Homozygous Knockout Research Articles

Abstract P2081: Defective Translation Of Mitochondrial Proteins Impairs Cardiac Metabolism In HFpEF

We recently reported that heart failure with preserved ejection fraction (HFpEF) in human myocardium has broadly reduced acylcarnitines and Krebs cycle intermediates, and that transcriptomics shows increased gene expression of oxidative-phosphorylation (OxPhos) related pathways. However, new preliminary data finds protein expression related to OxPhos is decreased. Here, we explored a key mechanism that could drive depressed mitochondrial protein translation in HFpEF. Elongation factor 5A (EIF5A) is a ribosomal subunit that is post-translationally modified on lysine 50 by the transfer of an aminobutyryl moiety from spermidine, called hypusination (Hyp). We find EIF5A Hyp and the mitochondrial proteome are diminished despite elevated gene transcripts in the myocardium of human and a mouse model of HFpEF (latter high-fat diet +L-NAME). Inhibiting EIF5A hypusination in neonatal rat cardiomyocytes results in ribosome stalling, disruption of OxPhos, and increased glycolysis.Proteomics of adult cardiomyocytes with inhibition of the rate-limiting hypusination enzyme deoxyhypusine synthase (DHPS) phenocopies depressed mitochondria as in human HFpEF. Cardiomyocyte-specific knockout of DHPS is lethal in pups within 24hrs after birth. These mice have significantly less myocardial EIF5A Hyp . Cardiac-specific DHPS+/- are viable and have no heart phenotype in unstressed situations, but display marked cardiac dilation with low ejection fraction with failure to hypertrophy in response to transverse aortic constriction.Four weeks following tamoxifen-inducible homozygous knockout of DHPS in cardiomyocytes of 8-week-old mice, we find impaired diastolic function (increased E/E’) with preserved ejection fraction and reduced exercise tolerance. Despite reduced hypusination in HFpEF, ornithine levels are elevated suggesting inhibition of hypusination at the hypusinating enzymes. Interestingly, DHPS levels are decreased in human HFpEF hearts.In summary, we have uncovered a novel role of polyamines in cardiomyocytes: the facilitation of translation of mitochondrial proteins by EIF5A hypusination. We find reduced hypusination resulting from defects in hypusine synthesizing enzymes contributes to mitochondrial defects in HFpEF.

Oocyte-specific Wee1-like protein kinase 2 is dispensable for fertility in mice.

Wee1-like protein kinase 2 (WEE2) is an oocyte-specific protein tyrosine kinase involved in the regulation of oocyte meiotic arrest in humans. As such, it has been proposed as a candidate for non-hormonal female contraception although pre-clinical models have not been reported. Therefore, we developed two novel knockout mouse models using CRISPR/Cas9 to test loss-of-function of Wee2 on female fertility. A frameshift mutation at the Wee2 translation start codon in exon 2 had no effect on litter size, litter production, or the ability of oocytes to maintain prophase I arrest. Because of the lack of a reproductive phenotype, we additionally generated a Wee2 allele with a large deletion by removing all coding exons. While there was no difference in the total number of litters produced, homozygous Wee2 female knockout mice with the larger deletion produced fewer pups than heterozygous littermates. Furthermore, there was no difference for key reproductive parameters measured in the mouse models, including ovarian weight, number of ovulated oocytes, or oocytes that underwent in vitro maturation. Therefore, as loss of Wee2 in mice shows only minor effects on overall fecundity, contraceptive development with WEE2 should consider exploiting alternative properties such as gain-of-function or protein-protein interactions, as Wee2 loss-of-function is likely complicated by biological redundancies with other proteins co-expressed in oocytes.

Gender Differences in a Mouse Model of Hepatocellular Carcinoma Revealed Using Multi-Modal Imaging.

