Osteogenesis imperfecta (OI) is a group of monogenic skeletal diseases caused by defect of type I collagen. There is no specific treatment strategy and targeted therapy is urgently needed. Dickkopf-1 (DKK1), is a Wnt antagonist, and inhibition of DKK1 stimulates Wnt signaling and osteogenic capacity. Here, we used DKK1 neutralizing antibody (DKK1 nAb) to identify its effect on OI and clarify its effects on different types of OI. Mesenchymal stem cells (MSCs) derived from OI patients with variants in COL1A1, COL1A2, SERPINF1, and WNT1 were treated with or without DKK1 nAb and induced toward osteoblast differentiation in vitro. In vivo, two Sprague-Dawley OI rat models carrying a Col1a2 (c.1000G > A) or Wnt1 (c.620G > A) mutation were administered DKK1 nAb for 2 and 4 weeks to assess skeletal therapeutic effects. In vitro, DKK1 nAb promoted osteogenic differentiation and mineralization and upregulated the mRNA levels of COL1A1, COL1A2, CTNNB1, and RUNX2 in MSCs from some, though not all, OI patients. This regulatory effect is independent of the genotypes of OI patients. In vivo, a 2-week DKK1 nAb treatment exerted no significant improvement in skeletal parameters in either Col1a2 or Wnt1 mutant rats. While 4-week treatment showed modest improvement in trabecular bone in Col1a2 mutant OI rats, whereas no significant changes were detected in Wnt1 mutant OI rats. The DKK1 nAb demonstrates a modest therapeutic effect in OI, indicating its potential for some individuals, although the limited sample size necessitates caution in interpretation, and further validation in larger cohorts is warranted.

Abstract Background Mitochondrial DNA (mtDNA) mutations are implicated in a wide range of diseases, underscoring the need to elucidate the relationship between mtDNA mutations and disease pathology. These diseases are often characterized by the presence of multiple mutations; however, research has been hampered by the lack of suitable animal models carrying multiplex mtDNA mutations. Such models cannot be produced through traditional breeding due to matrilineal inheritance of mtDNA. Methods Based on the TALE‐based mitochondrial genome editing tools, we generated rats harboring multiple mtDNA mutations by microinjecting mixed pairs of DdCBE plasmids into one‐cell‐stage zygotes. The efficiency of mtDNA editing and the potential off‐target effects were assessed through deep sequencing and long reads sequencing methodologies. Results In this study, we established double‐ and triple‐site mutant rats with the editing efficiencies of up to 58.5% and confirmed that these mutations can be stably transmitted through the germline. Moreover, our results demonstrated that DdCBE‐mediated mtDNA multi‐site editing of mtDNA exhibits minimal off‐target effects in both the mitochondrial and nuclear genomes in vivo. Conclusion This work represents the first successful generation of heritable multi‐site mtDNA mutant rats, providing a valuable model for elucidating the pathophysiological mechanisms of mitochondrial disorders and for developing potential therapeutics.

ABSTRACT Nipah virus (NiV) is a zoonotic paramyxovirus classified as a WHO priority pathogen, which causes severe respiratory disease and encephalitis in humans with high mortality. Currently, no licensed vaccines or specific antiviral therapies are available. A major obstacle in NiV research is the lack of immunocompetent rodent models that endogenously express human‐compatible entry receptors Ephrin‐B2 (EFNB2) and Ephrin‐B3 (EFNB3). Here, we report the generation and characterization of two novel knock‐in mouse models using the CRISPR/Cas9 system. The human EFNB2 or EFNB3 gene, driven by the human keratin 18 (K18) promoter, was site‐specifically integrated into the mouse Hipp11 ( H11 ) safe‐harbor locus. Founder mice exhibited stable germline transmission without obvious developmental abnormalities. These established models provide a physiologically relevant and genetically engineered platform for studying NiV entry mechanisms, pathogenesis, and the development of vaccines and antiviral therapeutics.

