Diabetes mellitus patients with pulmonary tuberculosis (DMTB) comorbidity has been recognized as a major obstacle towards achieving the World Health Organization goal of reducing the tuberculosis incidence rate by 90% in 2035. Host immune responses affected by diabetes can lead to increased susceptibility, severity and poor treatment outcomes in DMTB patients, and the underlying mechanisms have not yet been fully elucidated. This study aimed to identify key immunological and cellular components that contribute to increased morbidity and mortality in DMTB cases.We performed RNA-Seq of total RNA isolated from peripheral blood mononuclear cells from 3 TB, 3 diabetes mellitus, and 3 DMTB patients and healthy controls, and analyzed differential expression, pathway enrichment and clustering of differentially-expressed genes (DEGs) to identify biological pathways altered specifically in DMTB patients.Bioinformatic analysis of DEGs suggested that enhanced inflammatory responses, small GTPases, the protein kinase C signaling pathway, hemostasis and the cell cycle pathway are likely implicated in the pathogenesis of the DMTB comorbidity.The DMTB comorbidity is associated with an altered transcriptome and changes in various biological pathways. Our study provides new insights on the pathological mechanism that may aid the development of host-directed therapies for this increasingly prevalent disease in high TB burden countries. Although the global TB incidence rate and the number of TB deaths have been falling in recent years, the soaring prevalence of diabetes mellitus (DM) worldwide and the fact that DM greatly increases TB susceptibility and severity has meant that the diabetes mellitus patients with pulmonary tuberculosis (DMTB) comorbidity has become a major global public health concern and has posed a great challenge to TB control and eradication.–4 Countries with the highest TB burden tend to be those with the largest number of diabetes cases.–6 In 2019 an estimated 0.35 million TB patients  had diabetes. Diagnosis of TB in diabetic individuals is more challenging, and mounting evidence shows that TB infection in diabetes patients leads to more unfavorable outcomes than in non-diabetic individuals. A recent meta-analysis of 44 studies showed that type 2 diabetes, the most prevalent form of DM, increases the risk of developing TB by two- to four-fold.–10 Moreover, patients with DMTB comorbidity are more likely to show more low-field pulmonary lesions, more cavitary lesions and more extrapulmonary lesions.,12 Additionally, as reported by several observational studies, DM can cause adverse TB treatment outcomes, including delays in pathogen clearance, treatment failure, relapse and recurrence.–18 The rapidly rising prevalence of diabetes, the increased risk and severity of DMTB comorbidity, and poor treatment outcomes emphasize the critical need to target this population in TB control programs.As the world’s most deadly infectious disease, tuberculosis (TB) claims more than a million lives each year and affected 8.9–11.0 million people in 2019 according to the World Health Organization.,19 However, the underlying mechanisms have not yet been fully understood. Most evidence suggests that the abnormal accumulation of glucose intermediates and excessive reactive oxidative species are the major factors impacting on the function of the host immune system.,21 In order to investigate how DM adversely affects TB patients and identify specific immunological pathways or biological processes underpinning DMTB comorbidity, in the present study we analyzed enriched pathways in differentially-expressed genes (DEGs) that displayed increased expression from DM to DMTB and in DEGs that are uniquely present in the DMTB patients relative to HCs. Cell cycle pathway, small GTPases and molecules in the PKC signaling pathway were identified here for the first time to be potentially involved in the pathogenesis of DMTB comorbidity. Our findings may contribute to the elucidation of novel disease biomarkers, pathogenic mechanisms and therapeutic targets.Current view of the most likely cause of the increased susceptibility and severity of TB in DM patients is that hyperglycemic condition leads to compromised host immune responses and function.Adults with newly diagnosed, bacteriologically confirmed pulmonary TB with and without diabetes were recruited between April 2021 and June 2021 at Shenzhen Center for Chronic Disease Control and Prevention . Patients were classified as having DM if HbA1c was ≥6.5% or plasma glucose levels were ≥200 mg/dL 2 hour post-challenge in the 75 g oral glucose tolerance test. The active TB group included individuals who displayed TB-specific clinical symptoms, had abnormal chest radiography consistent with active TB, and acid-fast bacilli sputum smear positive and/or bacterial culture positive. All patients were either newly diagnosed or under anti-TB or anti-diabetic treatment for no longer than 2 weeks at the time of recruitment. Individuals taking immunosuppressants or with other comorbidities were excluded. Informed consent was obtained from all subjects. This study was approved by the Ethical Committee of Shenzhen Center for Chronic Disease Control and Prevention (NO: SZCCC-2021-030-01-YJ).6 cells/mL in TRIzol reagent (Thermo Fisher) and stored at –80°C until they were used for total RNA extraction.Peripheral blood (10 mL) from each participant was withdrawn from the median ubital vein of the antecubital fossa in Sodium citrate vacutainer tubes . PBMCs were separated by density gradient using Lympholyte Cell Separation Media  within 4 hours of blood withdrawal. The number of viable cells was counted using Automated Cell Counter  by trypan blue staining. The PBMCs from each subject were adjusted to a density of 1–4 × 10Total RNA was extracted from each sample using TRIzol reagent . The isolated RNAs were digested with Dnase I  to remove the residual DNA, and were suspended in 25 μL of DNase/RNase-free water. Total RNA from each sample was quantified and its quality was assessed by Agilent 2100 Bioanalyzer , NanoDrop , and 1% agarose gel electrophoresis. Total RNA (1 μg) with an RNA integrity number above 8 was used for library preparation.RNA libraries for next generation sequencing were constructed with nine patients and three HCs according to the manufacturer’s protocol . The rRNA was removed from the total RNA samples using Ribo-Zero™ rRNA removal Kit (Human/Mouse/Rat) (Illumina) and the remaining RNA was then fragmented and reverse-transcribed. The first strand cDNA was synthesized using ProtoScript II Reverse Transcriptase with random primers and actinomycin D. The second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix (including dACG-TP/dUTP). The double-stranded cDNA was purified by AxyPrep Mag PCR Clean-up (Axygen) and then treated with End Prep Enzyme Mix to repair both the ends. dA-tailing was performed in one reaction, which was followed by T-A ligation to add adaptors to both the ends. Size selection of adaptor-ligated DNA was then performed using AxyPrep Mag PCR Clean-up (Axygen), and ~360 bp fragments (with an approximate insert size of 300 bp) were recovered. Libraries with different indices were multiplexed and loaded on an Illumina HiSeq instrument according to the manufacturer’s instructions (Illumina). The libraries were sequenced on an Illumina HiSeq X Ten platform and 150 bp paired-end reads were generated. About 7G raw reads for each sample were generated. The transcriptome reads have been deposited in the NCBI SRA database with accession numbers PRJNA767116. Transcript assembly was performed using Cufflinks v2.1.1 with the default parameters. All transcripts were compared with gene models in the reference genome to identify novel genes expressed from previously intergenic regions (class code “u”) using Cuffcompare. The gene expression levels in all the samples were calculated using fragments per kilobase of exon model per million mapped reads, and the genes with more than 1 fragments per kilobase of exon model per million mapped reads in at least one sample were used for further analysis. An adjusted P value (FDR < 0.05) and fold change (FC) ratio (|Log2 FC| ≥ 1) were used to determine the DEGs between patients and health control using DESeq R package. Principal component analysis (PCA) was performed using the DESeq package for clustering the samples based on gene expression patterns. The heatmap of DEGs was clustered using pheatmap  package in software (version 3.6.1).To obtain high-quality reads, the reads containing adaptor sequences, more than 10% ambiguous bases (noted as N) and low-quality bases (Qphred ≤20 bases account for more than 50% of the entire read length of the reads) were filtered. The resulting clean reads were then aligned to the human reference genome (GRCh38 genome) using HISAT2 with the default values. (http://metascape.org) online database integrates over forty bioinformatics knowledge bases enabling the extraction of abundant annotations, as well as the identification of enriched pathways and the construction of protein–protein interaction networks from lists of gene and protein identifiers. Using the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) tools within Metascape, we analyzed a genes list containing DEGs genes to further explore the biological process of disease. The GO analysis included three categories: biological process (BP), cellular component, and molecular function. We undertook enrichment analysis of DEGs different comparison groups. Protein–protein interaction (PPI) analysis was carried out to predict the interaction of selected genes based on the STRING Database (V.11). Further, Molecular Complex Detection (MCODE) algorithm was used to check modules of the PPI network. KEGG pathway and GO terms with computed P values ˂ 0.05, were considered significantly enriched based on the hypergeometric distribution.The Metascape The software package implements a method for clustering short time-series expression data that can differentiate between real and random patterns of temporal gene expression changes and assigns each gene to the model profile that most closely matches the temporal gene expression profile for that gene as determined by the correlation coefficient. Mfuzz v.2.46.0 was used to detect different subclustering models of gene expression among the different groups and also incorporates GO enrichment functionality for biological interpretation of time-series gene expression data.Cluster analysis of gene expression data from the different patient was performed using Mfuzz v.2.46.0.t-test. Significance was defined as P < .05. All statistical analysis was performed using R software (version 3.6.1).The differentially expressed mRNAs between HC and the other three groups  were analyzed using the https://www.ncbi.nlm.nih.gov/sra/PRJNA767116).All raw and processed RNA files are available from the NCBI’s Sociological Research in America database . These filtered reads were then aligned to the human genome (GRCh38). This yielded a mean of 47.69 ± 1.2 million filtered reads (94.11%) that uniquely mapped to this human genome; a mean of 1.66 ± 0.19 million reads (3.47%) that mapped to multiple genomic locations. Filtering of the RNA-seq data using 12 samples  produced 17,242 genes suitable for downstream differential expression analysis among them, 13,269 genes were retained for analysis after filtering . Data exploration was carried out using principal components analysis and hierarchical cluster analysis. PCA of quantified genes  revealed that there were significant altered in Disease group  compared with those of the healthy controls  were presented in Figure Differential expression was tested by the pair-wise comparison: DM versus HC, TB versus HC and DMTB versus HC. The gene with significant changes in expression level in the pair-wise comparison , BP and Reactome gene sets were applied using Metascape Fig. A.And in comparison between TB and HC, we performed GO and KEGG analysis on the 845 DEGs. The enrichment results of DEGs that these genes are mainly involved in cellular immunity functions such as myeloid leukocyte activation, leukocyte differentiation and Cytokine Signaling in Immune system. The KEGG pathways that these genes were involved in were NF-kappa B signaling pathway, B cell receptor signaling pathway, and Th17 cell differentiation , of which 952 are only found in DMTB patients  zeta) plays an important regulatory role in the transcriptional response to infection.,42PLA2G6 encodes A2 phospholipase and activation of this enzyme is critically involved in the process of M. tuberculosis infection induced macrophage apoptosis. Specific pathological mechanisms of these molecules in the PKC signaling pathway in DMTB warrant further investigation. Last but not least, the cell cycle pathway was enriched in both cluster 3 and in the 952 DEGs uniquely found in DMTB profiles, implying that abnormal immune cell cycle progression may also underpin the DMTB comorbidity; the specific inflammatory or immunological components affected should be further investigated.Our clustering and enrichment analysis yielded several additional interesting findings. Firstly, “Ras protein signal transduction” was enriched in cluster 2 and “small GTPase signal transduction” was enriched in the DMTB unique DEGs, suggesting that certain small GTPases may play a role in the pathogenesis of the DMTB comorbidity. It has been reported that defects in the signaling of small G-proteins such as Rac1 and Cdc42 can affect insulin secretion,It should be noted that the sample size of our study was small, and confounding factors such as body mass index, and tobacco or alcohol consumption were not included or analyzed. Future studies with larger sample sizes and adjustments for more confounding factors will be needed to validate these findings.Our study investigated the impact of diabetes on the pathogenesis of the DMTB comorbidity through transcriptome sequencing, providing useful information for studying the etiological mechanisms and developing new therapeutics against the menace of coincident diabetes and TB.We would like to gratefully acknowledge TB healthcare Co., Ltd. for the kind technical help.Conceptualization: Wenxu Hong, Tao Liu.Data curation: Jing Gui.Formal analysis: Yu Fu.Funding acquisition: Wenxu Hong.Investigation: Tao Liu, Yaguo Wang.Methodology: Tao Liu, Yaguo Wang.Supervision: Chunli Ye.Validation: Xiangya Hong.Visualization: Ling Chen, Yuhua Li.Writing—original draft: Tao Liu, Yaguo Wang.Writing—review & editing: Wenxu Hong.