The worldwide incidence of hepatocellular carcinoma (HCC) continues to rise, in part due to poor diet, limited exercise, and alcohol abuse. Numerous studies have suggested that the loss or mutation of PTEN plays a critical role in HCC tumorigenesis through the activation of the PI3K/Akt signaling axis. The homozygous knockout of PTEN in the livers of mice results in the accumulation of fat (steatosis), inflammation, fibrosis, and eventually progression to HCC. This phenotype bears a striking similarity to non-alcoholic steatohepatitis (NASH) which is thought to occupy an intermediate stage between non-alcoholic fatty liver disease (NAFLD), fibrosis, and HCC. The molecular and physiological phenotypes that manifest during the transition to HCC suggest that molecular imaging could provide a non-invasive screening platform to identify the hallmarks of HCC initiation prior to the presentation of clinical disease. We have carried out longitudinal imaging studies on the liver-specific PTEN knockout mouse model using CT, MRI, and multi-tracer PET to interrogate liver size, steatosis, inflammation, and apoptosis. In male PTEN knockout mice, significant steatosis was observed as early as 3 months using both magnetic resonance spectroscopy (MRS) and computed tomography (CT). Enhanced uptake of the apoptosis tracer 18F-TBD was also observed in the livers of male PTEN homozygous knockout mice between 3 and 4 months of age relative to heterozygous knockout controls. Liver uptake of the inflammation tracer [18F]4FN remained relatively low and constant over 7 months in male PTEN homozygous knockout mice, suggesting the suppression of high-energy ROS/RNS with PTEN deletion relative to heterozygous males where the [18F]4FN liver uptake was elevated at early and late time points. All male PTEN homozygous mice developed HCC lesions by month 10. In contrast to the male cohort, only 20% (2 out of 10) of female PTEN homozygous knockout mice developed HCC lesions by month 10. Steatosis was significantly less pronounced in the female PTEN homozygous knockout mice relative to males and could not accurately predict the eventual occurrence of HCC. As with the males, the [18F]4FN uptake in female PTEN homozygous knockout mice was low and constant throughout the time course. The liver uptake of 18F-TBD at 3 and 4.5 months was higher in the two female PTEN knockout mice that would eventually develop HCC and was the most predictive imaging biomarker for HCC in the female cohort. These studies demonstrate the diagnostic and prognostic role of multi-modal imaging in HCC mouse models and provide compelling evidence that disease progression in the PTEN knockout model is highly dependent on gender.

Optical Imaging Reveals Liver Metabolic Perturbations in Mblac1 Knockout Mice.

Metabolic changes have been extensively documented in brain tissue undergoing neurodegeneration, including Parkinson's disease and Alzheimer's disease (AD). Mutations in the C. elegans swip-10 gene result in dopamine (DA) dependent motor dysfunction accompanied by DA neuron degeneration. Recently, the putative human ortholog of swip-10 (MBLAC1) was implicated as a risk factor in AD, that like PD, has been associated with mitochondrial dysfunction. Interestingly, the AD risk associated with MBLAC1 arises in subjects with cardiovascular morbidity, suggesting the possibility of a broader functional insult arising from reduced MBLAC1 protein expression, and one possibly linked to metabolic alterations. Our current studies, utilizing Mblac1 knockout (KO) mice, seeks to determine whether mitochondrial respiration is affected in peripheral tissues of these animals in this model. To initiate these studies, we quantified the levels of mitochondrial coenzymes, NADH, FAD, and their redox ratio (NADH/FAD, RR) in the livers of wild type (WT) mice and their homozygous KO littermates, using 3D optical cryo-imaging. We found that Mblac1 KO mice exhibited a greater oxidized redox state compared to WT mice. When compared to the WT group, the redox ratio of KO mice was decreased by 46.32%, driven predominantly by significantly lower NADH levels (more oxidized state). We speculate that, as seen with C. elegans swip-10 mutants, that loss of MBLAC1 protein results in deficits in tricarboxylic acid cycle (TCA) production of NADH and FAD TCA that leads to diminished cellular ATP production and oxidative stress. Such observations are consistent with changes that in the central nervous system (CNS) could support neurodegeneration and in the periphery account for comorbidities.