Messenger RNA (mRNA) delivery critically depends on the molecular architecture of ionizable lipids within lipid nanoparticles (LNPs). We report a modular platform of sterol-containing ionizable lipids designed to probe how sterol motifs and branched dialkyl tails govern in vivo performance. Each lipid incorporates a sterol-derived (e.g., cholesterol) tail and a dialkyl tail with tunable branching and spatial arrangement, enabling systematic control over packing behavior, protonation characteristics, and nanoparticle assembly. The resulting LNPs exhibited favorable apparent pKa values (6.2-6.8), encapsulation efficiencies higher than 90%, and strong transfection potency. Our lead LNP (L1-aCho-e3 LNP) exhibited higher transfection efficiency than the benchmark SM-102 LNP. Incorporation of cationic (DOTAP) or anionic (DOPA) lipids as fifth components further demonstrated reorienting biodistribution from liver to lung or spleen. Finally, mRNAH1N1@L1-aCho-e3 LNPs elicited robust antibody responses and full protection against lethal H1N1 challenge. These results reveal how rational sterol tail design can be applied to create ionizable lipids with tunable structural functional properties for next-generation mRNA therapeutics.

Kinesin family member 13B (KIF13B), a crucial motor protein, exerts multiple cellular biological functions. However, the implication of KIF13B in metabolic dysfunction-associated fatty liver disease (MAFLD) has not been explored yet. This study aimed to investigate KIF13B’s role and underlying mechanism in MAFLD and proposes it as a potential pharmacological target. We assessed KIF13B expression in MAFLD patients and rodent models. The roles of Kif13b in lipid metabolism and MAFLD were investigated using whole-body Kif13b knockout mice, hepatocyte-specific Kif13b-deficient mice and hamsters exposed to different diets. The underlying mechanisms by which Kif13b governed hepatic lipid homeostasis and MAFLD progression were explored in vitro. Finally, the Kif13b’s impact on atherosclerotic development was studied in the context of MAFLD. KIF13B expression was reduced in patients and murine models with MAFLD. Rodents with global or liver-specific knockout of the Kif13b gene exhibit spontaneous hepatic steatosis, which is further exacerbated by different overnutrition diets. Overexpression of human KIF13B by lentivirus effectively prevented metabolic dysfunction-associated steatohepatitis (MASH) in methionine-choline-deficient diet (MCD)-fed mice. Furthermore, Kif13b deficiency accelerates atherosclerosis in the context of MAFLD. Mechanistically, Kif13b depletion increases hepatic lipid synthesis and impairs mitochondrial oxidative phosphorylation. Further screening reveals that Kif13b interacts with AMP-activated catalytic subunit alpha 1 (AMPKα1) to regulate the phosphorylation of AMPKα1, governing mitochondrial homeostasis and suppressing sterol regulatory element binding protein 1 (Srebp1)-mediated de novo lipogenesis in the liver. This work establishes a causal relationship between KIF13B deficiency and MAFLD, emphasizing KIF13B as a potential therapeutic target for treating MAFLD.

Amyotrophic lateral sclerosis (ALS) is categorized into ~10% familial and ~90% sporadic cases. While familial ALS is caused by mutations in many genes of diverse functions, the underlying pathogenic mechanisms of ALS, especially in sporadic ALS (sALS), are largely unknown. Notably, about half of the cases with sALS showed defects in mitochondrial respiratory complex IV (CIV). To determine the causal role of this defect in ALS, we used transcription activator-like effector-based mitochondrial genome editing to introduce mutations in CIV subunits in rat neurons. Our results demonstrate that neuronal CIV deficiency is sufficient to cause a number of ALS-like phenotypes, including cytosolic TAR DNA-binding protein 43 redistribution, selective motor neuron loss and paralysis. These results highlight CIV deficiency as a potential cause of sALS and shed light on the specific vulnerability of motor neurons, marking an important advance in understanding and therapeutic development of sALS.

BACKGROUND:The Cre/loxP system is most popular in mice, but its application in rats has largely lagged far behind. The rat is vital laboratory animal, especially in toxicological and neurological studies. Generating genetic tools to manipulate neurons in rats could benefit neurological research. METHODS:Using the CRISPR/Cas9 system, we inserted a Cre cassette into endogenous Thy1 and NeuN loci. Thy1-Cre rats featured a downstream P2A-linked insertion, while NeuN-Cre was inserted at the transcriptional start site. The Cre activity was assessed by crossing with a Cre reporter (Rosa26imCherry) rat and through analyzing mCherry expression patterns. The specificity of cell type was further confirmed by immunofluorescence with NeuN antibody. Phenotypic consequences were assessed by crossing with ND1LSL rats to deplete ND1, followed by monitoring weight/survival and conducting motor function tests. RESULTS:We generated two neuron-specific rats (Thy1-Cre and NeuN-Cre), which exhibited high neuron-specific Cre expression in brain and spinal cord with minor leakage in other tissues. Thy1-Cre showed minor leakage in spleen, lung and kidney while NeuN-Cre showed minor leakage in spleen and kidney. ND1Thy1-Cre and ND1NeuN-Cre rats both showed decreased body weights and survival times. The ND1NeuN-Cre rats died within two weeks, while ND1Thy1-Cre rats lived longer with impaired motor function. CONCLUSIONS:We successfully generated two neuron-specific NeuN-Cre and Thy1-Cre rats, and systemically analyzed their expression pattern.