Mimicking bioactive conformations of peptide segments involved in the formation of protein-protein interfaces with small molecules is thought to represent a promising strategy for the design of protein-protein interaction (PPI) inhibitors. For compound design, the use of three-dimensional (3D) scaffolds rich in sp3-centers makes it possible to precisely mimic bioactive peptide conformations. Herein, we introduce DeepCubist, a molecular generator for designing peptidomimetics based on 3D scaffolds. Firstly, enumerated 3D scaffolds are superposed on a target peptide conformation to identify a preferred template structure for designing peptidomimetics. Secondly, heteroatoms and unsaturated bonds are introduced into the template via a deep generative model to produce candidate compounds. DeepCubist was applied to design peptidomimetics of exemplary peptide turn, helix, and loop structures in pharmaceutical targets engaging in PPIs. Many coPeptidomimetics are compounds designed to mimic the bioactive conformation of isolated peptides or specific peptide segments in proteins. While peptidomimetic design has a long tradition in drug discovery, it continues to be challenging, depending on the particular target and its ligand binding characteristics. This especially applies to the design of PPI inhibitors that are required to disrupt large protein-protein interfaces because in such cases, binding of a single small molecule must compensate for the free energy gained by large complementary protein surfaces forming many specific interactions.Grossmann proposed four different classes of peptidomimetics: Class A-modified peptides  ; Class B-modified peptides/foldamers (formed by amino acids with varying backbone and side chain iterations); Class C-structural mimetics (synthetic scaffolds with substitution sites corresponding to peptide side chain vectors); and Class D-mechanistic mimetics  . Class CScaffolds with new topology can in principle be obtained by computational enumeration of ring systems. For example, construction of a database called GDB4c containing 916,130 possible ring systems composed of up to four individual rings has been reported, enabling the discovery of new kinase inhibitors with previously unobserved chirality and shape [Herein, we introduce DeepCubist, a molecular generator relying on deep learning for designing peptidomimetics based on previously unobserved 3D scaffolds and report initial proof-of-concept applications. In practice, the best fitting 3D scaffolds can be identified for turns, loops, or helical segments in structures of target proteins of interest and chemically diversified to obtain peptidomimetic candidates for interfering with PPIs. Hence, the DeepCubist approach complements and further extends structure-based design of PPI inhibitors by generating peptidomimetics with varying chemical features.DeepCubist is conceptualized to include two design stages, as illustrated in Fig. tricyclic or tetracyclic bridged ring system consisting of 5- and/or 6-membered rings. This scaffold definition can be modified for different applications depending on the specific requirements. Our definition ensured that scaffold structures could be chemically diversified compared to, for example, bicyclic systems while restricting theoretically possible chemical complexity and hence increasing the likelihood of achieving synthetic accessibility. For our proof-of-concept investigation, so-defined scaffolds consisting of 10 to 14 carbon atoms were then systematically generated as illustrated in Fig. Construction of DeepCubist’s scaffold database began with defining a qualifying 3D scaffold as a 1) Six fused or bridged bicyclic systems consisting of 5- and/or 6-membered rings were computationally constructed as starting points (the number can be varied).m carbon atoms added to any pair of ring atoms. From the resulting tricyclic ring systems, tetracyclic structures were obtained by addition of fragments with n carbon atoms to every atom pair of the tricyclic systems. Hence,  fragment combination were defined to obtain target scaffolds with 10 to 14 carbon atoms, depending on the size of the original bicyclic system.2) Tricyclic ring systems were then exhaustively generated by extensions of bicyclic systems with fragments comprising 1 consisting of eight carbon atoms,  = {, } were used to exhaustively construct scaffolds with 10 carbons. As a result of these operations, a total of 1347 bridged ring systems were obtained at this stage.For example, for bicyclic ring system 3) The generated tri- and tetracyclic candidate structures were then filtered to collect chemically feasible 3D scaffolds with limited strain energy. Scaffold conformers were generated using the “ligand preparation” option of Discovery Studio 2020  and conf3D cyclic skeletons, following the hierarchical scaffold definition of Bemis & Murcko [4) Finally, combinations of three substituents were added to each 3D scaffold, in each case permitting the presence of at most one quaternary carbon for ease of synthesis (the number of substituents can vary). The introduction of substituent combinations resulted in a total of 28,440 unique carbon atom scaffolds with no chiral information. These carbon atom scaffolds can be classified as & Murcko . These stransformer model from natural language processing was selected [attention mechanisms that identify and highly weight the most important representation elements for achieving accurate predictions during the training phase [Once 3D carbon skeletons are generated, they must be converted into chemically meaningful scaffolds. For this purpose, DeepCubist employs a deep generative model based on SMILES strings  as a staselected . Differeng phase . As furttarget (output) structures were extracted from the original ChEMBL and COCONUT compounds by removing all exocyclic atoms from primary ring substituents and replacing removed fragments with a hydrogen atom , as illustrated in Fig. Source (input) structures were then obtained by converting target structures into cyclic skeletons through replacement of all heteroatoms with carbons and conversion of all bond orders to 1 (single bonds), as also illustrated in Fig. Drug- and natural product-like compounds were retrieved from ChEMBL version 30  and COCOFor converting scaffolds into compounds using SMILES-based deep generative models, substitution sites in input structures are often marked as wild-card sites such as “*” to enable chemical diversification –16. FurtPairs of source and target structures were randomly divided into 42,990 training (90%) and 4777 validation set (10%) instances. Following data separation, the SMILES augmentation and alignment steps were carried out. Original SMILES were iteratively augmented with randomized SMILES to obtain a total number of 168,137 pairs for training and 18,624 pairs for validation. A multi-head attention transformer model was constructed using Pytorch . SMILES Scripts for the calculations and the data can be obtained via the following link:https://www.dropbox.com/s/4gdhew9xjit43e4/DeepCubist_Materials.zip?dl=0.7 as input with the model trained for 30 epochs, only nine target structures were found to completely retain the ring systems and the positions of substituents of source structures. In many cases, output structures with different ring sizes were obtained. By contrast, when the model was trained over 30 epochs with augmented and aligned SMILES, structure retention significantly increased. Using the same skeleton 7 as input, 99 of 100 newly generated structures exactly matched the composition and topology of the source structures.Initially, the effect of minimizing the edit distance between source and target SMILES was assessed. For transformer models trained using pairs of canonical SMILES Fig.  A or augThe model was further evaluated using 100 randomly selected skeletons as input. In this case,  sampling of 100 new structures yielded ~ 94% string validity, ~ 99% input structure retention, and 100% output structure novelty Fig.  C.Having confirmed that the trained transformer model could effectively retain input structures during scaffold generation, DeepCubist was used to design exemplary peptidomimetics. To assess the general applicability of the approach, three types of peptide secondary structure were selected as starting points including peptide turns, helices, and loops. For each of the examples discussed below, a consistent number of 100 unique scaffolds was sampled. Furthermore, as an initial assessment of synthetic feasibility, the synthetic accessibility (SA) score according to Ertl & Schuffenhauer  were cal8 was discovered to best reproduce the side chain orientations of the tripeptide in its bioactive conformation with a sum of squared deviations (SSD) value of 0.944 Å2  is a transcription factor that interacts with steroid receptor co-activating factor-1 (SRC-1) via the LxxLL motif presented by an α-helical peptide structure . This Lx10 hat –to our knowledge– has thus far not been considered in drug discovery and design  and Kelch-like-ECH-associated protein 1 (KEAP1) has been identified as an attractive therapeutic strategy for interfering with oxidative stress-related diseases such as cancer, neurodegenerative, cardiovascular, metabolic, or inflammatory diseases . Nrf2 waign Fig. . The SSD3-rich compounds have often more limited synthetic accessibility thancombinations of popular aromatic ring systems. Therefore, future work will primarily concentrate on ensuring a high degree of synthetic feasibility of newly generated scaffolds, for which different methodological avenues such as template- or reaction-based design can be considered.In this study, we have introduced DeepCubist, a molecular generator for the design of peptidomimetics. DeepCubist includes a specialized database of complex sp3-rich skeletons as templates for design and a transformer model trained to convert preferred skeletons into viable 3D scaffolds including heteroatoms and unsaturated bonds. Minimizing the edit distance of input and output SMILES was found to be a simple and effective way to control overfitting and tune the transformer for the construction of chemically meaningful 3D scaffolds retaining input structures. To establish proof-of-concept for DeepCubist’s design capacity, we have reported peptidomimetic designs for different peptide secondary structure motifs in high-profile therapeutic targets. While mostly favorable synthetic accessibility scores are obtained so far for newly generated 3D scaffolds, sp


