Mouse DNA Research Articles

The reconstruction of evolutionary dynamics of processed pseudogenes indicates deep silencing of “retrobiome” in naked mole rat

Approximately half of mammalian genomes are occupied by retrotransposons, highly repetitive interspersed genetic elements expanded through the mechanism of reverse transcription. The evolution of this "retrobiome" involved a series of explosive amplifications, presumably associated with high mutation rates, interspersed with periods of silencing. A by-product of retrotransposon activity is the formation of processed pseudogenes (PPGs)-intron-less, promoter-less DNA copies of messenger RNA (mRNA). We examined the proportion of PPGs with varying degrees of deviation from their ancestor mRNAs as an indicator of the intensity of retrotranspositions at different times in the past. Our analysis revealed a high proportion of "young'' (recently acquired) PPGs in the DNA of mice and rats, indicating significant retrobiome activity during the recent evolution of these species. The ongoing process of new PPG entries in mouse germ line DNA was confirmed by identifying diversity in PPG content within the single strain of mice, C57BL/6. In contrast, the highly abundant PPGs of the naked mole rat (NMR) exhibited substantial deviation from their mRNAs, with a near-complete lack of PPGs without mutations, indicative of the silencing of the retrobiome in the most recent evolutionary past, preceded by a period of high activity. This distinctive feature of the NMR genome was confirmed through the analysis of a broad range of mammalian species. The peculiar evolutionary dynamics of PPGs in the NMR, an organism with exceptional longevity and resistance to cancer, may reflect the role played by the retrobiome in aging and cancer.

Genome-wide DNA methylation sequencing reveals the involvement of ferroptosis in hepatotoxicity induced by dietary exposure to food-grade titanium dioxide

BackgroundFollowing the announcement by the European Food Safety Authority that the food additive titanium dioxide (E 171) is unsafe for human consumption, and the subsequent ban by the European Commission, concerns have intensified over the potential risks E 171 poses to human vital organs. The liver is the main organ for food-grade nanoparticle metabolism. It is increasingly being found that epigenetic changes may play an important role in nanomaterial-induced hepatotoxicity. However, the profound effects of E 171 on the liver, especially at the epigenetic level, remain largely unknown.MethodsMice were exposed orally to human-relevant doses of two types of E 171 mixed in diet for 28 and/or 84 days. Conventional toxicology and global DNA methylation analyses were performed to assess E 171-induced hepatotoxicity and epigenetic changes. Whole genome bisulfite sequencing and further ferroptosis protein detection were used to reveal E 171-induced changes in liver methylation profiles and toxic mechanisms.ResultsExposed to E 171 for 28 and/or 84 days resulted in reduced global DNA methylation and hydroxymethylation in the liver of mice. E 171 exposure for 84 days elicited inflammation and damage in the mouse liver, whereas 28-day exposure did not. Whole-genome DNA methylation sequencing disclosed substantial methylation alterations at the CG and non-CG sites of the liver DNA in mice exposed to E 171 for 84 days. Mechanistic analysis of the DNA methylation alterations indicated that ferroptosis contributed to the liver toxicity induced by E 171. E 171-induced DNA methylation changes triggered NCOA4-mediated ferritinophagy, attenuated the protein levels of GPX4, FTH1, and FTL in the liver, and thereby caused ferroptosis.ConclusionsLong-term oral exposure to E 171 triggers hepatotoxicity and induces methylation changes in both CG and non-CG sites of liver DNA. These epigenetic alterations activate ferroptosis in the liver through NCOA4-mediated ferritinophagy, highlighting the role of DNA methylation and ferroptosis in the potential toxicity caused by E 171 in vivo.

Rapid detection of murine-derived ingredients in meat products using real-time enzymatic recombinase amplification.