BACKGROUND:The golden Syrian hamster is a valuable animal model for studying carcinogenesis, metabolic disorders, cardiovascular diseases, and viral infections due to its biological and pathological similarities to humans. However, the development of genetically engineered hamsters has lagged behind that of mice and rats, largely because of an embryonic development block at the two-cell stage in vitro. Although CRISPR/Cas9-mediated gene knockout has been achieved in hamsters, precise DNA fragment insertion or conditional knockout (cKO) models have not previously been reported, likely due to technical limitations in embryo manipulation and insufficient efficiency of homology-directed repair (HDR). METHODS:In this study, we generated conditional alleles of the ApoF gene in golden Syrian hamsters. A two-cut strategy was applied using Cas9 protein, two sgRNAs, and a single donor plasmid containing exon 2 flanked by loxP sites and two ~0.8 kb homology arms. A mixture of Cas9 protein, sgRNAs, and the donor plasmid was microinjected into the pronuclei of one-cell stage hamster embryos. RESULTS:The efficiency of CRISPR/Cas9-mediated loxP knock-in reached up to 27%, and the genetically modified floxed alleles were successfully transmitted through the germline. The functionality of the inserted loxP sites was validated by in vivo Cre-mediated recombination following local administration of AAV vectors, including AAV-cTnT-Cre in the heart and AAV-CMV-Cre in the brain. CONCLUSIONS:To our knowledge, this work represents the first successful establishment of a conditional knockout model in the golden Syrian hamster, providing a valuable tool for mechanistic studies of gene function and disease modeling.

BACKGROUND:Tripartite motif (TRIM46) is a relatively novel protein that belongs to tripartite motif family. TRIM46 organizes parallel microtubule arrays on the axons, which are important for neuronal polarity and axonal function. TRIM46 is highly expressed in the brain, but its biological function in adults has not yet been determined. RESULTS:Trim46 knockout (KO) rat line was established using CRISPR/cas9. Trim46 KO rats had smaller hippocampus sizes, fewer neuronal dendritic arbors and dendritic spines, and shorter and more distant axon initial segment. Furthermore, the protein interaction between endogenous TRIM46 and FK506 binding protein 5 (FKBP5) in brain tissues was determined; Trim46 KO increased hippocampal FKBP5 protein levels and decreased hippocampal protein kinase B (Akt) phosphorylation, gamma-aminobutyric acid type A receptor subunit alpha1 (GABRA1) and glutamate ionotropic receptor NMDA type subunit 1 (NMDAR1) protein levels. Trim46 KO rats exhibited hypoactive behavioral changes such as reduced spontaneous activity, social interaction, sucrose preference, impaired prepulse inhibition (PPI), and short-term reference memory. CONCLUSIONS:These results demonstrate the significant impact of Trim46 KO on brain structure and behavioral function. This study revealed a novel potential association of TRIM46 with dendritic development and neuropsychiatric behavior, providing new insights into the role of TRIM46 in the brain.

An animal model harboring pathogenic mitochondrial DNA (mtDNA) mutations is important to understand the biological links between mtDNA variation and mitochondrial diseases. DdCBE, a DddA-derived cytosine base editor, has been utilized in zebrafish, mice, and rats for tC sequence-context targeting and human mitochondrial disease modeling. However, human pathogenic mtDNA mutations other than the tC context cannot be manipulated. Here, we screened the combination of different DdCBE pairs at pathogenic mtDNA mutation sites with nC (n for a, g, or c) context and identified that the left-G1333C (L1333C) + right G1333N (R1333N) pair could mediate C & BULL;G-to-T & BULL;A conversion effectively at aC sites in rat C6 cells. The editing efficiency at disease-associated mtDNA mutation sites within aC context was further confirmed to be up to 67.89% in vivo. Also, the installed disease-associated mtDNA mutations were germline transmittable. Moreover, the edited rats showed impaired cardiac function and mito-chondrial function, resembling human mitochondrial disease symptoms. In summary, for the first time, we expanded the DdCBE targeting scope to an aC motif and installed the path-ogenic mutation in rats to model human mitochondrial dis-eases.

