In vivo CRISPR editing with no detectable genome-wide off-target mutations.

CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications1-6 but identifying unwanted off-target mutations is important for clinical translation7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.

Impact of Intestinal Concentration and Colloidal Structure on the Permeation-Enhancing Efficiency of Sodium Caprate in the Rat.

In this work, we set out to better understand how the permeation enhancer sodium caprate (C10) influences the intestinal absorption of macromolecules. FITC-dextran 4000 (FD4) was selected as a model compound and formulated with 50-300 mM C10. Absorption was studied after bolus instillation of liquid formulation to the duodenum of anesthetized rats and intravenously as a reference, whereafter plasma samples were taken and analyzed for FD4 content. It was found that the AUC and Cmax of FD4 increased with increasing C10 concentration. Higher C10 concentrations were associated with an increased and extended absorption but also increased epithelial damage. Depending on the C10 concentration, the intestinal epithelium showed significant recovery already at 60-120 min after administration. At the highest studied C10 concentrations (100 and 300 mM), the absorption of FD4 was not affected by the colloidal structures of C10, with similar absorption obtained when C10 was administered as micelles (pH 8.5) and as vesicles (pH 6.5). In contrast, the FD4 absorption was lower when C10 was administered at 50 mM formulated as micelles as compared to vesicles. Intestinal dilution of C10 and FD4 revealed a trend of decreasing FD4 absorption with increasing intestinal dilution. However, the effect was smaller than that of altering the total administered C10 dose. Absorption was similar when the formulations were prepared in simulated intestinal fluids containing mixed micelles of bile salts and phospholipids and in simple buffer solution. The findings in this study suggest that in order to optimally enhance the absorption of macromolecules, high (≥100 mM) initial intestinal C10 concentrations are likely needed and that both the concentration and total dose of C10 are important parameters.

In vivo genome and base editing of a human PCSK9 knock-in hypercholesterolemic mouse model.

BACKGROUND: Plasma concentration of low-density lipoprotein (LDL) cholesterol is a well-established risk factor for cardiovascular disease. Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which regulates cholesterol homeostasis, has recently emerged as an approach to reduce cholesterol levels. The development of humanized animal models is an important step to validate and study human drug targets, and use of genome and base editing has been proposed as a mean to target disease alleles. RESULTS: To address the lack of validated models to test the safety and efficacy of techniques to target human PCSK9, we generated a liver-specific human PCSK9 knock-in mouse model (hPCSK9-KI). We showed that plasma concentrations of total cholesterol were higher in hPCSK9-KI than in wildtype mice and increased with age. Treatment with evolocumab, a monoclonal antibody that targets human PCSK9, reduced cholesterol levels in hPCSK9-KI but not in wildtype mice, showing that the hypercholesterolemic phenotype was driven by overexpression of human PCSK9. CRISPR-Cas9-mediated genome editing of human PCSK9 reduced plasma levels of human and not mouse PCSK9, and in parallel reduced plasma concentrations of total cholesterol; genome editing of mouse Pcsk9 did not reduce cholesterol levels. Base editing using a guide RNA that targeted human and mouse PCSK9 reduced plasma levels of human and mouse PCSK9 and total cholesterol. In our mouse model, base editing was more precise than genome editing, and no off-target editing nor chromosomal translocations were identified. CONCLUSIONS: Here, we describe a humanized mouse model with liver-specific expression of human PCSK9 and a human-like hypercholesterolemia phenotype, and demonstrate that this mouse can be used to evaluate antibody and gene editing-based (genome and base editing) therapies to modulate the expression of human PCSK9 and reduce cholesterol levels. We predict that this mouse model will be used in the future to understand the efficacy and safety of novel therapeutic approaches for hypercholesterolemia.

Cardiac-Specific Overexpression of Oxytocin Receptor Leads to Cardiomyopathy in Mice.