Accurate identification of meat species is critical to prevent economic fraud and safeguard public health. The use of inappropriate meat sources, such as murine, poses significant health risks because of potential contamination with pathogens and allergens, leading to foodborne illnesses. The present study aimed to develop a novel real-time enzymatic recombinase amplification (ERA) method for the rapid and specific detection of murine DNA in meat products. A novel ERA primer and probe set was designed, targeting a murine-specific single-copy nuclear gene identified through bioinformatics analysis. The assay demonstrates high specificity, showing no amplification in commonly consumed meats, other animals or major crops. Additionally, it exhibits remarkable sensitivity, detecting as few as five copies of murine genomic DNA. For practical application, the ERA method could effectively identify mouse DNA in laboratory-prepared samples at concentrations as low as 0.5% and also quantify samples with mouse DNA content as low as 5%. It also accurately detects the presence of murine-derived ingredients in commercially available meat products. The detection process is straightforward, utilizing a simple isothermal device for incubation, blue light excitation and a smartphone camera for result interpretation. This rapid analysis can be completed within 20 min. The newly developed real-time ERA method provides a valuable tool for standardizing meat trade practices, promoting food safety and enhancing consumer confidence in the authenticity of meat products. © 2024 Society of Chemical Industry.

Immunogenic Comparison of Nucleic Acid-Based Vaccines Administered by Pyro-Drive Jet Injector.

mRNA vaccines were successfully developed and approved for emergency use to fight coronavirus disease 2019. However, the effect of DNA vaccines against SARS-CoV-2 is considerably lower than that of mRNA vaccines. A pyro-drive jet injector (PJI) efficiently delivers plasmid DNA intradermally into animal models. Here, we compared the immunogenic potential of DNA and mRNA vaccines in mice using the same platform. PJI was used to deliver naked mRNA and pDNA and their efficacy in inducing antigen expression and immune responses was assessed. Our results showed that PJI efficiently delivered mRNA into the skin, and a smaller effective dose than that of pDNA injection was required to achieve similar levels of antigen expression. The PJI-delivered CpG-free pDNA vaccine efficiently induced antigen-specific antibody production and a cell-mediated IFN-γ response compared to the mRNA vaccine, as well as the upregulation of inflammatory cytokines (IL-6, IFN-γ, and IL-1β) in the skin and lymph nodes. However, the intradermal mRNA vaccine was significantly less immunogenic than the standard intramuscular mRNA-lipid nanoparticle vaccine, despite equivalent mRNA dosages. Improvements in lipid nanoparticle and mRNA technology have revolutionized mRNA vaccines, and DNA vaccines can be similarly modified for higher clinical efficacy.

Abstract 2071: Identification Of Genetic Loci Influencing Lethal Atherothrombosis In Mice

Introduction: Atherothrombosis leading to myocardial infarction and stroke is a major cause of mortality. Ruptured atherosclerotic plaques release prothrombotic molecules such as tissue factor, which initiates thrombosis. Both genetic and environmental factors contribute to atherothrombosis. Combined complete deficiency of the Scavenger Receptor Class B type 1 (Scarb1) and Apolipoprotein E (ApoE) genes (dKO) on a mixed C57BL/6J (B6) and 129S1/SvImJ (129S1) background leads to an atherothrombotic phenotype and early death before 8 weeks of age. However, the dKO phenotype is dramatically different if the Scarb1 and ApoE deficient mice are backcrossed extensively to the B6 mouse strain, with no dKOs surviving to the weaning age. Hypothesis/Goals: We hypothesized that 129S1 genetic variants reduce atherothrombosis severity in dKO mice and used mouse genetics to identify these loci. Methods: To identify potential genetic factors, we produced and analyzed Scarb1 and ApoE dKO mice from 6 backcross/intercross generations with progressively more B6 genomic contribution. Weights were taken for 38 dKO mice and littermates from 3 weeks old until death of the dKO mice at 8 weeks of age. dKO mice from F1-F3 generations were divided into 3 distinct phenotypes based on their weights relative to their littermates. dKO mouse DNA was subjected to genome wide marker genotyping using the GigaMUGA platform. Quantitative trait loci analysis (QTL) was performed to identify loci responsible for the phenotypic weight differences. Results: dKO weights fell into 3 categories: indistinguishable from littermates, 14%-39% less than littermates, and greater than 39% less than littermates. QTL analyses identified significant interactions of Chr 12 with Chrs 1, 2, 3, and 14. Niemann Pick type C2 (Npc2), an intracellular cholesterol transporter, is within the interaction region on Chr 12 and is a prime candidate dKO modifier. Conclusion: We used weight data as a representative of overall health to categorize dKO phenotypes relative to non dKO littermates. We identified 6 significant genetic loci, with Npc2 as a candidate modifier gene for the Chromosome 12 locus. Identification of atherothrombosis modifier genes will provide novel therapeutic targets for this complex disease.