Smarca2 genetic ablation is phenotypically benign in a safety assessment of tamoxifen-inducible conditional knockout rats

SMARCA2 and SMARCA4 are the ATPases of the SWI/SNF chromatin remodeling complex, which play a significant role in regulating transcriptional activity and DNA repair in cells. SMARCA2 has become an appealing synthetic-lethal, therapeutic target in oncology, as mutational loss of SMARCA4 in many cancers leads to a functional dependency on residual SMARCA2 activity. Thus, for therapeutic development, an important step is understanding any potential safety target-associated liabilities of SMARCA2 inhibition. To best mimic a SMARCA2 therapeutic, a tamoxifen-inducible (TAMi) conditional knockout (cKO) rat was developed using CRISPR technology to understand the safety profile of Smarca2 genetic ablation in a model system that avoids potential juvenile and developmental phenotypes. As the rat is the prototypical rodent species utilized in toxicology studies, a comprehensive toxicological and pathological assessment was conducted in both heterozygote and homozygous knockout rats at timepoints up to 28 days, alongside relevant corresponding controls. To our knowledge, this represents the first TAMi cKO rat model utilized for safety assessment evaluations. No significant target-associated phenotypes were observed when Smarca2 was ablated in mature (11- to 15-week-old) rats; however subsequent induction of SMARCA4 was evident that could indicate potential compensatory activity. Similar to mouse models, rat CreERT2-transgene and TAMi toxicities were characterized to avoid confounding study interpretation. In summary, a lack of significant safety findings in Smarca2 cKO rats highlights the potential for therapeutics targeting selective SMARCA2 ATPase activity; such therapies are predicted to be tolerated in patients without eliciting significant on-target toxicities.

MTORC2 acts as a gatekeeper for mTORC1 deficiency-mediated impairments in ILC3 development.

Group 3 innate lymphoid cells (ILC3s) are mediators of intestinal immunity and barrier function. Recent studies have investigated the role of the mammalian target of rapamycin complex (mTOR) in ILC3s, whereas the mTORC1-related mechanisms and crosstalk between mTORC1 and mTORC2 involved in regulating ILC3 homeostasis remain unknown. In this study, we found that mTORC1 but not mTORC2 was critical in ILC3 development, IL-22 production, and ILC3-mediated intestinal homeostasis. Single-cell RNA sequencing revealed that mTORC1 deficiency led to disruption of ILC3 heterogeneity, showing an increase in differentiation into ILC1-like phenotypes. Mechanistically, mTORC1 deficiency decreased the expression of NFIL3, which is a critical transcription factor responsible for ILC3 development. The activities of both mTORC1 and mTORC2 were increased in wild-type ILC3s after activation by IL-23, whereas inhibition of mTORC1 by Raptor deletion or rapamycin treatment resulted in increased mTORC2 activity. Previous studies have demonstrated that S6K, the main downstream target of mTORC1, can directly phosphorylate Rictor to dampen mTORC2 activity. Our data found that inhibition of mTORC1 activity by rapamycin reduced Rictor phosphorylation in ILC3s. Reversing the increased mTORC2 activity via heterozygous or homozygous knockout of Rictor in Raptor-deleted ILC3s resulted in severe ILC3 loss and complete susceptibility to intestinal infection in mice with mTORC1 deficiency (100% mortality). Thus, mTORC1 acts as a rheostat of ILC3 heterogeneity, and mTORC2 protects ILC3s from severe loss of cellsand immune activity against intestinal infection when mTORC1 activity is diminished.

Improving Rice Leaf Shape Using CRISPR/Cas9-Mediated Genome Editing of SRL1 and Characterizing Its Regulatory Network Involved in Leaf Rolling through Transcriptome Analysis