Primordial germ cells (PGCs) are the germ cell progenitors that arise from the extraembryonic ectoderm, migrate through the hindgut endoderm, and reach the genital ridge before sex determination In mammals, both male and female gonads are derived from the bipotential gonadal primordium, +TAC1−) cell type in the Turner syndrome embryo. Alternatively, if the XO embryo is originated from an XY zygote , the phenotypes would suggest that the genes on Y chromosome are not essential for the development of germ cells and some types of gonadal somatic cells.Recently, Wang et al. In this study, Wang et al. SPARC), a master gene regulating cell migration. According to previous studies, germ cell migration occurs from the hindgut along the gut and across to dorsal mesentery to reach gonads at around 4–5 W in humans, thus most of the germ cells have already completed the migration process at around 5 W. However, the SPARC+ germ cells can be found in the gonads of human fetuses up to 23 W, suggesting that after entering the gonad, some of the germ cells may still maintain the migration potential and could migrate within the gonad to optimize their spatial distributions there.In addition, Wang et al. ALDH1A2 [a gene encoding a retinoic acid (RA)-synthesizing enzyme] in male germ cells at relatively late developmental stage (15 W). Further studies have demonstrated that the BMP signaling pathway plays vital roles in the gonocyte-to-spermatogonium transition (GST) process.Wang et al. ALDH1A3 (a gene encoding a RA-synthesizing enzyme) in all testicular somatic cell types, Sertoli cells, Leydig cells, and keratin 19-positive (KRT19+) cells, at both the early (7 W) and late (15 W) developmental stages. Notably, different gonadal somatic cells exhibit different responses to BMP inhibition. BMP and RA signaling pathways play critical roles in regulating the meiosis of germ cells This study has also revealed the functional crosstalk between the BMP signaling pathway and RA signaling pathway in male gonadal somatic cells. BMP signaling activity inhibits the expression of + cells in early developmental stages of male and female embryos may further differentiate into Leydig cells in testes and theca cells in ovaries. DLK1 was continually expressed in Leydig cells of the testes, while its expression disappeared in theca-like cells of ovaries. The origin of steroidogenic cells is a controversial question with a lot of debates in the literature. The results indicate that DLK1+ cells are probably a new type of progenitors of steroidogenic cells. They have also proposed that TAC1+ cells at 7 W may further differentiate into granulosa cells in later development stages. However, these conclusions need to be validated by lineage tracing experiments in other animal models.Furthermore, Wang et al. HSD17B3 during fetal and neonatal periods. Leydig cells do not express HSD17B3 until neonatal stages and the ratio of cells that express HSD17B3 gradually increases afterward. These results indicate that the androstenedione is produced by Sertoli cells, but not by fetal Leydig cells during fetal stages, which is consistent with the findings in mouse model Finally, Wang et al. + cell population is a progenitor population of the steroidogenic cell lineage and the TAC1+ cell population is a progenitor population of granulosa cells (Together, Wang et al. sa cells . NotablyMin Chen: Writing ‐ original draft, Writing ‐ review & editing. Fei Gao: Funding acquisition, Writing ‐ original draft, Writing ‐ review & editing. Both authors have read and approved the final manuscript.The authors have declared no competing interests.


The recent development of modular origami structures has ushered in an era for active metamaterials with multiple degrees of freedom (multi-DOF). Notably, no systematic inverse design approach for 3D curvilinear modular origami structures has been reported. Moreover, very few modular origami topologies have been studied to design active metamaterials with multi-DOF. Herein, we develop an inverse design method for constructing 3D reconfigurable architected structures — we synthesize modular origami structures whose unit cells can be volumetrically mapped into a prescribed 3D curvilinear shape followed by volumetric shrinkage to construct modules. After modification of the tubular geometry, we search through all the possible geometric and topological combinations of the modular origami structures to attain the target mobility using a topological reconstruction of modules. Our inverse design using geometric and topological reconstructions can provide an effective solution to construct 3D curvilinear reconfigurable structures with multi-DOF. Our work opens a path toward 3D reconfigurable systems based on volumetric inverse design, such as 3D active metamaterials and 3D morphing devices for automotive, aerospace, and biomedical engineering applications. Systematic inverse design for 3D curvilinear modular origami structures has not yet been reported. Here, the authors develop an inverse design method for 3D reconfigurable architected structures by geometric and topological reconstruction. Researchers are even pushing the limit of complex 3D structural design to motion structures that can change their shapes from one state to another, tuning their physical properties in adapted and active ways to varying physical environments23.With the advancement of conventional manufacturing in both additive24, given the potential of future intelligent reconfigurable structures. Starting from 2D flexible structures using a Miura-ori sheet25, Waterbomb base26, or Ron-Resch pattern27, researchers have recently explored the design of 3D architected structures using modular origami. Stacking multiple origami sheets29 and assembling foldable modules33 are the currently available methods to construct 3D architected structures. Stacked Miura-ori28 and Tachi–Miura polyhedron30 are deployable with flat-foldability, with a single origami sheet serving as the building block. Assembled structures with folding modules using prismatic polyhedrons33, tubular bellows35, and kirigami32 have demonstrated the potential of isotropic design using spatial symmetry while displaying single and multiple degrees of freedom (DOFs)33.Many groups have employed origami and kirigami techniques, the ancient folding and cutting artsAll these methods require the construction of a building block (unit cell) with crease patterns and spatial periodicity (tessellation). This bottom-up design approach is convenient for controlling the kinematic and kinetic properties of the overall 3D architected structures with only a unit-cell design. However, this bottom-up design approach has a critical limitation when constructing practical engineering and artistic structures, the shapes of which are mostly 3D curvilinear . Such structures require that the size and shape of the building block no longer be homogeneous in the design domain. Most rectangular or cubic engineering structures have curved edges for minimum stress concentration and safety. Because of the break in spatial periodicity at the boundary of curvilinear geometries, the kinematic and kinetic properties of the unit cell tend to differ from those of the tessellated structure, limiting the structural implementation of the architected origami materials.38. Very few studies have explored the inverse design of 3D architected structures39. The existing inverse-design methods can only be applied to 2D curvilinear surfaces with origami and kirigami47 and not to volumetric 3D spatial curvilinear geometries and their reconfigurability.The top-down approach to the design, often called ‘inverse design,’ of mechanical metamaterials has been explored to identify tessellated microstructures and target physical properties such as anisotropic stiffness. Topology optimization has been a typical approach to identify microstructuresIn this work, we explore an inverse design for 3D reconfigurable architected origami materials. Without tessellating a constant building block, our method produces volumetric gradient cells mapped into complex curvilinear 3D geometries, followed by topological reconstruction of modules. We develop a top-down approach making unit cells map into a sphere, hyperboloid, cone, twisted cylinder, torus, and any combined curvilinear shapes with reconfigurability.16, we implement the synthesis principle to 3D curvilinear shapes with nonperiodic tiling of irregular polyhedrons. Figure 48 for a target number of tessellations, e.g., Inspired by the pioneering work on the space-filling tessellation to construct 3D modular origami with prismatic tubes16, where the connecting direction was normal to the regular polyhedrons; instead, we determine the connecting direction by bridging the centroids of the deformed polyhedrons in the template. We obtain the length of the connection Next, we spatially shrink the deformed polyhedrons in the template while implementing a scaling constraint, as illustrated in Fig. Next, we connect the shrunken polyhedrons by extruding prismatic tubes on each shared face inside the template, as shown in Fig. mentclass2pt{minimWe can build other complex nonperiodic modular origami structures using the geometric reconstruction with the volumetric mapping into an arbitrary target shape and volumetric shrinkage of deformed polyhedrons, as illustrated in Fig. 49. Note that a module denotes a single origami unit constructed by the extruded tubes from the shrunken polyhedrons after geometric reconstruction, as illustrated in Fig. Although the geometric reconstruction generates arbitrary 3D curvilinear structures through volumetric mapping and volumetric shrinkage of unit cells, the constructed origami structures hardly produce reconfigurability. From a macroscopic perspective, the fully connected origami modules with fully extruded tubes produce spatial loops, constraining the motion of the modulesTo release the microscopic immobility, extruded tubes generated by the geometric reconstruction in Fig. The objective function in Eq.  minimizeentclass1pt{minimaentclass1pt{minimaEquation  adjusts Equation  constraientclass1pt{minimaFigure After the geometric modification, we search through all the possible geometric and topological combinations of each module, applying the graph theory in the assembly. A graph consists of vertices and edges representing modules and tubular connections, as shown in Fig. entclass1pt{minimaFigure Depending on the topologically reconstructed modules selected, e.g., Fig. entclass1pt{minimaFigure 50, where We implement transformation by obtaining the independent dihedral angles, as demonstrated in Figs. By calculating the reduced row echelon form of 52, we obtain sphere reconfigurable structures for various target DOFs. Our algorithm provides multiple solutions, e.g., multiple combinations of geometry and topology for a target Figure 16.We plot the DOFs with varying connecting topology while demonstrating various deformation modes, including uniaxial extension and volumetric shrinkage, as shown in Fig. The same unit cell, e.g., a cubic unit cell, provides different reconfigurability depending on the variation of the topological connections, which is controlled by the mapping size. Small-sized polyhedrons with a 53, which is convenient for structural design. However, this bottom-up approach has a critical limitation when constructing curvilinear 3D engineering and artistic structures, where the size and shape of the building blocks are no longer homogeneous in the design domain. The reconstruction of geometry and topology in this study provides a solution for designing non-periodic and curvilinear 3D structures and their reconfigurability. The geometric reconstruction via volumetric mapping of polyhedrons followed by volumetric shrinkage can be used to construct a stress-free 3D curvilinear structure. The fully connected irregular modules produce spatial loops and constrain inter-and intra-modular motions, delivering a rigid yet stiff curvilinear structure. The topology reconstruction after geometric modification can provide solutions for an inverse design of reconfigurable structures with multi-DOF. The reconfigurable motion is controlled by the topology of the spatial connection of modules, not by the geometry of the templates. For advanced reconfigurability, one may need an optimization of shape transformation, which is beyond the scope of this work but can be explored near future.3D curvilinear structures are ubiquitous in automotive, aerospace, and ocean engineering structures. To date, the design of architected materials has relied on the bottom-up design approach, using planar and orthogonal periodic tessellation of modules46. Because of the instability  of the out-of-plane deformation during reconfiguration, relatively high local energy is needed for the transformation40. The bottom-up approach with periodic modular origami structures can construct a curved shape by filling building blocks inside the curvilinear