DTYMK is an essential gene in mice and heterozygosity does not cause neural tube defects

Regulation of nucleotide biosynthesis is necessary for maintaining cellular processes including DNA replication and repair. A key enzyme in this process is deoxythymidylate kinase (dTYMK), which catalyzes the initial step in the production of dTTP from dTMP. This gene constitutes the first merged step of dTTP synthesis from the de novo and salvage pathways which regulate dTMP biosynthesis. Decreased de novo dTMP biosynthesis causes dysregulated dTTP:dUTP pools, and leads to increased uracil in DNA and neural tube closure defect (NTD) development in mice. The goal of this research was to investigate if dTYMK, the downstream enzyme in dTTP production, is an essential gene in mice and if impairments in dTYMK play a causal role in development including NTD pathology in mice. Dtymk+/− C57BL/6J females were weaned onto either a control, excess folic acid, or folic acid deficient diet and timed breeding was performed after 8 weeks on diet. The offspring were analyzed for NTDs and other reproductive outcomes at embryonic day 12.5 (E12.5). Dtymk–/– mice were confirmed to be embryonic lethal before E12.5, and Dtymk+/− mice on all three experimental diets did not show the presence of open neural tube defects, spina bifida or exencephaly. However, the expression of dTYMK in Dtymk+/− mouse embryos was confirmed to be decreased by approximately 3-fold compared to Dtymk+/+ embryos. Although dTYMK was demonstrated to be an essential gene in mice and is required for the regulation of nucleotide pools in vitro, there was no evidence of increased risk of NTDs because of a reduction in expression of this enzyme during embryonic development. It is possible that a further reduction in expression may be required to see developmental anomalies in C57BL/6J mice.

Mitochondrial DNA as a target for analyzing the biodistribution of cell therapy products.

Biodistribution tests are crucial for evaluating the safety of cell therapy (CT) products in order to prevent unwanted organ homing of these products in patients. Quantitative polymerase chain reaction (qPCR) using intronic Alu is a popular method for biodistribution testing owing to its ability to detect donor cells without modifying CT products and low detection limit. However, Alu-qPCR may generate inaccurate information owing to background signals caused by the mixing of human genomic DNA with that of experimental animals. The aim of this study was to develop a test method that is more specific and sensitive than Alu-qPCR, targeting the mitochondrial DNA (mtDNA) sequence that varies substantially between humans and experimental animals. We designed primers for 12S, 16S, and cytochrome B in mtDNA regions, assessed their specificity and sensitivity, and selected primers and probes for the 12S region. Human adipose-derived stem cells, used as CT products, were injected into the tail vein of athymic NCr-nu/nu mice and detected, 7 d after administration, in their lungs at an average concentration of 2.22 ± 0.69 pg/μg mouse DNA, whereas Alu was not detected. Therefore, mtDNA is more specific and sensitive than Alu and is a useful target for evaluating CT product biodistribution.

TIME-seq reduces time and cost of DNA methylation measurement for epigenetic clock construction.