Leaf rolling is a crucial agronomic trait to consider in rice (Oryza sativa L.) breeding as it keeps the leaves upright, reducing interleaf shading and improving photosynthetic efficiency. The SEMI-ROLLED LEAF 1 (SRL1) gene plays a key role in regulating leaf rolling, as it encodes a glycosylphosphatidylinositol-anchored protein located on the plasma membrane. In this study, we used CRISPR/Cas9 to target the second and third exons of the SRL1 gene in the indica rice line GXU103, which resulted in the generation of 14 T0 transgenic plants with a double-target mutation rate of 21.4%. After screening 120 T1 generation plants, we identified 26 T-DNA-free homozygous double-target mutation plants. We designated the resulting SRL1 homozygous double-target knockout as srl1-103. This line exhibited defects in leaf development, leaf rolling in the mature upright leaves, and a compact nature of the fully grown plants. Compared with the wild type (WT), the T2 generation of srl1-103 varied in two key aspects: the width of flag leaf (12.6% reduction compared with WT) and the leaf rolling index (48.77% increase compared with WT). In order to gain a deeper understanding of the involvement of SRL1 in the regulatory network associated with rice leaf development, we performed a transcriptome analysis for the T2 generation of srl1-103. A comparison of srl1-103 with WT revealed 459 differentially expressed genes (DEGs), including 388 upregulated genes and 71 downregulated genes. In terms of the function of the DEGs, there seemed to be a significant enrichment of genes associated with cell wall synthesis (LOC_Os08g01670, LOC_Os05g46510, LOC_Os04g51450, LOC_Os10g28080, LOC_Os04g39814, LOC_Os01g71474, LOC_Os01g71350, and LOC_Os11g47600) and vacuole-related genes (LOC_Os09g23300), which may partially explain the increased leaf rolling in srl1-103. Furthermore, the significant downregulation of BAHD acyltransferase-like protein gene (LOC_Os08g44840) could be the main reason for the decreased leaf angle and the compact nature of the mutant plants. In summary, this study successfully elucidated the gene regulatory network in which SRL1 participates, providing theoretical support for targeting this gene in rice breeding programs to promote variety improvement.

Low ceruloplasmin levels exacerbate retinal degeneration in a hereditary hemochromatosis model.

In a previous report, a 39-year-old patient with high serum iron levels from hereditary hemochromatosis (HH) was diagnosed with a form of retinal degeneration called bull's eye maculopathy. This is atypical for patients with HH, so it was theorized that the low serum levels of ferroxidase ceruloplasmin (CP) of this patient coupled with the high iron levels led to the retinal degeneration. CP, by oxidizing iron from its ferrous to ferric form, helps prevent the oxidative damage caused by ferrous iron. To test this, a hepcidin knockout (KO) mouse model of HH was combined with Cp KO to test whether the combination would lead to more severe retinal degeneration. Monthly in vivo retinal images were acquired and, after 11 months, mice were euthanized for further analyses. Both heterozygous and homozygous Cp KO increased the rate and severity of retinal degeneration. These results demonstrate the protective role of CP, which is most likely owing to its ferroxidase activity. The findings suggest that CP levels may influence the severity of retinal degeneration, especially in individuals with high serum iron.

Molecular Basis of Transglutaminase-2 and Muscarinic Cholinergic Receptors in Experimental Myopia: A Target for Myopia Treatment.

Myopia, a prevalent refractive error disorder worldwide, is characterized by the elongation of the eye, leading to visual abnormalities. Understanding the genetic factors involved in myopia is crucial for developing therapeutic and preventive measures. Unfortunately, only a limited number of genes with well-defined functionality have been associated with myopia. In this study, we found that the homozygous TGM2-deleted gene in mice protected against the development of myopia by slowing down the elongation of the eye. The effectiveness of gene knockdown was confirmed by achieving a 60 percent reduction in TGM-2 transcript levels through the use of TGM-2-specific small interfering RNA (siRNA) in human scleral fibroblasts (SFs). Furthermore, treating normal mouse SFs with various transglutaminase inhibitors led to the down-regulation of TGM-2 expression, with the most significant reduction observed with specific TGM-2 inhibitors. Additionally, the study found that the pharmacological blockade of muscarinic receptors also slowed the progression of myopia in mice, and this effect was accompanied by a decrease in TGM-2 enzyme expression. Specifically, mice with homozygous mAChR5, mAChR1, and/or mAChR4 and knockout mice exhibited higher levels of TGM-2 mRNA compared to mice with homozygous mAChR2 and three knockout mice (fold changes of 5.8, 2.9, 2.4, -2.2, and -4.7, respectively; p < 0.05). These findings strongly suggest that both TGM-2 and muscarinic receptors play central roles in the development of myopia, and blocking these factors could potentially be useful in interfering with the progression of this condition. In conclusion, targeting TGM-2 may have a beneficial effect regarding myopia, and this may also be at least partially be the mechanism of anti-muscarinic drugs in myopia. Further studies should investigate the interaction between TGM-2 and muscarinic receptors, as well as the changes in other extracellular matrix genes associated with growth during the development of myopia.