BACKGROUND: Oxytocin (Oxt) and its receptor (Oxtr) gene system has been implicated in cardiomyogenesis and cardioprotection; however, effects of chronic activation of Oxtr are not known. We generated and investigated transgenic (TG) mice that overexpress Oxtr specifically in the heart. METHODS AND RESULTS: Cardiac-specific overexpression of Oxtr was obtained by having the α-major histocompatibility complex promoter drive the mouse Oxtr gene (α-Mhc-Oxtr). Left ventricular (LV) function and remodeling were assessed by magnetic resonance imaging and echocardiography. In α-Mhc-Oxtr TG mice, LV ejection fraction was severely compromised at 14 weeks of age compared with wild-type (WT) littermates (25 ± 6% vs 63 ± 3%; P < .001). LV end-diastolic volume was larger in the TG mice (103 ± 6 µL vs 67 ± 5 µL; P < .001). α-Mhc-Oxtr TG animals displayed cardiac fibrosis, atrial thrombus, and increased expression of pro-fibrogenic genes. Mortality of α-Mhc-Oxtr TG animals was 45% compared with 0% (P < .0001) of WT littermates by 20 weeks of age. Most cardiomyocytes of α-Mhc-Oxtr TG animals but not WT littermates (68.0 ± 12.1% vs 5.6 ± 2.4%; P = .008) were positive in staining for nuclear factor of activated T cells (NFAT). To study if thrombin inhibitor prevents thrombus formation, a cohort of 7-week-old α-Mhc-Oxtr TG mice were treated for 12 weeks with AZD0837, a potent thrombin inhibitor. Treatment with AZD0837 reduced thrombus formation (P < .05) and tended to attenuate fibrosis and increase survival. CONCLUSIONS: Cardiac-specific overexpression of Oxtr had negative consequences on LV function and survival in mice. The present findings necessitate further studies to investigate potential adverse effects of chronic Oxt administration. We provide a possible mechanism of Oxtr overexpression leading to heart failure by nuclear factor of activated T cell signaling. The recapitulation of human heart failure and the beneficial effects of the antithrombin inhibitor render the α-Mhc-Oxtr TG mice a promising tool in drug discovery for heart failure.

Nonproliferative and Proliferative Lesions of the Rat and Mouse Endocrine System.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Project (www.toxpath.org/inhand.asp) is a joint initiative among the Societies of Toxicological Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the endocrine organs (pituitary gland, pineal gland, thyroid gland, parathyroid glands, adrenal glands and pancreatic islets) of laboratory rats and mice, with color photomicrographs illustrating examples of the lesions. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for endocrine lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Molecular Consequences of the SERPINH1/HSP47 Mutation in the Dachshund Natural Model of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a heritable connective tissue disease characterized by bone fragility and increased risk of fractures. Up to now, mutations in at least 18 genes have been associated with dominant and recessive forms of OI that affect the production or post-translational processing of procollagen or alter bone homeostasis. Among those, SERPINH1 encoding heat shock protein 47 (HSP47), a chaperone exclusive for collagen folding in the ER, was identified to cause a severe form of OI in dachshunds (L326P) as well as in humans (one single case with a L78P mutation). To elucidate the disease mechanism underlying OI in the dog model, we applied a range of biochemical assays to mutant and control skin fibroblasts as well as on bone samples. These experiments revealed that type I collagen synthesized by mutant cells had decreased electrophoretic mobility. Procollagen was retained intracellularly with concomitant dilation of ER cisternae and activation of the ER stress response markers GRP78 and phospho-eIF2α, thus suggesting a defect in procollagen processing. In line with the migration shift detected on SDS-PAGE of cell culture collagen, extracts of bone collagen from the OI dog showed a similar mobility shift, and on tandem mass spectrometry, the chains were post-translationally overmodified. The bone collagen had a higher content of pyridinoline than control dog bone. We conclude that the SERPINH1 mutation in this naturally occurring model of OI impairs how HSP47 acts as a chaperone in the ER. This results in abnormal post-translational modification and cross-linking of the bone collagen.

Tailored Pig Models for Preclinical Efficacy and Safety Testing of Targeted Therapies.