Epigenetic 'clocks' based on DNA methylation have emerged as the most robust and widely used aging biomarkers, but conventional methods for applying them are expensive and laborious. Here we develop tagmentation-based indexing for methylation sequencing (TIME-seq), a highly multiplexed and scalable method for low-cost epigenetic clocks. Using TIME-seq, we applied multi-tissue and tissue-specific epigenetic clocks in over 1,800 mouse DNA samples from eight tissue and cell types. We show that TIME-seq clocks are accurate and robust, enriched for polycomb repressive complex 2-regulated loci, and benchmark favorably against conventional methods despite being up to 100-fold less expensive. Using dietary treatments and gene therapy, we find that TIME-seq clocks reflect diverse interventions in multiple tissues. Finally, we develop an economical human blood clock (R > 0.96, median error = 3.39 years) in 1,056 demographically representative individuals. These methods will enable more efficient epigenetic clock measurement in larger-scale human and animal studies.

Glabridin improves autoimmune disease in Trex1-deficient mice by reducing type I interferon production

BackgroundThe cGAS-STING signaling pathway is an essential section of the natural immune system. In recent years, an increasing number of studies have shown a strong link between abnormal activation of the cGAS-STING signaling pathway, a natural immune pathway mediated by the nucleic acid receptor cGAS, and the development and progression of autoimmune diseases. Therefore, it is important to identify an effective compound to specifically downregulate this pathway for disease.MethodsThe effect of Glabridin (Glab) was investigated in BMDMs and Peripheral blood mononuclear cell (PBMC) by establishing an in vitro model of cGAS-STING signaling pathway activation. An activation model stimulated by DMXAA was also established in mice to study the effect of Glab. On the other hand, we investigated the possible mechanism of action of Glab and the effect of Glab on Trex1-deficient mice.ResultsIn this research, we report that Glab, a major component of licorice, specifically inhibits the cGAS-STING signaling pathway by inhibiting the level of type I interferon and inflammatory cytokines (IL-6 and TNF-α). In addition, Glab has a therapeutic effect on innate immune diseases caused by abnormal cytoplasmic DNA in Trex1-deficient mice. Mechanistically, Glab can specifically inhibit the interaction of STING with IRF3.ConclusionGlab is a specific inhibitor of the cGAS-STING signaling pathway and may be used in the clinical therapy of cGAS-STING pathway-mediated autoimmune diseases.

The Expression Kinetics and Immunogenicity of Lipid Nanoparticles Delivering Plasmid DNA and mRNA in Mice.

In recent years, lipid nanoparticles (LNPs) have emerged as a revolutionary technology for vaccine delivery. LNPs serve as an integral component of mRNA vaccines by protecting and transporting the mRNA payload into host cells. Despite their prominence in mRNA vaccines, there remains a notable gap in our understanding of the potential application of LNPs for the delivery of DNA vaccines. In this study, we sought to investigate the suitability of leading LNP formulations for the delivery of plasmid DNA (pDNA). In addition, we aimed to explore key differences in the properties of popular LNP formulations when delivering either mRNA or DNA. To address these questions, we compared three leading LNP formulations encapsulating mRNA- or pDNA-encoding firefly luciferase based on potency, expression kinetics, biodistribution, and immunogenicity. Following intramuscular injection in mice, we determined that RNA-LNPs formulated with either SM-102 or ALC-0315 lipids were the most potent (all p-values < 0.01) and immunogenic (all p-values < 0.05), while DNA-LNPs formulated with SM-102 or ALC-0315 demonstrated the longest duration of signal. Additionally, all LNP formulations were found to induce expression in the liver that was proportional to the signal at the injection site (SM102: r = 0.8787, p < 0.0001; ALC0315: r = 0.9012, p < 0.0001; KC2: r = 0.9343, p < 0.0001). Overall, this study provides important insights into the differences between leading LNP formulations and their applicability to DNA- and RNA-based vaccinations.