Objective To explore the effect of the Trim44 gene in the nervous system, Trim44 conditional knockout rats were used to analyze behavioral abnormalities and pathological changes in the brain. Methods TRIM44 expression in brain tissue was detected by Western blot and immunohistochemistry. Neuronal apoptosis was determined by TUNEL staining. Behavioral changes of knockout rats were assessed by Morris Y, Morris water, and food maze tests. Abnormal expression of apoptosis-related proteins was detected by Western blot. Results TRIM44 was highly expressed in brain tissues of adult rats. The knockout efficiency of Trim44 in brain tissues was 67%. Trim44 knockout decreased learning and memory abilities, increased neuronal apoptosis, and expression of active caspase 3 and 9 proteins in aged rats. Conclusions Trim44 knockout promotes neuronal apoptosis and impairs learning and memory abilities in rats. These result suggest that TRIM44 plays an important role in the nervous system, and the Trim44 knockout rat model can be used for further mechanism exploration.

Hundreds of pathogenic variants of mitochondrial DNA (mtDNA) have been reported to cause mitochondrial diseases, which still lack effective treatments. It is a huge challenge to install these mutations one by one. We repurposed the DddA-derived cytosine base editor to incorporate a premature stop codon in the mtProtein-coding genes to ablate mitochondrial proteins encoded in the mtDNA (mtProteins) instead of installing pathogenic variants and generated a library of both cell and rat resources with mtProtein depletion. In vitro, we depleted 12 of 13 mtProtein-coding genes with high efficiency and specificity, resulting in decreased mtProtein levels and impaired oxidative phosphorylation. Moreover, we generated six conditional knockout rat strains to ablate mtProteins using Cre/loxP system. Mitochondrially encoded ATP synthase membrane subunit 8 and NADH:ubiquinone oxidoreductase core subunit 1 were specifically depleted in heart cells or neurons, resulting in heart failure or abnormal brain development. Our work provides cell and rat resources for studying the function of mtProtein-coding genes and therapeutic strategies.

ABSTRACT When pathological hypertrophy progresses to heart failure (HF), the prognosis is often very poor. Therefore, it is crucial to find new and effective intervention targets. Here, myocardium-specific Trim44 knockout rats were generated using CRISPR-Cas9 technology. Cardiac phenotypic observations revealed that Trim44 knockout affected cardiac morphology at baseline. Rats with Trim44 deficiency exhibited resistance to cardiac pathological changes in response to stimulation via isoproterenol (ISO) treatment, including improvement of cardiac remodeling and dysfunction by morphological and functional observations, reduced myocardial fibrosis and reduced expression of molecular markers of cardiac stress. Furthermore, signal transduction validation associated with growth and hypertrophy development in vivo and in vitro demonstrated that Trim44 deficiency inhibited the activation of signaling pathways involved in myocardial hypertrophy, especially response to pathological stress. In conclusion, the present study indicates that Trim44 knockout attenuates ISO-induced pathological cardiac remodeling through blocking the AKT/mTOR/GSK3β/P70S6K signaling pathway. This is the first study to demonstrate the function and importance of Trim44 in the heart at baseline and under pathological stress. Trim44 could be a novel therapeutic target for prevention of cardiac hypertrophy and HF.