space39. However, the bottom-up approach cannot yield a smooth surface; it only provides discrete curves, whereas the geometric reconstruction in this work can produce a smooth 3D curvilinear structure. Notably, our inverse design of reconfigurability produces multiple solutions depending on the loop topology and geometry of modules.The inverse design of 2D origami structures can generate a curvilinear surface with local control of the variable curvature7 and micro-stereolithography11 can be applied to our reconfiguration systems on the microscale, e.g., for the design of battery electrodes54 and microelectronic mechanical systems55.Our top-down approach can expand the design space of modular origami to 3D non-periodic structures and 3D curvilinear geometries — spheres, hyperboloids, cones, twisted cylinders, and toruses, overcoming the limitation of planar and spatial tessellations for periodic structural design. Unlike 2D reconfigurable curvilinear origami structures, our 3D motion structures can be reconfigured into other shapes with multi-DOF and are frustration-free. This work will advance the design of 3D architected materials with reconfigurability and disrupt traditional periodic-tessellation-based 3D metamaterial design. The volumetric shrinkage of space-filled polyhedrons can be applied for both orthogonal and curvilinear coordinates, meaning that our inverse design method is universal and can be applied to both periodic and non-periodic structures. Our physical prototypes of 3D curvilinear structures fabricated using additive manufacturing validate the tunable motion, demonstrating untethered actuation by embedded hard magnets and moving external magnetic fields We prototype a rigid model using multi-material inkjet 3D printing  and a MATLAB script to build an origami mesh model with a prescribed wall thickness. We fabricate reconfigurable prototypes with an assembly of paperboards (Silhouette Cameo) and additive manufacturing by stereolithography (SLA) by Form 3 (Formlabs). We print modular origamis and selectively embedded permanent magnets (NdFeB) to demonstrate a reconfigurable structure with remote control. We apply a rotational uniform magnetic field by a Halbach array composed of a circumferential array of permanent magnets. To validate our numerical simulation of reconfigurability with experiments, we fabricate a reconfigurable structure and use a 3D scanner (Einscan Pro) to capture the transformed shapes. We show the quantitative comparison of the independent dihedral angles of the reconfigurable structure between numerical simulation and 3D scanned measurement in Supplementary Note Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5


CLas inhibits callose deposition in the sieve pores and the accumulation of reactive oxygen species to favor its cell-to-cell movement. Dear Editor,Candidatus Liberibacter asiaticus (CLas). The study of CLas–phloem interaction has been hampered because of the low and unequal distribution of CLas inside trees, and the difficulty in isolating vasculature from trees. Previous studies reached the conclusion that infection increases sieve pores callose levels and the production of H2O2, blocking the phloem and causing a toxic build-up leading to programmed death  and “Duncan” grapefruit (Citrus paradisi) seeds, which highly accumulate CLas  is a severe disease in citrus that is associated with ed death , but eveate CLas . We showC. sinensis CalS and phloem protein 2 (CsPP2) genes increased, and the phloem pore opening decreased, but these sieve elements (SEs) had very low levels of CLas  and callose levels were lower  in leaves and seed vasculatures from healthy and infected samples  indicate that CLas inhibits H2O2 production inside cells.Previous research demonstrated that reactive oxygen species (ROS) were also elevated in d leaves . We meas samples . Infecteles .kiac346_Supplementary_DataClick here for additional data file.


Scientific Reports 10.1038/s41598-023-27379-9, published online 11 January 2023Correction to: The original version of this Article contained errors in the Abstract.“To evaluate the prevalence of adverse pregnancy outcomes in TA. Searches were conducted on PubMed, Cochrane Library, Scopus and Cinahl databases from inception to 25 May 2022.”now reads:“The aim of this study was to evaluate the prevalence of adverse pregnancy outcomes in TA. Searches were conducted on PubMed, Cochrane Library, Scopus and Cinahl databases from inception to 25 May 2022.”Additionally,“Trial registration: There was no registration for this systematic review. The aim of this study was to evaluate etc in order to be correct by syntax.”now reads:“Trial registration: There was no registration for this systematic review.”The original Article has been corrected.


Ultrasmallnanosized silicate grains are likely to be highly abundantin the interstellar medium. From sporadically absorbing energy fromultraviolet photons, these nanosilicates are subjected to significantinstantaneous temperature fluctuations. These stochastically heatednanograins subsequently emit in the infrared. Previous estimates ofthe extent of the heating and emission have relied on empirical fitsto bulk silicate heat capacities. The heat capacity of a system dependson the range of available vibrational modes, which for nanosized solidsis dramatically affected by the constraints of finite size. Althoughexperimental vibrational spectra of nanosilicates is not yet available,we directly take these finite size effects into account by using accuratevibrational spectra of low-energy nanosilicate structures from quantumchemical density functional theory calculations. Our results indicatethat the heat capacities of ultrasmall nanosilicates are smaller thanpreviously estimated, which would lead to a higher temperature andmore intense infrared emission during stochastic heating. Specifically,we find that stochastically heated grains ultrasmall nanosilicatescould be up to 35–80 K hotter than previously predicted. Ourresults could help to improve the understanding of infrared emissionfrom ultrasmall nanosilicates in the ISM, which could be observedby the James Webb Space Telescope. However, the numberof dust grains with respect to size approximately follows an inversepower law (ng(r) = cr–3.5),2 whichmassively favors higher populations of smaller grains. It has longbeen realized that the low time-averaged temperature of very smallgrains in the ISM would not capture the large temperature fluctuationsdue to transient heating of such species when interacting with theradiation field of the ISM.3 The firstcalculations of this effect showed that dust grains with diametersof only a few nanometres are particularly susceptible to sporadictemperature spikes.4 This effect, now commonlyreferred to as stochastic heating, involves the relatively infrequentabsorption of energy from ultraviolet (UV) photons, which leads toabove average grain temperatures and a subsequently rapid readjustmentto ambient ISM temperatures via emission in the infrared (IR). Stochasticheating is now appreciated to be an important mechanism for generatingIR emission in a number of astrophysical environments. The temperatureand emissivity of stochastically heated nanograins on the rates ofUV energy absorption and subsequent IR emission, which, in turn, isintimately linked to their specific heat capacity.6 Mostmodern calculations of stochastic heating follow the approach by Draineand Li, which used a statistical mechanical treatment.5 In this work the temperature dependent heat capacity, C(T), of a nanosized dust grain is directlycalculated from its distribution of available vibrational modes (forinducing temperature increase) with respect to their frequency . We follow this approach and show how it can be enhanced forthe smallest grain sizes by using vibrational modes evaluated fromdirect and accurate quantum chemical calculations using atomisticallydetailed stable nanograin structures.Dust particles areprevalent throughout the interstellar medium(ISM), where they reveal themselves through their interaction withelectromagnetic radiation.3) or olivine (Mg2SiO4) composition.7 Considering observationsof IR emission in the ISM, it has been estimated that ultrasmall nanosilicates(r ≤ 1.5 nm) could be highly abundant andaccount for ∼10% of all silicon in the dust population.8 Nanosilicates can be formed from the processingof larger grains in the ISM,9 where theyare a likely source of the anomalous microwave emission.12 and can assist in the formation of H214 and ices.15 With respect to the latter,studies have shown that stochastically heated silicate grains with r ≈ 0.005 μm (5 nm) exhibit temperature fluctuationsup to 20–40 K, which is predicted to have a significant impacton their role in astrochemical processes.17 For even smaller nanosilicates possessing only a few 10s of atoms(approximately with r ≤ 0.5 nm), the fluctuationsin temperature from stochastic heating have been estimated to be upto ∼1000 K5 with, as yet, unknownconsequences for astrochemistry.Herein, we consider silicatedust, which is observed in a widerange of astronomical environments and typically has a magnesium-richpyroxene  has since been employed in other studiesof stochastic heating of small silicate dust grains.17 For nanosilicates, their extreme small size and resultant non-bulk-likeand noncrystalline structures leads to their vibrational spectra beingquite unlike those of bulk silicates.18 Thus, although the general BFM approach represents a solid microscopicframework for calculating heat capacities of small silicate dust grains,the original implementation has a few shortcomings: (1) The referencebulk materials  are not compositionallygood matches for the composition of Mg-rich silicate dust grains inthe ISM; (2) The fit of the spectrum to the bulk C(T) versus T curves is purely empiricaland does not consider the direct physical origin of the vibrationalmodes in silicate dust grains; (3) The fit is with respect to themeasured C(T) of bulk samples . Herein, we address all these three issues usingan accurate quantum chemical approach in which we directly calculatethe vibrational modes in atomically detailed and energetically stablenanosilicates having Mg-rich olivinic and pyroxenic compositions.As heat capacities of nanosilicates are not yet experimentally available,our approach provides a direct and accurate alternative to relyingon empirical fits to bulk heat capacity data. Our resulting calculatedheat capacities and energy-dependent temperatures of ultrasmall nanosilicategrains are compared with those derived from the BFM approximation.In ref  the heat18 Therein,we confirmed that the IR vibrational spectra of these species, accuratelyobtained using quantum chemical density functional theory (DFT) basedcalculations, are quite distinct from those of bulk silicates. Herein,we use DFT-based calculations to obtain accurate full vibrationaldistributions of selected nanosilicate grains and use this data todirectly derive temperature-dependent heat capacities. Our resultsshow that BFM estimates of the distribution of vibrational modes tendto overestimate the heat capacity of nanosilicate grains, especiallyat temperatures around 50–300 K. As a result, for a typicalrange of energies for photon absorption, our refined heat capacityderivations predict that ultrasmall nanosilicates in the ISM couldbe heated by 35–80 K more than previously expected.In previous work, we derived the atomistically detailed structuresof low energy nanosilicate clusters for astronomically relevant Mg-richpyroxene and olivine compositions.19 Our results should allow formore accurate predictions of the IR emission characteristics of stochasticallyheated nanosilicate grains and could thus be important for