Genotoxic Impacts of Gluten on Chromosomes and DNA of Mice

Genotoxic Impacts of Gluten on Chromosomes and DNA of Mice

Evaluation of Genotoxic and Cytotoxic Effects of Gluten in Male Albino Mice

Gluten is a protein commonly found in daily diets in the form of wheat, barley, rye, and other grains. It serves as the structural component in flour, providing the binding qualities that maintain the shape and texture of food items. This study aimed to investigate the genotoxic and cytotoxic effects of gluten on bone marrow chromosomes and DNA of male albino mice. The animals were divided into four groups: a control group, a negative control group that received an oral dose of 0.02M glacial acetic acid, and two groups that were treated with gluten dissolved in 0.02M glacial acetic acid at doses of 1.5 g/kg and 3.0 g/kg body weight. The treated animals received oral doses with non-consecutively three times a week for a period of four weeks. The study evaluated chromosomal aberrations in the bone marrow, micronucleus test, and DNA damage using the comet assay. The results of the study showed that treatment with 1.5 and 3.0g/kg body weight of gluten induced chromosomal aberrations and damage in DNA content, with an increase in the severity of effects at a higher dose of gluten. The chromosomal aberrations seen included deletion, fragment, centromeric attenuation, centric fusion, ring formation, end to end association, chromosomal gap, beaded chromosomes, and polyploidy. The micronucleus test revealed toxicity in the bone marrow, as shown by appearance of micronuclei in polychromatic erythrocytes and a reduction in the ratio of polychromatic erythrocytes. The comet assay showed a significant increase of DNA damage in the tail length of the comet cells. This study concluded that the treatment with gluten has detrimental effects on the bone marrow chromosomes and DNA of mice, as demonstrated by the increased chromosomal aberrations, micronuclei, and DNA damage observed in the treated mice. So, the use of gluten should be within an acceptable and safe range.

Targeting STING oligomerization with small-molecule inhibitors.

Stimulator of interferon genes (STING) is an essential adaptor protein required for the inflammatory response to cytosolic DNA. dsDNA activates cGAS to generate cGAMP, which binds and activates STING triggering a conformational change, oligomerization, and the IRF3- and NFκB-dependent transcription of type I Interferons (IFNs) and inflammatory cytokines, as well as the activation of autophagy. Aberrant activation of STING is now linked to a growing number of both rare as well as common chronic inflammatory diseases. Here, we identify and characterize a potent small-molecule inhibitor of STING. This compound, BB-Cl-amidine inhibits STING signaling and production of type I IFNs, IFN-stimulated genes (ISGs) and NFκB-dependent cytokines, but not other pattern recognition receptors. In vivo, BB-Cl-amidine alleviated pathology resulting from accrual of cytosolic DNA in Trex-1 mutant mice. Mechanistically BB-Cl-amidine inhibited STING oligomerization through modification of Cys148. Collectively, our work uncovers an approach to inhibit STING activation and highlights the potential of this strategy for the treatment of STING-driven inflammatory diseases.

Genetic Divergence and Evolutionary Transition/Transversion Rate Bias in the Control Region of Mitochondrial DNA of Palearctic Mice (Murinae)

Genetic Divergence and Evolutionary Transition/Transversion Rate Bias in the Control Region of Mitochondrial DNA of Palearctic Mice (Murinae)

Astragalus Mongolians' Suppression of HBV DNA Level in ChronicHepatitis B (CHB) Patients with Moderately Increased AlanineAminotransferase (ALT)

This paper aims to examine the impact of Astragalus on the quantity of HBV DNA in chronic hepatitis B (CHB) patientswith slightly raised ALT and the effect on IFN-G. Moreover, the work predicted that IFN-gamma would increase aftertaking Astragalus Mongolians in CHB mouse patients with mildly raised ALT and effectively suppress the amount ofHBV DNA. Three groups of mice were set up, including a positive control group, a negative control group, and a groupof mice with mildly elevated ALT, which are more significant than one to two times the upper limit of normal (1-2 timesULN) and carrying CHB. Astragalus was administered to mice previously infected with HBV in increasing doses andfor longer lengths of time. Entecavir was a positive control, while PBS + DMSO was the negative control. IFN-g in theblood was detected using an ELISA test, and HBV DNA was determined using RT-PCR. The six possible results in thispaper correspond to the reaction of CHB-carrying mice with mildly elevated ALT to Astragalus. Whether the amount ofIFN-G and HBV DNA was elevated or reduced. The results of this paper will effectively predict the effect of Astragaluson the amount of HBV DNA in CHB-carrying mice with mildly raised ALT with several different outcomes. This paperalso analyzes the different outcomes and compares the changes in IFN-G; corresponding conclusions will be drawnbased on the different outcomes.