Generation of myoglobin (MB)-knockout human embryonic stem cell (hESC) line (KAIMRCe002-A-1S) using CRISPR/Cas9 technology

Myoglobin (MB) is a cytoplasmic hemoprotein that is predominantly expressed in the heart and oxidative myofibers of skeletal muscle. It has been demonstrated that MB binds to oxygen and promotes its diffusion for energy production in the mitochondria. Recently, MB was found to be expressed in different forms of malignant tumors and cancer cell lines. Further studies using gene disruption technology will enhance the understanding of MB’s role in human cardiovascular biology and cancers. Here, we describe the generation of a homozygous MB knockout in human embryonic stem cells (hESC-MB−/−) via CRISPR/Cas9 to study MB function in human biology and diseases.

P-028 Loss of the m6A methyltransferase METTL5 impairs spermiogenesis and male fertility

Abstract Study question Whether and how METTL5 regulates germ cell development during spermatogenesis. Summary answer METTL5 is required for spermiogenesis. Loss of METTL5 resulted in teratozoospermia and male infertility via reduced translation of acrosome and flagellum formation proteins. What is known already The roles of m6A modifications on mRNA in spermatogenesis have been extensively studied. It was reported that METTL5 knockout mice showed the brain development defect and sterility of 16-week male mice. However, the detailed mechanism of METTL5 affecting male fertility remains elusive. Study design, size, duration Mettl5 KO mice were kindly gifted from Prof. Shuibin Lin of The First Affiliated Hospital of Sun Yat-sen University. Heterozygotes of Mettl5 mice were used to generate Mettl5 homozygous knockout mice. The phenotype of KO mice was assessed. Ribosomal sequencing, proteomics analysis and further validation of protein translation were performed to explore the mechanism. Participants/materials, setting, methods Fertility assessment, sperm parameter analyses, sperm nuclear morphology analysis, Transmission electron microscopy (TEM), tissue Collection and histological analysis, protein extraction and western blot analysis, immunofluorescence studies, cDNA synthesis and qRT-PCR, in vitro fertilization (IVF) were used in this study. Main results and the role of chance Here we reported that methyltransferase-like 5 (METTL5) is involved in spermiogenesis as a methyltransferase mediating m6A modification on rRNA. Mettl5 knockout mice were infertile with teratozoospermia. The acrosome in the sperm head was absent with reduced sperm motility. Furthermore, the fertilization ability of sperm in the IVF experiment failed. Mechanistically, the level of rRNA m6A modification was significantly decreased in the testes of Mettl5 KO mice. The translational efficiency and protein levels of acrosome and flagellum formation proteins such as FSIP2, ODF2, GK2, PGK2, and AKAP4 were significantly reduced when METTL5 was depleted. Limitations, reasons for caution The METTL5 mutation in the patient with teratozoospermia was not examined in the present study Wider implications of the findings The rRNA m6A modification is also involved in regulating spermatogenesis by METTL5. This study highlights the critical role of rRNA epigenetic modifications during spermatogenesis and provides novel theoretical explanations for the m6A modifications. Trial registration number Not applicable

SINE RNA of the imprinted miRNA clusters mediates constitutive type III interferon expression and antiviral protection in hemochorial placentas

Hemochorial placentas have evolved defense mechanisms to prevent the vertical transmission of viruses to the immunologically underdeveloped fetus. Unlike somatic cells that require pathogen-associated molecular patterns to stimulate interferon production, placental trophoblasts constitutively produce type III interferons (IFNL) through an unknown mechanism. We demonstrate that transcripts of short interspersed nuclear elements (SINEs) embedded in miRNA clusters within the placenta trigger a viral mimicry response that induces IFNL and confers antiviral protection. Alu SINEs within primate-specific chromosome 19 (C19MC) and B1 SINEs within rodent-specific microRNA cluster on chromosome 2 (C2MC) produce dsRNAs that activate RIG-I-like receptors (RLRs) and downstream IFNL production. Homozygous C2MC knockout mouse trophoblast stem (mTS) cells and placentas lose intrinsic IFN expression and antiviral protection, whereas B1 RNA overexpression restores C2MCΔ/Δ mTS cell viral resistance. Our results uncover a convergently evolved mechanism whereby SINE RNAs drive antiviral resistance in hemochorial placentas, placing SINEs as integral players in innate immunity.