Despite enormous advances in translational biomedical research, there remains a growing demand for improved animal models of human disease. This is particularly true for diseases where rodent models do not reflect the human disease phenotype. Compared to rodents, pig anatomy and physiology are more similar to humans in cardiovascular, immune, respiratory, skeletal muscle, and metabolic systems. Importantly, efficient and precise techniques for genetic engineering of pigs are now available, facilitating the creation of tailored large animal models that mimic human disease mechanisms at the molecular level. In this article, the benefits of genetically engineered pigs for basic and translational research are exemplified by a novel pig model of Duchenne muscular dystrophy and by porcine models of cystic fibrosis. Particular emphasis is given to potential advantages of using these models for efficacy and safety testing of targeted therapies, such as exon skipping and gene editing, for example, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system. In general, genetically tailored pig models have the potential to bridge the gap between proof-of-concept studies in rodents and clinical trials in patients, thus supporting translational medicine.

Tissue Sampling Guides for Porcine Biomedical Models.

This article provides guidelines for organ and tissue sampling adapted to porcine animal models in translational medical research. Detailed protocols for the determination of sampling locations and numbers as well as recommendations on the orientation, size, and trimming direction of samples from ∼50 different porcine organs and tissues are provided in the Supplementary Material. The proposed sampling protocols include the generation of samples suitable for subsequent qualitative and quantitative analyses, including cryohistology, paraffin, and plastic histology; immunohistochemistry;in situhybridization; electron microscopy; and quantitative stereology as well as molecular analyses of DNA, RNA, proteins, metabolites, and electrolytes. With regard to the planned extent of sampling efforts, time, and personnel expenses, and dependent upon the scheduled analyses, different protocols are provided. These protocols are adjusted for (I) routine screenings, as used in general toxicity studies or in analyses of gene expression patterns or histopathological organ alterations, (II) advanced analyses of single organs/tissues, and (III) large-scale sampling procedures to be applied in biobank projects. Providing a robust reference for studies of porcine models, the described protocols will ensure the efficiency of sampling, the systematic recovery of high-quality samples representing the entire organ or tissue as well as the intra-/interstudy comparability and reproducibility of results.

Pharmacological inhibition of DGAT1 induces sebaceous gland atrophy in mouse and dog skin while overt alopecia is restricted to the mouse.

Diacylglycerol O-acyltransferase 1 (DGAT1) plays an important role in synthesizing lipids, and inhibitors of DGAT1 have been investigated as potential treatments for diabetes and metabolic diseases. DGAT1 knockout (-/-) mice are resistant to obesity, have increased sensitivity to insulin, and exhibit sebaceous gland atrophy and alopecia. Prolonged pharmacological inhibition of DGAT1 with AZD7687 in mice results in the same skin phenotype, including sebaceous gland atrophy and alopecia, as seen in the skin of DGAT1 (-/-) mice. AZD7687-mediated effects on the skin were dose- and time-dependent and reversible. They occurred only at substantial levels of continuous DGAT1 inhibition. Prolonged treatment of dogs with AZD7687 also resulted in sebaceous gland atrophy but did not result in the more adverse skin changes of hair loss and skin lesions. Our findings highlight a significant risk of generating the same lesions that were seen in mouse skin during clinical development of DGAT1 inhibitors in humans and also reveal a species difference in the effects on the skin, indicating that the mouse may be an especially sensitive species. Therefore, although human therapeutic doses may not have the same influence on skin morphology as seen in mice, monitoring of skin changes will be essential in clinical trials with DGAT1 inhibitors.

