with longer follow-up times need to be collected in order to verify these preliminary observations.

In late 2022, SARS-CoV-2 Omicron subvariants have become highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged through the recombination of two cocirculating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022. XBB.1 is the variant most profoundly resistant to BA.2/5 breakthrough infection sera to date and is more fusogenic than BA.2.75. The recombination breakpoint is located in the receptor-binding domain of spike, and each region of the recombinant spike confers immune evasion and increases fusogenicity. We further provide the structural basis for the interaction between XBB.1 spike and human ACE2. Finally, the intrinsic pathogenicity of XBB.1 in male hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provides evidence suggesting that XBB is the first observed SARS-CoV-2 variant to increase its fitness through recombination rather than substitutions., (© 2023. The Author(s).)

In late 2022, various Omicron subvariants emerged and cocirculated worldwide. These variants convergently acquired amino acid substitutions at critical residues in the spike protein, including residues R346, K444, L452, N460, and F486. Here, we characterize the convergent evolution of Omicron subvariants and the properties of one recent lineage of concern, BQ.1.1. Our phylogenetic analysis suggests that these five substitutions are recurrently acquired, particularly in younger Omicron lineages. Epidemic dynamics modelling suggests that the five substitutions increase viral fitness, and a large proportion of the fitness variation within Omicron lineages can be explained by these substitutions. Compared to BA.5, BQ.1.1 evades breakthrough BA.2 and BA.5 infection sera more efficiently, as demonstrated by neutralization assays. The pathogenicity of BQ.1.1 in hamsters is lower than that of BA.5. Our multiscale investigations illuminate the evolutionary rules governing the convergent evolution for known Omicron lineages as of 2022., (© 2023. The Author(s).)

Introduction: Chronic lymphocytic leukemia (CLL) is characterized by an aberrant cytokine network that can support tumor growth by triggering janus kinase (JAK)/STAT pathways. Targeting cytokine-signaling should then be a rational therapeutic strategy but the JAK inhibitor ruxolitinib failed to control and seemingly accelerated the disease in clinical trials., Methods: The effect of ruxolitinib on primary human CLL cells was studied in vitro and in vivo ., Results: Ruxolitinib increased phosphorylation of IRAK4, an important toll-like receptor (TLR)- signaling intermediate, in circulating CLL cells in vitro . It also enhanced p38 and NFKB1 phosphorylation while lowering STAT3 phosphorylation in CLL cells activated with TLR-7/8 agonists and IL-2. Among the cytokines made by activated CLL cells, high levels of IL-10 contributed strongly to STAT3 phosphorylation and inhibited TLR7 activity. Ruxolitinib limited TLR-mediated IL10 transcription and markedly reduced IL-10 production in vitro . It also decreased blood levels of IL-10 while increasing TNFα along with phospho-p38 expression and gene sets associated with TLR-activation in CLL cells in vivo . The bruton's tyrosine kinase inhibitor ibrutinib decreased IL-10 production in vitro but, in contrast to ruxolitinib, blocked initial IL10 transcription induced by TLR-signaling in vitro, decreased TNFα production, and deactivates CLL cells in vivo ., Discussion: These findings suggest the possible benefits of inhibiting growth factors with JAK inhibitors in CLL are outweighed by negative effects on potential tumor suppressors such as IL-10 that allow unrestrained activation of NFκB by drivers such as TLRs. Specific inhibition of growth-promoting cytokines with blocking antibodies or infusing suppressive cytokines like IL-10 might be better strategies to manipulate cytokines in CLL., Competing Interests: Author DS reports grants from Novartis to support the submitted work and personal fees from Janssen outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Spaner, Luo, Wang, Schreiber, Harari and Shi.)