Thymic lymphoma detection in RORγ knockout mice using 5-hydroxymethylcytosine profiling of circulating cell-free DNA

A high incidence of thymic lymphoma has been noted in mice deficient of retinoid-related orphan receptor γ2 (RORγ2), which is required for differentiation of naïve CD4+ T cells into TH17 cells. Using a RORγ homozygous knockout (KO) mouse model of thymic lymphoma, we characterized this tumor progression and investigated the utility of 5-hydroxymethylcytosine (5hmC) signatures as a non-invasive circulating biomarker for early prediction of malignancy. No evidence for malignancy was noted in the wild-type mice, while primary thymic lymphoma with multi-organ metastasis was observed microscopically in 97% of the homozygous RORγ KO mice. The severity of thymic lymphoma was not age-dependent in the KO mice of 2 to 4 months old. Differential enrichment of 5hmC in thymic DNA and plasma cell-free DNA (cfDNA) was compared across different stages of tumor progression. Random forest modeling of plasma cfDNA achieved good predictivity (AUC = 0.74) in distinguishing early non-metastatic thymic lymphoma compared to cancer-free controls, while perfect predictivity was achieved with advanced multi-organ metastatic disease (AUC = 1.00). Lymphoid-specific genes involved in thymocyte selection during T cell development (Themis, Tox) were differentially enriched in both plasma and thymic tissue. This could help in differentiating thymic lymphoma from other tumors commonly detected in rodent carcinogenicity studies used in pharmaceutical drug development to inform human malignancy risk. Overall, these results provide a proof-of-concept for using circulating cfDNA profiles in rodent carcinogenicity studies for early risk assessment of novel pharmaceutical targets.

Generation of a homozygous RANGRF knockout hiPSC line by CRISPR/Cas9 system

The RAN Guanine Nucleotide Release Factor (RANGRF) gene encodes the protein MOG1, which binds to Nav1.5 and facilitates its transport to the cell membrane. Nav1.5 mutations have been linked to various cardiac arrhythmias and cardiomyopathy. To investigate the role of RANGRF in this process, we utilized the CRISPR/Cas9 gene editing system to generate a homozygous RANGRF knockout hiPSC line. The availability of the cell line will prove to be an invaluable asset in the study of disease mechanisms and the testing of gene therapies for cardiomyopathy.

CRISPR/Cas9-based functional characterization of PxABCB1 reveals its roles in the resistance of Plutella xylostella (L.) to Cry1Ac, abamectin and emamectin benzoate

The identification of functional midgut receptors for pesticidal proteins produced by Bacillus thuringiensis (Bt) is critical for deciphering the molecular mechanism of Bt resistance in insects. Reduced expression of the PxABCB1 gene was previously found to be associated with Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.). To directly validate the potential receptor role of PxABCB1 and its contribution to Bt Cry1Ac toxicity in P. xylostella, we used CRISPR/Cas9 to generate a homozygous knockout ABCB1KO strain with a 5-bp deletion in exon 3 of its gene. The ABCB1KO strain exhibited a 63-fold resistance to Cry1Ac toxin compared to the parental DBM1Ac-S strain. Intriguingly, the ABCB1KO strain also exhibited significant increases in susceptibility to abamectin and emamectin benzoate. No changes in susceptibility to various other Bt Cry proteins or synthetic insecticides were observed. The knockout strain exhibited no significant fitness costs. Overall, our study indicates that PxABCB1 can protect the insect against avermectin insecticides on one hand, while on the other hand it facilitates the toxic effect of the Bt Cry1Ac toxin. The results of this study will help to inform integrated pest management approaches against this destructive pest.