Aims: Multiple mitochondrial dysfunction (MMD) can lead to complex damage of mitochondrial structure and function, which then lead to the serious damage of various metabolic pathways including cerebral abnormalities. However, the effects of MMD on heart, a highly mitochondria-dependent tissue, are still unclear. In this study, we use iron-sulfur cluster assembly 1 (Isca1), which has been shown to cause MMD syndromes type 5 (MMDS5), to verify the above scientific question. Main methods: We generated myocardium-specific Isca1 knockout rat (Isca1(flox/flox)/alpha-MHC-Cre) using CRISPR-Cas9 technology. Echocardiography, magnetic resonance imaging (MRI), histopathological examinations and molecular markers detection demonstrated phenotypic characteristics of our model. Immunoprecipitation, immunofluorescence co-location, mitochondrial activity, ATP generation and iron ions detection were used to verify the molecular mechanism. Key findings: This study was the first to verify the effects of Isca1 deficiency on cardiac development in vivo, that is cardiomyocytes suffer from mitochondria damage and iron metabolism disorder, which leads to myocardial oncosis and eventually heart failure and body death in rat. Furthermore, forward and reverse validation experiments demonstrated that six-transmembrane epithelial antigen of prostate 3 (STEAP3), a new interacting molecule for ISCA1, plays an important role in iron metabolism and energy generation impairment induced by ISCA1 deficiency. Significance: This result provides theoretical basis for understanding of MMDS pathogenesis, especially on heart development and the pathological process of heart diseases, and finally provides new clues for searching clinical therapeutic targets of MMDS.

BACKGROUND:Inflammation is a complex physiological and pathological process. Although many types of inflammation are well characterized, their physiological functions are largely unknown. tRNA aspartic acid methyltransferase 1 (TRDMT1) has been implicated as a stress-related protein, but its intrinsic biological role is unclear.METHODS:We constructed a Trdmt1 knockout rat and adopted the LPS-induced sepsis model. Survival curve, histopathological examination, expression of inflammatory factors, and protein level of TLR4 pathway were analyzed.RESULTS:Trdmt1 deletion had no obvious impact on development and growth. Trdmt1 deletion slightly increased the mortality during aging. Our data showed that Trdmt1 strongly responded in LPS-treated rats, and Trdmt1 knockout rats were vulnerable to LPS treatment with declined survival rate. We also observed more aggravated tissue damage and more cumulative functional cell degeneration in LPS-treated knockout rats compared with control rats. Further studies showed upregulated TNF-α level in liver, spleen, lung, and serum tissues, which may be explained by enhanced p65 and p38 phosphorylation.CONCLUSIONS:Our data demonstrated that Trdmt1 plays a protective role in inflammation by regulating the TLR4-NF-κB/MAPK-TNF-α pathway. This work provides useful information to understand the TRDMT1 function in inflammation.

Self-grooming is a complex behavior with important biological functions and pathological relevance. How the brain coordinates with the spinal cord to generate the repetitive movements of self-grooming remains largely unknown. Here, we report that in the caudal part of the spinal trigeminal nucleus (Sp5C), neurons that express Cerebellin-2 (Cbln2+) form a neural circuit to the cervical spinal cord to maintain repetitive orofacial self-grooming. Inactivation of Cbln2+ Sp5C neurons blocked both sensory-evoked and stress-induced repetitive orofacial self-grooming. Activation of these neurons triggered short-latency repetitive forelimb movements that resembled orofacial self-grooming. The Cbln2+ Sp5C neurons were monosynaptically innervated by both somatosensory neurons in the trigeminal ganglion and paraventricular hypothalamic neurons. Among the divergent projections of Cbln2+ Sp5C neurons, a descending pathway that innervated motor neurons and interneurons in the cervical spinal cord was necessary and sufficient for repetitive orofacial self-grooming. These data reveal a brain-to-spinal sensorimotor loop for repetitive self-grooming in mice.

Dilated cardiomyopathy (DCM) is a major cause of heart failure. LMNA variants contribute to 6-10% DCM cases, but the underlying mechanisms remain incompletely understood. Here, we reported two patients carrying the LMNA c.1621C > T/ p.R541C variant and generated a knock-in mouse model (Lmna(RC)) to study the role of this variant in DCM pathogenesis. We found Lmna(RC/RC) mice exhibited ventricular dilation and reduced systolic functions at 6 months after birth. The Lmna(RC/RC) cardiomyocytes increased in size but no nuclear morphology defects were detected. Transcriptomic and microscopic analyses revealed suppressed gene expression and perturbed ultrastructure in Lmna(RC/RC) mitochondria. These defects were associated with increased heterochromatin structures and epigenetic markers including H3K9me2/3. Together, these data implied that the LMNA c.1621C > T/ p.R541C variant enhanced heterochromatic gene suppression and disrupted mitochondria functions as a cause of DCM.