A missense mutation in the SERPINH1 gene in Dachshunds with osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a hereditary disease occurring in humans and dogs. It is characterized by extremely fragile bones and teeth. Most human and some canine OI cases are caused by mutations in the COL1A1 and COL1A2 genes encoding the subunits of collagen I. Recently, mutations in the CRTAP and LEPRE1 genes were found to cause some rare forms of human OI. Many OI cases exist where the causative mutation has not yet been found. We investigated Dachshunds with an autosomal recessive form of OI. Genotyping only five affected dogs on the 50 k canine SNP chip allowed us to localize the causative mutation to a 5.82 Mb interval on chromosome 21 by homozygosity mapping. Haplotype analysis of five additional carriers narrowed the interval further down to 4.74 Mb. The SERPINH1 gene is located within this interval and encodes an essential chaperone involved in the correct folding of the collagen triple helix. Therefore, we considered SERPINH1 a positional and functional candidate gene and performed mutation analysis in affected and control Dachshunds. A missense mutation (c.977C>T, p.L326P) located in an evolutionary conserved domain was perfectly associated with the OI phenotype. We thus have identified a candidate causative mutation for OI in Dachshunds and identified a fifth OI gene.

Subcutaneous delivery of FGF21 mRNA therapy reverses obesity, insulin resistance, and hepatic steatosis in diet-induced obese mice.

Fibroblast growth factor 21 (FGF21) is a promising therapeutic agent for treatment of type 2 diabetes (T2D) and non-alcoholic steatohepatitis (NASH). We show that therapeutic levels of FGF21 were achieved following subcutaneous (s.c.) administration of mRNA encoding human FGF21 proteins. The efficacy of mRNA was assessed following 2-weeks repeated s.c. dosing in diet-induced obese (DIO), mice which resulted in marked decreases in body weight, plasma insulin levels, and hepatic steatosis. Pharmacokinetic/pharmacodynamic (PK/PD) modelling of several studies in both lean and DIO mice showed that mRNA encoding human proteins provided improved therapeutic coverage over recombinant dosed proteins in vivo. This study is the first example of s.c. mRNA therapy showing pre-clinical efficacy in a disease-relevant model, thus, showing the potential for this modality in the treatment of chronic diseases, including T2D and NASH.

Holistic Characterization of a Salmonella Typhimurium Infection Model Using Integrated Molecular Imaging.

A more complete and holistic view on host-microbe interactions is needed to understand the physiological and cellular barriers that affect the efficacy of drug treatments and allow the discovery and development of new therapeutics. Here, we developed a multimodal imaging approach combining histopathology with mass spectrometry imaging (MSI) and same section imaging mass cytometry (IMC) to study the effects of Salmonella Typhimurium infection in the liver of a mouse model using the S. Typhimurium strains SL3261 and SL1344. This approach enables correlation of tissue morphology and specific cell phenotypes with molecular images of tissue metabolism. IMC revealed a marked increase in immune cell markers and localization in immune aggregates in infected tissues. A correlative computational method (network analysis) was deployed to find metabolic features associated with infection and revealed metabolic clusters of acetyl carnitines, as well as phosphatidylcholine and phosphatidylethanolamine plasmalogen species, which could be associated with pro-inflammatory immune cell types. By developing an IMC marker for the detection of Salmonella LPS, we were further able to identify and characterize those cell types which contained S. Typhimurium.

Functionalized lipid nanoparticles for subcutaneous administration of mRNA to achieve systemic exposures of a therapeutic protein.

Lipid nanoparticles (LNPs) are the most clinically advanced delivery system for RNA-based drugs but have predominantly been investigated for intravenous and intramuscular administration. Subcutaneous administration opens the possibility of patient self-administration and hence long-term chronic treatment that could enable messenger RNA (mRNA) to be used as a novel modality for protein replacement or regenerative therapies. In this study, we show that subcutaneous administration of mRNA formulated within LNPs can result in measurable plasma exposure of a secreted protein. However, subcutaneous administration of mRNA formulated within LNPs was observed to be associated with dose-limiting inflammatory responses. To overcome this limitation, we investigated the concept of incorporating aliphatic ester prodrugs of anti-inflammatory steroids within LNPs, i.e., functionalized LNPs to suppress the inflammatory response. We show that the effectiveness of this approach depends on the alkyl chain length of the ester prodrug, which determines its retention at the site of administration. An unexpected additional benefit to this approach is the prolongation observed in the duration of protein expression. Our results demonstrate that subcutaneous administration of mRNA formulated in functionalized LNPs is a viable approach to achieving systemic levels of therapeutic proteins, which has the added benefits of being amenable to self-administration when chronic treatment is required.