A Novel Repetition Frequency-Based DNA Encoding Scheme to Predict Human and Mouse DNA Enhancers with Deep Learning.

Recent studies have shown that DNA enhancers have an important role in the regulation of gene expression. They are responsible for different important biological elements and processes such as development, homeostasis, and embryogenesis. However, experimental prediction of these DNA enhancers is time-consuming and costly as it requires laboratory work. Therefore, researchers started to look for alternative ways and started to apply computation-based deep learning algorithms to this field. Yet, the inconsistency and unsuccessful prediction performance of computational-based approaches among various cell lines led to the investigation of these approaches as well. Therefore, in this study, a novel DNA encoding scheme was proposed, and solutions were sought to the problems mentioned and DNA enhancers were predicted with BiLSTM. The study consisted of four different stages for two scenarios. In the first stage, DNA enhancer data were obtained. In the second stage, DNA sequences were converted to numerical representations by both the proposed encoding scheme and various DNA encoding schemes including EIIP, integer number, and atomic number. In the third stage, the BiLSTM model was designed, and the data were classified. In the final stage, the performance of DNA encoding schemes was determined by accuracy, precision, recall, F1-score, CSI, MCC, G-mean, Kappa coefficient, and AUC scores. In the first scenario, it was determined whether the DNA enhancers belonged to humans or mice. As a result of the prediction process, the highest performance was achieved with the proposed DNA encoding scheme, and an accuracy of 92.16% and an AUC score of 0.85 were calculated, respectively. The closest accuracy score to the proposed scheme was obtained with the EIIP DNA encoding scheme and the result was observed as 89.14%. The AUC score of this scheme was measured as 0.87. Among the remaining DNA encoding schemes, the atomic number showed an accuracy score of 86.61%, while this rate decreased to 76.96% with the integer scheme. The AUC values of these schemes were 0.84 and 0.82, respectively. In the second scenario, it was determined whether there was a DNA enhancer and, if so, it was decided to which species this enhancer belonged. In this scenario, the highest accuracy score was obtained with the proposed DNA encoding scheme and the result was 84.59%. Moreover, the AUC score of the proposed scheme was determined as 0.92. EIIP and integer DNA encoding schemes showed accuracy scores of 77.80% and 73.68%, respectively, while their AUC scores were close to 0.90. The most ineffective prediction was performed with the atomic number and the accuracy score of this scheme was calculated as 68.27%. Finally, the AUC score of this scheme was 0.81. At the end of the study, it was observed that the proposed DNA encoding scheme was successful and effective in predicting DNA enhancers.

Co-delivery of Cas9 mRNA and guide RNAs edits hepatitis B virus episomal and integration DNA in mouse and tree shrew models

With 296 million chronically infected individuals worldwide, hepatitis B virus (HBV) causes a major health burden. The major challenge to cure HBV infection lies in the fact that the source of persistence infection, viral episomal covalently closed circular DNA (cccDNA), could not be targeted. In addition, HBV DNA integration, although normally results in replication-incompetent transcripts, considered as oncogenic. Though several studies evaluated the potential of gene-editing approaches to target HBV, previous in vivo studies have been of limited relevance to authentic HBV infection, as the models do not contain HBV cccDNA or feature a complete HBV replication cycle under competent host immune system. In this study, we evaluated the effect of in vivo codelivery of Cas9 mRNA and guide RNAs (gRNAs) by SM-102-based lipid nanoparticles (LNPs) on HBV cccDNA and integrated DNA in mouse and a higher species. CRISPR nanoparticle treatment decreased the levels of HBcAg, HBsAg and cccDNA in AAV-HBV1.04 transduced mouse liver by 53%, 73% and 64% respectively. In HBV infected tree shrews, the treatment achieved 70% reduction of viral RNA and 35% reduction of cccDNA. In HBV transgenic mouse, 90% inhibition of HBV RNA and 95% inhibition of DNA were observed. CRISPR nanoparticle treatment was well tolerated in both mouse and tree shrew, as no elevation of liver enzymes and minimal off-target was observed. Our study demonstrated that SM-102-based CRISPR is safe and effective in targeting HBV episomal and integration DNA in vivo. The system delivered by SM-102-based LNPs may be used as a potential therapeutic strategy against HBV infection.