Interferon regulatory factor 3 (IRF3) is an essential transductor for initiation of many immune responses. Here, we show that lncRNA-ISIR directly binds IRF3 to promote its phosphorylation, dimerization, and nuclear translocation, along with enhanced target gene productions. In vivo lncRNA-ISIR deficiency results in reduced IFN production, uncontrolled viral replication, and increased mortality. The human homolog, AK131315, also binds IRF3 and promotes its activation. More important, AK131315 expression is positively correlated with type I interferon (IFN-I) level and severity in patients with lupus. Mechanistically, in resting cells, IRF3 is bound to suppressor protein Flightless-1 (Fli-1), which keeps its inactive state. Upon infection, IFN-I-induced lncRNA-ISIR binds IRF3 at DNA-binding domain in cytoplasm and removes Fli-1's association from IRF3, consequently facilitating IRF3 activation. Our results demonstrate that IFN-I-inducible lncRNA-ISIR feedback strengthens IRF3 activation by removing suppressive Fli-1 in immune responses, revealing a method of lncRNA-mediated modulation of transcription factor (TF) activation.

Background:Multiple mitochondrial dysfunction syndromes (MMDS) presents as complex mitochondrial damage, thus impairing a variety of metabolic pathways. Heart dysplasia has been reported in MMDS patients; however, the specific clinical symptoms and pathogenesis remain unclear. More urgently, there is a lack of an animal model to aid research. Therefore, we selected a reported MMDS causal gene, Isca1, and established an animal model of MMDS complicated with cardiac dysplasia.Methods:The myocardium-specific Isca1 knockout heterozygote (Isca1 HET) rat was obtained by crossing the Isca1 conditional knockout (Isca1 cKO) rat with the α myosin heavy chain Cre (α-MHC-Cre) rat. Cardiac development characteristics were determined by ECG, blood pressure measurement, echocardiography and histopathological analysis. The responsiveness to pathological stimuli were observed through adriamycin treatment. Mitochondria and metabolism disorder were determined by activity analysis of mitochondrial respiratory chain complex and ATP production in myocardium.Results:ISCA1 expression in myocardium exhibited a semizygous effect. Isca1 HET rats exhibited dilated cardiomyopathy characteristics, including thin-walled ventricles, larger chambers, cardiac dysfunction and myocardium fibrosis. Downregulated ISCA1 led to deteriorating cardiac pathological processes at the global and organizational levels. Meanwhile, HET rats exhibited typical MMDS characteristics, including damaged mitochondrial morphology and enzyme activity for mitochondrial respiratory chain complexes Ⅰ, Ⅱ and Ⅳ, and impaired ATP production.Conclusion:We have established a rat model of MMDS complicated with cardiomyopathy, it can also be used as model of myocardial energy metabolism dysfunction and mitochondrial cardiomyopathy. This model can be applied to the study of the mechanism of energy metabolism in cardiovascular diseases, as well as research and development of drugs.

Chemokine production by epithelial cells is important for neutrophil recruitment during viral infection, the appropriate regulation of which is critical for restraining inflammation and attenuating subsequent tissue damage. Epithelial cell expression of long noncoding RNAs (lncRNAs), RNA-binding proteins, and their functional interactions during viral infection and inflammation remain to be fully understood. Here, we identified an inducible lncRNA in the Cxcl2 gene locus, lnc-Cxcl2, which could selectively inhibit Cxcl2 expression in mouse lung epithelial cells but not in macrophages. lnc-Cxcl2-deficient mice exhibited increased Cxcl2 expression, enhanced neutrophils recruitment, and more severe inflammation in the lung after influenza virus infection. Mechanistically, nucleus-localized lnc-Cxcl2 bound to Cxcl2 promoter, recruited a ribonucleoprotein La, which inhibited the chromatin accessibility of chemokine promoters, and consequently inhibited Cxcl2 transcription in cis However, unlike mouse lnc-Cxcl2, human lnc-CXCL2-4-1 inhibited multiple immune cytokine expressions including chemokines in human lung epithelial cells. Together, our results demonstrate a self-protecting mechanism within epithelial cells to restrain chemokine and neutrophil-mediated inflammation, providing clues for better understanding chemokine regulation and epithelial cell function in lung viral infection.