A MALT1 inhibitor suppresses human myeloid DC, effector T-cell and B-cell responses and retains Th1/regulatory T-cell homeostasis.

The paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) regulates nuclear-factor-kappa-B (NF-κB) activation downstream of surface receptors with immunoreceptor tyrosine-based activation motifs (ITAMs), such as the B-cell or T-cell receptor and has thus emerged as a therapeutic target for autoimmune diseases. However, recent reports demonstrate the development of lethal autoimmune inflammation due to the excessive production of interferon gamma (IFN-É£) and defective differentiation of regulatory T-cells in genetically modified mice deficient in MALT1 paracaspase activity. To address this issue, we explored the effects of pharmacological MALT1 inhibition on the balance between T-effector and regulatory T-cells. Here we demonstrate that allosteric inhibition of MALT1 suppressed Th1, Th17 and Th1/Th17 effector responses, and inhibited T-cell dependent B-cell proliferation and antibody production. Allosteric MALT1 inhibition did not interfere with the suppressive function of human T-regulatory cells, although it impaired de novo differentiation of regulatory T-cells from naïve T-cells. Treatment with an allosteric MALT1 inhibitor alleviated the cytokine storm, including IFN-É£, in a mouse model of acute T-cell activation, and long-term treatment did not lead to an increase in IFN-É£ producing CD4 cells or tissue inflammation. Together, our data demonstrate that the effects of allosteric inhibition of MALT1 differ from those seen in mice with proteolytically inactive MALT1, and thus we believe that MALT1 is a viable target for B and T-cell driven autoimmune diseases.

Development of an ObLiGaRe Doxycycline Inducible Cas9 system for pre-clinical cancer drug discovery.

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.

Monosodium iodoacetate-induced monoarthritis develops differently in knee versus ankle joint in rats.

OBJECTIVE: Disability and movement-related pain are major symptoms of joint disease, motivating the development of methods to quantify motor behaviour in rodent joint pain models. We compared effects on behaviour, assessed the levels of biochemical mediators and made a detailed histopathological evaluation after induction of rat monoiodoacetate (MIA) monoarthritis into the ankle or knee joint. DESIGN: Twenty-seven male Lewis rats were used. Before and up to 28 days after induction, they were tested for weight bearing during walking (dynamic), and standing (static), and for mechanical sensitivity. At termination synovial fluid was taken from ankle and/or knee joints for analysis of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), macrophage inflammatory protein 3 alpha (MIP-3α), keratinocyte chemoattractant (KC)/human growth-regulated oncogene (GRO) and L(+)-lactate, and from separate rats joints were collected for histopathological assessment. RESULTS: MIA ankle joint injection gave a marked reduction of dynamic weight bearing during the first days, not seen in rats with knee joint injection. At three weeks, it was decreased in the group with knee injection, but not in those with ankle injection. However, the different injection sites caused similar reductions in static weight bearing during the early phase, which was normalized in the group with ankle injection but continued and was strengthened with time in the knee injected group. Histopathological assessment, biochemical mediators and joint swelling confirmed the disparate profiles. CONCLUSIONS: This work shows that ankle versus knee joint injection of MIA resulted in different profiles in rats, which may mirror what has been found in human patients with osteoarthritis.

IDOL regulates systemic energy balance through control of neuronal VLDLR expression.