Potent immunogenicity and broad-spectrum protection potential of microneedle array patch-based COVID-19 DNA vaccine candidates encoding dimeric RBD chimera of SARS-CoV and SARS-CoV-2 variants

ABSTRACT Breakthrough infections by SARS-CoV-2 variants pose a global challenge to COVID-19 pandemic control, and the development of more effective vaccines of broad-spectrum protection is needed. In this study, we constructed pVAX1-based plasmids encoding receptor-binding domain (RBD) chimera of SARS-CoV-1 and SARS-CoV-2 variants, including pAD1002 (encoding RBDSARS/BA1), pAD1003 (encoding RBDSARS/Beta) and pAD131 (encoding RBDBA1/Beta). Plasmids pAD1002 and pAD131 were far more immunogenic than pAD1003 in terms of eliciting RBD-specific IgG when intramuscularly administered without electroporation. Furthermore, dissolvable microneedle array patches (MAP) greatly enhanced the immunogenicity of these DNA constructs in mice and rabbits. MAP laden with pAD1002 (MAP-1002) significantly outperformed inactivated SARS-CoV-2 virus vaccine in inducing RBD-specific IFN-γ+ effector and memory T cells, and generated T lymphocytes of different homing patterns compared to that induced by electroporated DNA in mice. In consistence with the high titer neutralization results of MAP-1002 antisera against SARS-CoV-2 pseudoviruses, MAP-1002 protected human ACE2-transgenic mice from Omicron BA.1 challenge. Collectively, MAP-based DNA constructs encoding chimeric RBDs of SARS-CoV-1 and SARS-CoV-2 variants, as represented by MAP-1002, are potential COVID-19 vaccine candidates worthy further translational study.

Abstract 3208: Bone-derived TGF-ß release is associated with cardiac dysfunction due to oxidation of SR calcium gatekeepers, Ca2+ leak and mitochondrial dysfunction

Abstract Cancer patients with bone metastases are associated with cardiac dysfunction. Heart attack was observed in 3.8% of patients within 30 days of diagnosis. Here, we report that bone turnover marker β-CTX (P=0.002) and platelet-poor plasma TGF-ß (P=0.036) from patients with bone metastasis were significantly higher than those without bone metastasis. In patients, serum β-CTX concentration was positively correlated with TGF-ß (R2=0.67, P&amp;lt;0.0001). Excess TGF-ß release caused skeletal muscle weakness by oxidation of the sarcoplasmic reticulum (SR) Ca2+ ryanodine receptor (RyR), induction of SR and oxidative stress in breast cancer patients with bone metastasis. Thus, we hypothesized that the systemic effects of TGF-ß could have similar effects on cardiac muscle to induce heart failure. Mechanistically, we showed that TGF-β1 increased transcription of NADPH oxidases, aberrant Ca2+ signaling, SR stress and ROS production in adult cardiomyocytes. We tested this in a transgenic mouse model of Camurati-Engelmann Disease (CED) in which mutant TGF-ß1 (H222D) is overexpressed in osteoblasts and causes accelerated bone destruction. We found that CED mice had increased cardiac size, dilated left ventricle and reduced ejection fraction, by echocardiography, compared to littermate controls. This was associated with increased phosphorylation of Smad2/3, myocardial necrosis and fibrosis in cardiac tissues. Similarly, SR Ca2+ channel RyR2 and SERCA2 showed a significant increase in oxidation and nitrosylation and reduced binding of the stabilizing subunits as well as upregulated iNOS and NADPH oxidases consistent with depressed SR Ca2+ re-uptake, RyR-mediated Ca2+ leak and oxidative stress in CED mice left ventricular. Mitochondria isolated from ventricular samples showed increased mitochondrial Ca2+ overload, ROS production, release of apoptotic proteins, reduced mitochondrial DNA and ATP reproduction in CED mice. Thus, a feed-forward cycle of pathological Ca2+ handling is created between SR mitochondria, which underlies TGF-ß-driven cardiac muscle dysfunction. These findings are consistent with mitochondrial calcium toxicity induced by SR Ca2+ re-uptake and leak, and heart failure with reduced ATP production. The data suggest that excess TGF-ß release by pathological bone destruction could have significant impact on cardiac muscle to induce oxidative stress, SR Ca2+ leak and heart failure and could explain heart disease observed clinically with bone metastasis. Targeting this pathway may improve survival in patients with bone metastases. Citation Format: Lei Shi, Truc Kuo, Jade Martinez, Sukanya Suresh, Trupti Trivedi, Gabriel M. Pagnotti, Leah D. Guerra, Xu Cao, Theresa A. Guise. Bone-derived TGF-ß release is associated with cardiac dysfunction due to oxidation of SR calcium gatekeepers, Ca2+ leak and mitochondrial dysfunction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3208.