accuratelyinterpreting upcoming JWST observations of the ISM.Untilnow it has been impossible to directly confirm the presenceof ultrasmall nanosilicates in the ISM. However, the James Webb SpaceTelescope (JWST) now has highly sensitive instruments covering anideal range of wavelengths to observe nanosilicate IR emission inthe ISM.20 In previous studies it has been shown that thisbasis set has a quality comparable to valence triple-ζ plusa polarization Gaussian-type orbital basis set.22 The PBE023 exchange-correlation functionalwas used in all the calculations. Previous works have shown that thisfunctional is able to accurately reproduce the IR spectra of bulkcrystalline olivine24 (forsterite) andpyroxene25 (enstatite), and the IR spectraof both pyroxene and olivine monomers.26Optimized nanosilicate structures and their harmonicvibrationalspectra were obtained using DFT calculations with an all-electronlight-tier 1 numerical atom-centered orbital basis set employing theFritz Haber Institute ab initio molecular simulations package (FHI-AIMS).T, and use standard statistical mechanics27 to obtain the average energy, E̅(T), fromN is the number ofvibrational frequencies of the grain, νj are the vibrational frequencies, and kB is the Boltzmann constant. By differentiatingthe expression for average energy in 2 with10 atoms) and olivine 2 with 14 atoms), (b) 35-atom nanograins of pyroxene 7) and olivine 5), and (c) 70-atom nanograins of pyroxene 14) and olivine 10). The structures of all nanosilicate clusterswere found by dedicated global optimization searches following theapproach described in our previous work.18 Specifically, for the 35-atom and 70-atom nanosilicates, we usedthe lowest energy structure from these searches. For the pyroxeneand olivine dimer species, we employed both the previously reportedglobal minima and also the next two higher energy isomers in eachcase. We note that the low energy 70-atom pyroxene structure and themetastable dimer species were not reported previously, and their structuresare provided in the Supporting Information.In this work we study three sizes of ultrasmall silicate grains:(a) dimers of pyroxene  with respectto our more realistic model, in which the vibrations are more evenlydistributed throughout the whole frequency range. This effect is observedin both pyroxene and olivine dimers, but is more noticeable in thecase of pyroxene. In both cases, around 66% of the vibrational modesare found between 0 and 400 cm–1 in the BFM spectra.However, in our directly calculated spectra, we do not reach 66% ofthe modes until a frequency of ∼700 cm–1.This difference can be more explicitly seen by summing the numberof frequencies found in regular bins of 100 cm–1 and comparing the number of frequencies in each bin for each spectrum.From the resulting histograms  can induce small conformational structural changes in such ultrasmallsilicate species.13 Higher temperatures will leadto a more significant structural isomerization and eventually totalmelting. To estimate the effect of isomerization, we compare the vibrationalspectra of the global minima silicate dimers , our calculated spectra, for allnanosilicatesizes, and the BFM spectra match quite well. Olivine species showa tendency to have slightly more modes at lower frequencies (approximately<700 cm–1) than the pyroxene species of the samesize and vice versa at higher frequencies. However, both pyroxeneand olivine nanosilicates have a significantly lower number of modesin this frequency range than the BFM spectra.From –1, as used in the BFM spectra. It is of note, however, that for thelargest size of 70 atoms (r ≈ 0.5/0.6 nm),we observe some indications of an emerging change in slope of thecumulative vibrational spectra near to 700 cm–1 (12.5μm). We speculate that this may be linked to the emergence ofthe separate 10 and 18 μm IR vibrational bands observed in largersilicate dust particles.7Generally, our calculatedvibrational mode spectra for all pyroxeneand olivine nanosilicates show a relatively smooth monotonic increasein the number of modes with increasing frequency. In particular, noneof our nanosilicates display a clear two-phase pattern to their cumulativevibrational spectra, with an abrupt change at ∼300 cm26 Assuminga relatively large anharmonic downshift of all our harmonic frequenciesby 5%, we find that our final results regarding the stochastic heatingare essentially unchanged (see SI for details).Finally,we note that all our vibrational spectra are derived usingthe harmonic approximation. Any vibrational modes exhibiting anharmonicitytend to shift to slightly lower frequencies (with respect to purelyharmonic modes) with increasing temperature. We have previously foundthat this effect is small in nanosilicates and only becomes apparentabove at approximately 400 K.C(T), of a grain can be derived from the spectrumof its vibrational modes as described above (see C(T) values. As this vibrational differenceis especially notable in the low frequency region, we expect thatthe corresponding largest C(T) differenceswould be at lower temperatures. In C(T) curves derived from the vibrational spectra shown in C(T) curves for 35-atom and 70-atom nanosilicates, where we also observethe corresponding differences with the BFM as found in the case ofthe dimers.The heat capacity, bove see 2. As we The differences between the calculated specific heat capacitiesin 6 Our resultsshow that the BFM vibrational spectra lead to overestimates of C(T) with respect to heat capacities derivedfrom accurate directly computed vibrational spectra for ultrasmallsilicates. For such species, the immediate temperature rise inducedby absorbing a UV photon in the ISM can be calculated using 26The heat capacity plays an important role incalculating how theseultrasmall silicates absorb and emit radiation in the ISM and thushow they are affected by stochastic heating.4 cm–1/∼1.2 eV). In sucha scenario, our results show that BFM-based calculations underestimatethe photon-induced instantaneous temperature increase by 50–80K  approximations to the BFM-derived C(T).17 In C(T) data with the low temperature BFM-likefitted C(T) used in refs  for nanopyroxenes.Perhaps surprisingly, however, this fitted expression appears to holdfor very low temperature stochastic heating of even ultrasmall olivinenanosilicates.Finally, we note that for larger nanograinswith radii of a fewnanometers, stochastic heating is predicted to produce relativelymilder heating of a few 10s of Kelvin. in refs  and 17 fin refs  and 17 rC(T) curves for bulk silicates(BFM), for example, we find significant differences, especially atlow frequencies (≤700 cm–1). Using our abinitio vibrational spectra, we also compute heat capacities of a rangeof nanosilicates from standard statistical mechanics. Generally, our C(T) values are found to be lower thanthose derived using the BFM, especially around 150 K. These differencesimply that the instantaneous photon-induced temperatures of relativelyhot stochastically heated small nanograins in the ISM would be 35–80K higher than previously expected. Our accurately derived C(T) values could also have implicationsfor relatively low-temperature stochastic heating of larger nanosilicategrains. Generally, our study shows the power of an accurate bottom-upquantum chemical approach for directly calculating the propertiesof nanoscale dust grains. Our results may provide useful insightsfor improving the modeling of the IR emission of the likely high populationof ultrasmall nanosilicates in the ISM, which hopefully will be confirmedby upcoming JWST observations.We calculate accuratevibrational spectra for ultrasmall nanosilicates(10–70 atoms) with stable low energy structures and with bothMg-rich pyroxene and olivine stoichiometries using quantum chemicalDFT calculations. Such spectra are known to be significantly affectedby the extreme small size of these species and quite distinct fromthe vibrational spectra of bulk silicates. Comparing our data withvibrational mode spectra that were empirically fitted to reproducebulk-like


Onasemnogene abeparvovec-related DRG findings were not associated with electrophysiology changes and were not ameliorated by prednisolone or rituximab plus everolimus coadministration. The pathogenesis is possibly a consequence of increased vector genome transduction and/or transgene expression.Intravenous onasemnogene abeparvovec is approved for the treatment of spinal muscular atrophy in children < 2 years. For later-onset patients, intrathecal onasemnogene abeparvovec may be advantageous over intravenous administration. Recently, microscopic dorsal root ganglion (DRG) changes were observed in nonhuman primates (NHPs) following intrathecal onasemnogene abeparvovec administration. To characterize these DRG findings, two NHP studies evaluating intrathecal onasemnogene abeparvovec administration were conducted: a 12-month study with a 6-week interim cohort and a 13-week study with a 2-week interim cohort. The latter investigated the potential impact of prednisolone or rituximab plus everolimus on DRG toxicity. An additional 6-month, single-dose, intravenous NHP study conducted in parallel evaluated onasemnogene abeparvovec safety (including DRG toxicity) with or without prednisolone coadministration. Intrathecal onasemnogene abeparvovec administration was well tolerated and not associated with clinical observations. Microscopic onasemnogene abeparvovec-related changes were observed in the DRG and trigeminal ganglion (TG) and included mononuclear cell inflammation and/or neuronal degeneration, which was colocalized with high vector transcript expression at 6 weeks postdose. Incidence and severity of DRG changes were generally decreased after 52 weeks compared with 6 weeks postdose. Other onasemnogene abeparvovec-related microscopic findings of axonal degeneration, mononuclear cell infiltrates and/or gliosis in the spinal cord, dorsal spinal nerve root/spinal nerves, and/or peripheral nerves were absent or found at decreased incidences and/or severities after 52 weeks. DRG and/or TG microscopic findings following intravenous onasemnogene abeparvovec dosing included minimal to slight neuronal degeneration and mononuclear cell inflammation at 6 weeks and 6 months postdose. Nervous system microscopic findings following intrathecal onasemnogene abeparvovec (≥1.2 × 10 SMA is an autosomal recessive, neurodegenerative disease resulting from biallelic SMN1 gene mutations that causes a survival motor neuron (SMN) protein deficiency, leading to motor neuron degeneration and early mortality.1 SMA is classified into four phenotypes based on age of onset and greatest motor function achieved.Onasemnogene abeparvovec is an approved one-time intravenous infusion therapy indicated for the treatment of patients with 5q spinal muscular atrophy (SMA) with a biallelic mutation in the 3 SMA type 1 patients present symptomatically within the first 6 months of life, never attain independent sitting, and die or require permanent ventilation before 2 years of age, if untreated.4–6 SMA type 2 manifests within 18 months of age, and patients are able to sit unassisted but never walk independently. These patients experience significant cumulative morbidity related to worsening contractures, spinal curvature, muscle atrophy, and slow but progressive motor function loss over time.7 SMA type 2 patients have a life expectancy of 20–40 years. SMA type 3 patients attain the ability to walk unaided. SMA type 4 is an adult-onset form.Type 0 represents severe, antenatal-onset SMA, which is not compatible with life.8–10 Furthermore, intravenous or intrathecal delivery of functional SMN via AAV gene therapy can abrogate the SMA phenotype in mouse models of disease.11–14Nonclinical studies have consistently