Liver X receptors limit cellular lipid uptake by stimulating the transcription of Inducible Degrader of the LDL Receptor (IDOL), an E3 ubiquitin ligase that targets lipoprotein receptors for degradation. The function of IDOL in systemic metabolism is incompletely understood. Here we show that loss of IDOL in mice protects against the development of diet-induced obesity and metabolic dysfunction by altering food intake and thermogenesis. Unexpectedly, analysis of tissue-specific knockout mice revealed that IDOL affects energy balance, not through its actions in peripheral metabolic tissues (liver, adipose, endothelium, intestine, skeletal muscle), but by controlling lipoprotein receptor abundance in neurons. Single-cell RNA sequencing of the hypothalamus demonstrated that IDOL deletion altered gene expression linked to control of metabolism. Finally, we identify VLDLR rather than LDLR as the primary mediator of IDOL effects on energy balance. These studies identify a role for the neuronal IDOL-VLDLR pathway in metabolic homeostasis and diet-induced obesity.

Lack of detectable West Nile virus RNA in brains and kidneys of dogs and cats with immunohistological precipitates using virus-specific antibodies.

Five dogs and four cats from Germany suffering from encephalitis revealed positive immunoreactivity using two West Nile virus (WNV) specific monoclonal antibodies in brain and in kidney. However, WNV-infection could not be confirmed by additional PCR analyses. This study indicated that positive immunoreactivity for WNV in dogs and cats must be interpreted cautiously and should be confirmed by a second virus-specific technique.

Therapeutic Genome Editing With CRISPR/Cas9 in a Humanized Mouse Model Ameliorates α1-antitrypsin Deficiency Phenotype.

α1-antitrypsin (AAT) is a circulating serine protease inhibitor secreted from the liver and important in preventing proteolytic neutrophil elastase associated tissue damage, primarily in lungs. In humans, AAT is encoded by the SERPINA1 (hSERPINA1) gene in which a point mutation (commonly referred to as PiZ) causes aggregation of the miss-folded protein in hepatocytes resulting in subsequent liver damage. In an attempt to rescue the pathologic liver phenotype of a mouse model of human AAT deficiency (AATD), we used adenovirus to deliver Cas9 and a guide-RNA (gRNA) molecule targeting hSERPINA1. Our single dose therapeutic gene editing approach completely reverted the phenotype associated with the PiZ mutation, including circulating transaminase and human AAT (hAAT) protein levels, liver fibrosis and protein aggregation. Furthermore, liver histology was significantly improved regarding inflammation and overall morphology in hSERPINA1 gene edited PiZ mice. Genomic analysis confirmed significant disruption to the hSERPINA1 transgene resulting in a reduction of hAAT protein levels and quantitative mRNA analysis showed a reduction in fibrosis and hepatocyte proliferation as a result of editing. Our findings indicate that therapeutic gene editing in hepatocytes is possible in an AATD mouse model.

MMP-12, MMP-3, and TIMP-1 are markedly upregulated in chronic demyelinating theiler murine encephalomyelitis.

Theiler murine encephalomyelitis (TME) represents a highly relevant viral model for multiple sclerosis. Matrix metalloproteinases (MMPs) degrade extracellular matrix molecules and are involved in demyelination processes. To elucidate their impact on demyelination in TME, spinal cords of TME virus (TMEV)-infected SJL/J mice were taken at 9 different time points postinfection (pi) ranging from 1 hour to 196 days pi and investigated for the expression of TMEV, MMP-2, -3, -7, -9, -10, -11, -12, -13, -14, -15, -24, and TIMP-1 to -4 by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). High TMEV RNA levels were detectable throughout the observation period using RT-qPCR. In addition, TMEV RNA was visualized within demyelinated lesions by in situ hybridization. MMP-3 mRNA was significantly upregulated at 1 day pi and again in the late phase of infection. TIMP-1 mRNA was significantly elevated throughout the observation period. MMP-12 mRNA was most prominently upregulated in the late phase of infection and MMP-12 protein was localized in intralesional microglia/macrophages and astrocytes by immunohistochemistry. In summary, in early TMEV infection, MMP-3 and TIMP-1 mRNA upregulation might be directly virus-induced, whereas persistent TMEV infection directly or indirectly stimulated MMP-12 production in microglia/macrophages and astrocytes and might account for ongoing demyelination in TME.