Abstract 40: NCI patient derived models repository: PDX, organoid and cell lines from the same patient - bridging the translational pipeline

Abstract The National Cancer Institute’s Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) has developed a national repository of Patient-Derived Models (PDMs) comprised of patient-derived xenografts (PDXs), in vitro patient-derived tumor cell cultures (PDCs) and cancer associated fibroblasts (CAFs) as well as patient-derived organoids (PDOrg). These PDMs are clinically annotated with molecular information available in an easily accessible database for the extramural community. A key effort in developing these models is to develop matched models sets allowing for larger scale screening efforts using 2D or 3D models to prioritize selection of PDX models for preclinical translational research. To date, over 220 model sets with a PDX, PDOrg, and PDC from a single patient have been developed; 40 of these have matched CAF models allowing for exploration of research questions focused on tumor microenvironment. The largest model sets are in colorectal cancer (COADREAD, n=76), gynecologic cancers (n=33), pancreatic adenocarcinoma (PAAD, n=29), melanoma (MEL, n=19), and head and neck squamous cell carcinomas (HNSCC, n=17). Every model undergoes several quality control assessments that serve as go/no-go criteria including pathology assessment, STR validation, NGS concordance assessment and for PDXs, human:mouse DNA content assessment. It should be noted that not every model is successful in the development or QC phase so additional model sets with only one or two of the model types are also available for researcher requests, for example there are over 125 PDX/PDOrg matched model sets. The NCI is currently performing parallel preclinical screening of PDXs and PDCs or PDOrgs to determine the ability of the in vitro lines to predict in vivo activity. Genetic and histopathologic assessment of these matched model sets have demonstrated a high degree of stability by somatic mutation, copy number alteration (CNA) and gene expression data. Gene expression correlation analysis shows that mean of Spearman r between PDXs 0.89, between matched PDC/PDXs 0.79, and between matched PDOrg/PDXs 0.82. As expected, some variation at the gene expression level when comparing PDX to in vitro cultures by t-SNE can be observed, likely due to the differences in culture conditions. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Li Chen, Sergio Alcoser, Gareth Bliss, Carrie Bonomi, Suzanne Borgel, John Carter, Ting-Chia Chang, Alice Chen, Kevin Cooper, Biswajit Das, Kelly Dougherty, Lindsay Dutko, Marion Gibson, Michelle M. Ahalt-Gottholm, Tara Grinnage-Pulley, Keegan Kalmbach, Chris Karlovich, Kimberly Klarmann, Shahanawaz Jiwani, Tiffanie Miner, Michael Mullendore, Matthew Murphy, Kevin Plater, Gloryvee Rivera, Jessica Steed, Luke Stockwin, Cindy R. Timme, Dianne L. Newton, Paul Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. NCI patient derived models repository: PDX, organoid and cell lines from the same patient - bridging the translational pipeline [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 40.