demonstrated that intravenous or intrathecal administration of self-complementary adeno-associated virus serotype 9 (scAAV9)-SMN or scAAV9–green fluorescent protein targets spinal motor neurons.SMN gene. The SMN gene is under the control of the cytomegalovirus enhancer/chicken-β-actin hybrid promoter as well as two AAV inverted terminal repeats derived from AAV2. Nonclinical studies, predominantly conducted in murine species, provided the foundation for the development of onasemnogene abeparvovec, the first approved systemic gene therapy administered as a single dose via intravenous infusion. Intravenous onasemnogene abeparvovec in patients is well tolerated, with an adverse event profile that is monitorable and largely manageable.15Onasemnogene abeparvovec is a recombinant biological product that comprised a nonreplicating, recombinant, scAAV9 capsid shell containing a functional copy of the human e.g., liver) and may result in an improved systemic safety profile.16The intrathecal route of onasemnogene abeparvovec administration may be the preferred approach for treatment of later-onset SMA patients. This approach reduces the dose in the central nervous system (CNS) by ∼10-fold relative to the approved intravenous dose and results in onasemnogene abeparvovec distribution throughout the spinal cord. In addition, intrathecal administration reduces the viral vector distribution in non-CNS tissues  partial clinical hold for clinical trials of intrathecal onasemnogene abeparvovec administration.In a preliminary 2-week biodistribution study with cynomolgus monkeys (nonhuman primates [NHPs]) to support the development of intrathecal onasemnogene abeparvovec administration, an unexpected change was observed in the dorsal root ganglion (DRG) of onasemnogene abeparvovec–treated NHPs using a dose of 3 × 1015 These findings were asymptomatic and unassociated with in vivo clinical observations.15Results from this NHP study demonstrated microscopic changes in the DRG, including mononuclear cell inflammatory infiltrate that was occasionally accompanied by neuronal satellitosis or neuronal necrosis at mild to marked severity.15 were not sufficient to characterize the progression of the observed DRG toxicity and evaluate long-term safety of intrathecally administered onasemnogene abeparvovec. Therefore, to characterize the finding further and investigate potential underlying mechanisms causing DRG changes, two nonclinical toxicity studies were conducted in NHPs administered onasemnogene abeparvovec by the intrathecal route of administration. The first intrathecal study was a 12-month, single-dose, good laboratory practice (GLP)‒compliant toxicity study with a 6-week interim cohort at dose concentrations of 1.2 × 1013, 3.0 × 1013, or 6.0 × 1013 vg/animal to explore dose response, characterize microscopic DRG findings, and assess the extent of any related or secondary microscopic findings in the nervous system of NHPs.Data from this 2-week study13 vg/animal. This study investigated whether the microscopic DRG findings following intrathecal onasemnogene abeparvovec administration could be prevented or mitigated by coadministration of prednisolone or rituximab plus everolimus.The doses selected for this study were based on the anticipated clinical dose. The low dose was the anticipated clinical dose, and the mid and high doses were 2.5 × and 5 × the anticipated clinical dose given the narrow safety margin of gene therapy products. This study also evaluated safety of intrathecal administration, assessing the persistence or reversibility of any effects as well as the progression or resolution of any nervous system findings. The second study was a 13-week, single-dose, mechanistic, non-GLP toxicity study with a 2-week interim cohort at a dose of 3.0 × 1017–22 The dosages and dose regimens were based on our internal experience and use in monkeys, published literature, and/or consultation with clinicians. In addition, the selected doses were intended to represent feasible anti-inflammatory and immunosuppressant doses, which would not result in adverse toxicity but would result in pharmacodynamic responses relevant to assessing whether immune suppression has any effect on DRG toxicity.23–29These pharmacologic agents were selected for their anti-inflammatory  and/or immunosuppressive (reduction of B and T cell expansion and/or depletion) properties in NHPs at dose concentrations that would not lead to adverse effects.After initiation of the 12-month intrathecal study, a 6-month, single-dose, intravenous toxicity study with a 6-week interim cohort was conducted in parallel to the intrathecal NHP studies to evaluate the safety (including DRG toxicity) of intravenous onasemnogene abeparvovec with or without coadministration of prednisolone in juvenile NHPs following additional feedback from the US FDA.The present publication focuses on microscopic findings in the sensory ganglia  following intrathecal onasemnogene abeparvovec administration in NHPs and related biomarkers and study investigations, including molecular localization and electrophysiology (nerve conduction velocity) assessments. Information regarding intravenous dosing in NHPs, including limited microscopic sensory ganglia findings, is also provided. Immunogenicity (anti-AAV9 antibody) and biodistribution assessments by ddPCR were included in the studies but are outside the scope of this publication and will be addressed in separate publications. However, limited data related to these assessments are included to aid in the interpretation of certain immunogenicity and biodistribution data.Macaca fascicularis) were acclimated to the test facility for up to 10 weeks before dosing. For the 12-month intrathecal and 6-month intravenous studies, animals were 13–19 months old . NHPs were 25–34 months old  for the 13-week mechanistic study.Cynomolgus monkeys . Monkeys were screened for the presence of serum total anti-AAV9 antibodies using an enzyme-linked immunosorbent assay method (data not shown). Monkeys with the lowest anti-AAV9 antibody response were selected for inclusion in the study. Because of the endemic presence of anti-AAV9 antibodies in cynomolgus monkeys and the current shortage of monkeys for use in biomedical research, selecting only seronegative monkeys was not possible.To include an appropriate number of monkeys in the study, the monkeys with highest anti-AAV9 antibody response(s) at screening were assigned to the control group. All groups of animals that were administered onasemnogene abeparvovec had a similar range of anti-AAV9 antibody responses as measured by an electrochemiluminescence immunoassay in samples collected immediately before dose administration (data not shown).n = 5/sex/group; n = 3/sex/group and n = 2/sex/group for interim and terminal euthanasia, respectively) of Asian origin were administered a single dose of 1.2 × 1013, 3.0 × 1013, or 6.0 × 1013 vg/animal onasemnogene abeparvovec  on day 1 of the dosing phase.In the 12-month study, three groups of NHPs (n = 5/sex) was administered vehicle control article. The test or control article was administered at a dose volume of 0.80 mL, preceded by 0.20 mL Omnipaque™ 180 contrast agent. The study included an interim (6 weeks postdose) and a terminal evaluation .A control group of NHPs  of NHPs of Mauritius origin were administered a single dose of 3 × 1013 vg/animal onasemnogene abeparvovec via slow push intrathecal (lumbar) bolus injection on day 1 (see n = 5/sex) was administered vehicle control article by intrathecal lumbar puncture on day 1. The test or control article was administered at a dose volume of 0.80 mL, preceded by 0.20 mL Omnipaque 180 contrast agent. Two different anti-inflammatory or immunosuppressive regimens were evaluated: oral prednisolone or a combination of intravenous rituximab and oral everolimus  was administered separately and immediately before the test article (onasemnogene abeparvovec) to be consistent with the clinical trial procedure to confirm proper placement in the intrathecal space .n = 6/sex/group; n = 3/sex/group; and n = 3/sex/group for interim and terminal euthanasia, respectively), DRG was evaluated microscopically as part of a complete nonclinical toxicity study after 6 weeks or 6 months of observation following a single dose of onasemnogene abeparvovec (1.1 × 1014 vg/kg). A single anti-inflammatory regime of prednisolone at a dosage concentration of 1 mg/kg/day via oral administration once daily starting on day −1 and continuing through day 29, and then on days 31, 33, 35, 37, 39, and 41 of the observation phase was examined (see Supplementary Appendix for details of methods and results).In the GLP-compliant 6-month intravenous study in NHPs of Asian origin  for at least 10 min following intrathecal dosing.Blood (plasma or serum) and CSF were collected for clinical pathology and analysis of neuronal injury biomarker (neurofilament light chain [NfL]). Tissue samples were collected , and extPeripheral nerve sampling differed slightly between the three studies. Specifically, peripheral nerves from the hindlimb  were sampled and evaluated in the 12-month intrathecal study. Those same hindlimb nerves, as well as radial and ulnar nerves from the forelimb, were collected and evaluated in the 13-week non-GLP mechanistic intrathecal study.Finally, in the GLP-compliant 6-month intravenous study, hindlimb  and forelimb  peripheral nerves were collected and evaluated.in situ hybridization (ISH), immunohistochemistry, and biodistribution.Nonclinical assessments included clinical observations, body weight and qualitative food consumption assessments, ophthalmologic examinations, electrocardiography, and neurologic examinations. Respiration rate assessments, recording of body temperatures, body condition scoring, biodistribution, clinical pathology, and complete macroscopic and microscopic evaluations were obtained. Nonclinical assessments also included, depending on the study design, soluble biomarker for neuronal injury, electrophysiology (neurography or nerve conduction velocity) evaluation, terminal organ weight assessment, molecular localization by e.g., liver or the Schwann cell component of peripheral nerves). For this study, the term “resolution” was applied in the CNS and sensory ganglia to indicate a lack of progression evidenced by the general absence of, and/or decreased incidence and/or severity of, degenerative, necrotic, or inflammatory findings in the neuron components of these tissues, which generally lack a regenerative or neuronogenic response to injury or cell loss in mammals such as NHPs. The presence of aggregates of satellite glial cells represented normal tissue remodeling/glial cell response (scar) subsequent to the loss of degenerate neurons.Typically, in a nonclinical study, reversibility is assessed based on the absence or decreased incidence, and/or severity of findings in tissues considered to have a regenerative capacity .For DRG microscopic findings, in which multiple specimens from each geographic region of the vertebral column  were evaluated, the greatest severity of the finding for that region was recorded, rather than a composite score representing the total area evaluated or the number of normal ganglia versus those with microscopic findings. This decision reflected study objectives to characterize the toxicity of the onasemnogene abeparvovec test article.Molecular localization analysis to detect onasemnogene abeparvovec antisense and sense sequences was undertaken using ISH and paraffin-embedded tissue sections (∼5-μm thick) from selected animals and blocks of spinal cord and DRG at 6 weeks postdose (see Supplementary Appendix for details on methods).Peripheral nerve function was assessed by neurography (nerve conduction velocity) at 2- and 13-weeks postdose in the 13-week mechanistic study (see Supplementary Appendix for details on methods).Prestudy serum anti-AAV9 antibody titers in the three studies are provided in The concentrations of onasemnogene abeparvovec DNA in DRG samples in the three studies are provided in Data are presented as means and standard deviations. No statistical analysis was performed because of the small number of animals per sex and per group.Data from all onasemnogene abeparvovec clinical trials, irrespective of route of administration , were evaluated through May 2021 using the ARGUS safety database. This database contained all postmarketing safety reports received through commercial, managed access program, and early access use. Clinical trial data were also evaluated through May 2021.13 vg/animal. No onasemnogene abeparvovec-related changes in qualitative food consumption or body weight, ophthalmic observations, electrocardiography parameters, neurologic findings, respiration rates, body temperatures, cardiac troponin I, organ weights, and/or macroscopic findings were observed. All animals survived to scheduled euthanasia. Similar results were obtained following intravenous administration of onasemnogene abeparvovec in NHPs.Intrathecal onasemnogene abeparvovec was clinically well tolerated at all doses tested and was not associated with any clinical findings, including neurobehavioral changes, up to 52 weeks following dose administration of up to 6.0 × 1013 vg/animal. These changes were transient and not observed at later time points. No onasemnogene abeparvovec-related CSF changes were observed.In the 12-month intrathecal NHP study, onasemnogene abeparvovec-related hematology and coagulation changes included minimally to mildly increased absolute monocyte and large unstained cell counts and fibrinogen concentration on days 1 and/or 14 for animals administered ≥3.0 × 1013 vg/animal with or without coadministration of prednisolone and/or rituximab plus everolimus , coagulation, and CSF changes included minimally to mildly increased fibrinogen concentrations on days 10, 11, or 12, and/or increased CSF total protein and microalbumin  on day 92 postdose at 3.0 × 10erolimus . FibrinoPrednisolone-related changes included minimal to mild decrease in total lymphocytes and B and T cells on day 1, and rituximab plus everolimus-related changes included mild decrease in total lymphocytes on days 1, and 10, 11, or 12, mild decreases in T cells on day 1, and marked (100% less than pretest) decrease in B cells throughout observation period indicative of immunosuppression.13 vg/animal with and without coadministration of prednisolone. No onasemnogene abeparvovec-related coagulation or CSF changes were observed. Changes in monocyte counts, large unstained cell counts, and platelets were transient and not observed at later time points.In the 6-month intravenous study, onasemnogene abeparvovec-related hematology changes included transient minimally to mildly increased absolute monocyte and large unstained cell counts and mildly to moderately lower platelet counts on days 3–8 at 3.0 × 10Any clinical chemistry changes from these studies were outside the scope of this article and will be reported in a different publication.R2 = 0.9044; In the 12-month intrathecal NHP study, onasemnogene abeparvovec-related trends toward increased serum and CSF NfL, a marker of axonal injury, were observed in individual animals from all dose groups on day 22, with trends toward predose and vehicle control concentrations by day 163 and resolution by day 365 . A correIn the 13-week intrathecal NHP study, individual animal increases in plasma and CSF NfL concentrations were observed following administration of onasemnogene abeparvovec alone or with immunosuppressants (prednisolone or rituximab/everolimus) at different intervals between days 12 and 92 postdose . Similar NfL trends were observed in the 6-month intravenous studies .In the 2-week interim cohort, onasemnogene abeparvovec-related nervous system microscopic findings were limited to the DRG and included minimal to marked mononuclear cell inflammation, minimal to moderate neuronal degeneration, and minimally to slightly increased satellite glial cells. Mononuclear cell inflammation was characterized by variable and increased numbers of mixed mononuclear cells associated with areas of tissue and/or neuronal degeneration and/or the presence of cellular debris. Neuronal degeneration was characterized by small and/or irregularly shrunken neuronal cell bodies with scalloped borders and an indistinct or absent nucleus with or without cellular debris, typically surrounded by mononuclear cells.Secondary findings included minimal to slight axon degeneration and minimal mononuclear cell infiltrates in the adjacent spinal root and/or spinal nerve .13 vg/animal onasemnogene abeparvovec alone or with prednisolone or rituximab/everolimus. At 13 weeks postdose, onasemnogene abeparvovec-related microscopic findings were observed in the DRG and/or TG  (Axon degeneration was characterized variably by one or more of the following: dilated spaces (formerly occupied by myelin sheaths and axons), swollen axons, cellular debris, and/or macrophages. Mononuclear cell infiltrates were characterized by increased number of mononuclear cells that were not associated with tissue degeneration or damage compared with mononuclear cell inflammation. In general, these findings were reported as similar in incidence and severity in animals administered 3 × 10ganglia) .Microscopic findings in these tissues were the same as observed at 2 weeks, and included neuronal cell loss, minimal to slight satellite glial cell aggregates, minimal to moderate mononuclear cell infiltrates, and/or slight axonal degeneration. The incidence and severity of the DRG and/or TG microscopic findings at 2- and 13-weeks postdose were comparable.13 vg/animal .After 52 weeks of observation postintrathecal dosing, microscopic DRG and/or TG findings were limited to minimal neuronal degeneration, mononuclear cell inflammation, and/or satellite glial cell aggregates in animals administered ≥1.2 × 10Satellite glial cell aggregates  were characterized by small clusters of closely packed, disorganized glial cells in the ganglia and were consistent with normal tissue remodeling in the nervous system and reflective of a glial response (scar) related to resolution in this tissue following neuronal degeneration that resulted in loss of the affected neuron. The incidence and/or severity of these microscopic findings was generally lower compared with animals examined at 6 weeks postdose . These c14 vg/kg) in NHPs at 6 weeks and 6 months postdose . At 13 weeks postdose, onasemnogene abeparvovec-related microscopic findings in the spinal cord and dorsal spinal nerve root/spinal nerves that were considered secondary to the DRG findings observed at the 2 week postdose necropsy consisted of minimal to moderate axonal degeneration in the dorsal (posterior) funiculus of the spinal cord and/or the dorsal spinal cord nerve roots, minimal gliosis in the dorsal funiculus, and minimal to slight mononuclear cell infiltrates in the dorsal spinal nerve roots  of the cervical, thoracic, and/or lumbar spinal cord of one male and two females administered onasemnogene abeparvovec alone or with prednisolone. Slight or moderate severity neuronal degeneration and gliosis in the spinal cord were sampled at the location of the intrathecal injection site.These findings were considered separate to the DRG findings, but may also be related to greater transduction of some cells in this tissue. Microscopic findings in the ventral motor neurons were not associated with any in-life or neurologic observations and may be of similar pathogenesis to the DRG findings in that they are subsequent to greater transduction of the AAV construct and/or greater expression of the transgene product. However, whether the response is specific to the transgene protein product is not clear.At 13 weeks postdose, onasemnogene abeparvovec-related microscopic findings in peripheral nerves consisted of minimal to marked axonal degeneration and minimal mononuclear cell infiltrates. Peripheral nerve axon degeneration was generally considered secondary to DRG neuronal degeneration.13 vg/animal  of the spinal cord in a few animals administered ≥3.0 × 10g/animal .13 vg/animal males at 6 weeks postdose rendered injury because of intrathecal injection unlikely. Therefore, these findings were considered onasemnogene abeparvovec related. No correlative in-life clinical or neurologic observations were reported in these animals.Although this finding was uncommon and was distributed more often in the lumbar or sacral spinal cord , the distribution of degenerate neurons in the ventral gray column, but not the dorsal gray column, in multiple sections of the spinal cord in one of two 6 × 1013 vg/animal  and minimal gliosis in the spinal cord white matter at 6 weeks postdose.Minimal axon degeneration was observed at a similar incidence and severity in concurrent female controls and onasemnogene abeparvovec-dosed females at the interim necropsy, with no onasemnogene abeparvovec-related increase in incidence or severity in females at this 6-week time point. At the end of the 6-month observation period, axon degeneration in the spinal cord was still present at similar  or lower  incidence and severity compared with the end of the 6-week observation period, supporting a lack of progression of the findings.No clinical observations, neurologic examination findings, or other correlations were observed for these minimal or slight axon findings in the ventral or lateral funiculi.No onasemnogene abeparvovec-related microscopic finding was noted in peripheral nerves  following intravenous administration of onasemnogene abeparvovec at either 6-weeks or 6-months postdose.13 vg/animal and two concurrent controls  to localize vector nucleic acid. Analysis included appropriate positive and tissue quality control (macaca mulatta peptidyl-prolyl cis-trans isomerase B) and negative control (dihydrodipicolinate reductase) genes.ISH for onasemnogene abeparvovec antisense and sense gene probes was conducted on selected sections of spinal cord and selected DRG from two males administered intrathecally 6.0 × 10ISH for onasemnogene abeparvovec using the antisense probe on sections of spinal cord from the two males with microscopic neuronal degeneration/satellitosis in the ventral gray matter noted at 6 weeks postdose detected moderate to high signal concentrations consistent with high concentrations of vector transcript in a subset of scattered individual neurons with morphologic features of degeneration and satellitosis compared with adjacent unaffected neurons .ISH with onasemnogene abeparvovec sense probes conducted on sequential sections of spinal cord detected lesser concentrations of cytoplasmic signal consistent with the presence of double-stranded ribonucleic acid. In regions of the spinal cord without microscopic findings, lesser concentrations of antisense and sense signals were observed and characterized by small, punctate nuclear and cytoplasmic signals. Sections from concurrent control animals did not detect signal from antisense or sense onasemnogene abeparvovec probes. Positive and negative control probes returned expected results.In DRG from the two males with microscopic neuronal degeneration at 6 weeks postdose, ISH for onasemnogene abeparvovec using the antisense probe detected moderate to high signal concentrations consistent with high concentrations of vector transcript in individual neurons with morphologic features of degeneration compared with adjacent unaffected neurons . ISH for13 vg/animal) with and without immunosuppression (prednisolone or rituximab and everolimus) in the 13-week mechanistic study demonstrated that mean peripheral sensory nerve conduction was within normal functional range at 2- and 13-weeks postdose. No physiologically relevant decreases were observed compared with concurrent controls in the sural, peroneal, and saphenous sensory nerves. No velocity decreases in the tibial motor function and ascending motor pathways were observed compared with concurrent controls (14 vg/kg) in NHPs (see Supplementary Appendix for details of results).Neuroelectrophysiologic evaluations assessing peripheral nerve function following intrathecal onasemnogene abeparvovec . A rapid and robust humoral immune response to the AAV capsid was observed in both serum and CSF (data not shown).As of May 2021, 102 patients received intravenous onasemnogene abeparvovec and 32 patients received intrathecal onasemnogene abeparvovec in clinical trials. In clinical trials, pediatric neuromuscular experts were trained to conduct neurologic assessments for all children, including infants documented in serial comprehensive examinations, to evaluate for signs or symptoms of ganglionopathy. Also, all reported that adverse events were reviewed to evaluate for signs or symptoms of ganglionopathy.Although self-reporting may be limited as these patients were infants, parental reports and investigator examinations were obtained to assess for ganglionopathy. No events suggestive of ganglionopathy were reported, and a review of documented neurologic and physical examination data in clinical studies demonstrated no findings consistent with ganglionopathy. Based on a review of safety data from the ARGUS safety database, no events of ganglionopathy have been reported as of May 2021.13 vg/animal was well tolerated and not associated with any in-life clinical or neurologic observations up to 52 weeks postdose. Adverse microscopic findings in the DRG and TG, including neuron degeneration, were considered nonprogressive with a trend toward resolution based on lower incidence and minimal severity of microscopic observations after 52 weeks of observation. In addition, microscopic findings, including aggregates of satellite glial cells, were considered consistent with tissue remodeling over time following loss of the affected (degenerate) neurons.For juvenile cynomolgus monkeys, intrathecal onasemnogene abeparvovec administration at doses up to 6.0 × 1032–36Neuronal cell degeneration/neuron loss is generally not considered a reversible finding, because neurons are not thought to have regenerative capacity in NHPs (compared with reported neurogenesis in dogs and rodents).Instead, in the 12-month NHP study, “resolution” was used to connote a decrease in the incidence and/or severity of the microscopic neuronal degeneration in the DRG and TG as well as the absence of related/secondary axonal degeneration in the examined peripheral nerves and central axonal projections of this sensory neuron population .Satellite glial cell aggregates represented a tissue remodeling/glial cell response (scar) subsequent to the loss of degenerative neurons and were not considered adverse in and of themselves but rather interpreted as evidence of quiescence/nonprogression of the microscopic neuronal and/or inflammatory findings in the sensory ganglia (DRG and TG) after 52 weeks of observation without any in-life neurologic or clinical onasemnogene abeparvovec-related findings.37At the interim necropsy, 6 weeks after the single intravenous dose, microscopic spinal cord findings of minimal, or rarely slight, severity axon degeneration were increased in incidence and severity  in onasemnogene abeparvovec-dosed males compared with concurrent controls, but were similar in controls and onasemnogene abeparvovec–dosed females. At the terminal necropsy, 6 months after dosing, incidence in females was lower compared with the interim necropsy, while incidence in males was similar. These findings were of uncertain pathogenesis, because minimal axon degeneration may be observed in undosed NHPs.38–40 As with the present studies, these microscopic findings were typically without corresponding clinical signs and did not demonstrate a dose response.39The onasemnogene abeparvovec-related microscopic DRG/TG findings observed in the NHP studies were comparable with those observed with several other intrathecally or intravenously administered AAV  gene therapies that used assorted transgenes, confirming that these findings were onasemnogene abeparvovec related.Our primary microscopic findings consisted of degeneration of DRG neurons with mononuclear inflammation. Associated axon degeneration and related microscopic findings  were observed in structures composed, at least in part, of the axon branches from the sensory DRG neurons that form the dorsal nerve roots, ascending spinal and bulbar nerve tracts, and sensory axons in peripheral nerves. This degeneration occurred within 2–6 weeks of intrathecal administration.41 These findings were nonprogressive and demonstrated morphologic features of expected tissue remodeling .The clinically silent nature of this microscopic finding was considered related to the limited, multifocal distribution and the relatively low extent and magnitude of the affected sensory ganglia (DRG and TG). Only a small section of the overall general proprioceptive and somatic afferent sensory ganglia was affected, in a system with substantial redundancy, given the evolutionary importance of these key sensory inputs for survival.39AAV-mediated DRG changes were likely multifactorial and potentially driven by increased sensitivity to greater transduction of individual DRG neurons by some AAV serotypes, most notably AAV9 and close analogs, although a contribution by specific transgene or transgene products may impact some circumstances.13 vg/animal  for antisense and sense onasemnogene abeparvovec probes and routine light microscopic findings from NHP studies also support this hypothesis, demonstrating high transgene vector expression in the affected (degenerate) DRG sensory neurons  of two males administered onasemnogene abeparvovec at a dose of 6 × 10g/animal . High co13 vg/animal.Together, these patterns of high vector transcript demonstrated by ISH collocated with morphologic findings of degeneration, necrosis, satellitosis, and/or mononuclear cell inflammation are consistent with a common pathogenesis driven by high concentrations of vector transduction and transgene expression, resulting in neuronal degeneration in the DRG in animals administered ≥1.2 × 1043 In fact, the sense probes for onasemnogene abeparvovec performed on sequential sections of nervous tissue by ISH detected cytoplasmic signal consistent with the presence of double-stranded ribonucleic acid, which may elicit an innate immune response through retinoic acid-inducible gene I-like receptor signaling pathways. Sensory neurons in the DRG were surrounded by satellite glial cells, scant numbers of resident tissue macrophages, and T and B lymphocytes,44–46 which were associated with AAV-mediated microscopic DRG findings.39Although several other factors, including vector dose, route of administration, and age at dosing, may play a role in AAV9-mediated microscopic DRG findings in NHPs, activation of the innate immune response in transduced cells was not excluded.13 vg/animal by intrathecal lumbar injection, suggesting that primary adaptive immune responses may not be a critical mediator in the pathogenesis of microscopic DRG (or TG) findings. The microscopic and molecular localization findings reported here are consistent with a published study, in which immunosuppression (mycophenolate mofetil and rapamycin) administered to NHPs treated with unmodified AAV9 did not alleviate DRG damage.42Systemic immunosuppression, including 100% B cell depletion at all time points, did not prevent, attenuate, and/or mitigate the incidence or severity of onasemnogene abeparvovec-related microscopic DRG findings in NHPs dosed with 3 × 1047 Minor to moderate transient increases in serum or CSF NfL concentrations were observed at different time points between days 12 and 92 postdose, decreasing toward baseline, with eventual resolution by day 365 following intrathecal onasemnogene abeparvovec administration in NHPs. There appears to be a correlation between the transient NfL increases and the onset of microscopic DRG findings observed at necropsy in NHPs in nonclinical safety studies. Compared with patients with neurodegenerative conditions, such as SMA, species used in nonclinical safety assessments are outbred, clinically healthy animals.NfL has been used in the clinic to monitor for neuronal/axonal injury and has recently been considered for monitoring of clinical response to molecular therapies.This enables identification of test article-related findings in the absence of concurrent disease or comorbid conditions, which is not possible in a human population. Serum or CSF NfL findings associated with administration of intrathecal onasemnogene abeparvovec administration in NHPs as reported here should be considered in the context of this nonclinical, nondiseased test system, and appropriate caution should be applied for any potential translation to human patients.No neuroelectrophysiologic changes  were reported following intrathecal or intravenous onasemnogene abeparvovec administration in NHPs. Mean peripheral sensory nerve conduction remained within the normal functional range, indicating no correlation between the DRG changes and peripheral nerve function. This finding is consistent with the lack of clinical or neurologic observations during in-life in animals. The generally low percentage of affected neurons in the DRG with onasemnogene abeparvovec-related microscopic findings, and the degree of redundancy and overlap associated with innervation of the dermatomes of the sensory system, may further explain the lack of in-life findings.SOD1) gene who received a single intrathecal infusion of AAV-miR-SOD1 (4.2 × 1014 vg).48 However, the published information was limited, and the authors were not able to determine if the findings were because of gene therapy or the underlying disease state. This case along with another unpublished case is presented in more detail in the Supplemental Results. In the present study, evaluation of available clinical safety data after intravenous and intrathecal onasemnogene abeparvovec dosing in clinical trials reported no clinical sensory neuropathy findings as of May 2021.The translational relevance of DRG pathology in humans is currently unknown. The medical literature includes one reported case of putative microscopic DRG findings in a 22-year-old male with amyotrophic lateral sclerosis and mutations in the superoxide dismutase 1  following a 52-week observation period. These observations support the nonprogressive nature of the nervous system findings, which were not associated with clinically detectable neurologic findings or interpretations. In addition, microscopic DRG findings were not mitigated with coadministration of anti-inflammatory or immunosuppressive regimens. The pathogenesis is possibly a consequence of increased vector genome transduction and/or transgene expression.15 did not reveal any clinical findings of ganglionopathy. Therefore, the clinical relevance of the DRG findings in NHP studies associated with intrathecal or intravenous administration of AAV vector gene therapies remains unknown. This potential risk will be monitored through usual clinical safety surveillance.For translational applicability to patients, analysis of clinical data in the onasemnogene abeparvovec clinical trials, using the intrathecal or intravenous route of administration, as well as review of postmarketing safety data,