MutSα and MutSβ as size-dependent cellular determinants for prime editing in human embryonic stem cells

Precise genome editing in human pluripotent stem cells (hPSCs) has potential applications in isogenic disease modeling and exvivo stem cell therapy, necessitating diverse genome editing tools. However, unlike differentiated somatic cells, hPSCs have unique cellular properties that maintain genome integrity, which largely determine the overall efficiency of an editing tool. Considering the high demand for prime editors (PEs), it is imperative to characterize the key molecular determinants of PE outcomes in hPSCs. Through homozygous knockout (KO) of MMR pathway key proteins MSH2, MSH3, and MSH6, we reveal that MutSα and MutSβ determine PE efficiency in an editing size-dependent manner. Notably, MSH2 perturbation disrupted both MutSα and MutSβ complexes, dramatically escalating PE efficiency from base mispair to 10 bases, up to 50 folds. Similarly, impaired MutSα by MSH6 KO improved editing efficiency from single to three base pairs, while defective MutSβ by MSH3 KO heightened efficiency from three to 10 base pairs. Thus, the size-dependent effect of MutSα and MutSβ on prime editing implies that MMR is a vital PE efficiency determinant in hPSCs and highlights the distinct roles of MutSα and MutSβ in its outcome.

Heterozygous midnolin knockout attenuates severity of nonalcoholic fatty liver disease in mice fed a Western-style diet high in fat, cholesterol, and fructose.

Although midnolin has been studied for over 20 years, its biological roles in vivo remain largely unknown, especially due to the lack of a functional animal model. Indeed, given our recent discovery that the knockdown of midnolin suppresses liver cancer cell tumorigenicity and that this antitumorigenic effect is associated with modulation of lipid metabolism, we hypothesized that knockout of midnolin in vivo could potentially protect from nonalcoholic fatty liver disease (NAFLD) which has become the most common cause of chronic liver disease in the Western world. Accordingly, in the present study, we have developed and now report on the first functional global midnolin knockout mouse model. Although the overwhelming majority of global homozygous midnolin knockout mice demonstrated embryonic lethality, heterozygous knockout mice were observed to be similar to wild-type mice in their viability and were used to determine the effect of reduced midnolin expression on NAFLD. We found that global heterozygous midnolin knockout attenuated the severity of NAFLD in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism. Collectively, our results support a role for midnolin in regulating cholesterol/lipid metabolism in the liver. Thus, midnolin may represent a novel therapeutic target for NAFLD. Finally, our observation that midnolin was essential for survival underscores the broad importance of this gene beyond its role in liver biology.NEW & NOTEWORTHY We have developed and now report on the first functional global midnolin knockout mouse model. We found that global heterozygous midnolin knockout attenuated the severity of nonalcoholic fatty liver disease (NAFLD) in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism.

PIEZO1Deletion in CD11b+ Cells Suppresses Rhabdomyosarcoma Tumor Rejection and Generates a Tumor-Permissive Immune Landscape

Abstract In several cancers, including the aggressive sarcoma, rhabdomyosarcoma (RMS), tumor-associated myeloid cells (TAMCs) are the largest immune populations and are correlated with poor survival outcomes. TAMCs are a mixed and ‘plastic’ population that can sense changes in their environment, including intra-tumoral ECM stiffness, and change their functioning accordingly. Despite the clinical influences these cells have and the detrimental effects that elevated tumor microenvironment (TME) stiffness has on immune cells’ phenotype, little is known about the proteins that sense and mediate these changes. We propose inhibition of mechanosensitive cation channel, PIEZO1, promotes a pro-tumor phenotype in TAMCs while its activation skews this population to anti-tumor functioning thereby enhancing tumor rejection.PIEZO1’s influence on tumor rejection was assessed by inoculating a novel murine CD11b-conditional homozygous PIEZO1 knockout strain with syngeneic RMS cells and performing several analyses. Tumor volume and survival analyses found that RMS-bearing CD11b-conditional PIEZO1 KO mice developed larger tumors and had diminished overall survival as compared to wild-type mice. Flow cytometric analysis of tumors revealed increased pro-tumor macrophage frequency in tumor and expansion of MDSCs in the TME and tumor-draining lymph node (TDLN). Interestingly, despite PIEZO1 KO tumors having increased T cells, they had lowerPD-1+ CD4 and CD8 T cell frequencies; a trend also observed in the spleen and TDLN. These findings suggest without PIEZO1 expression, TAMCs predominantly polarize to a ‘pro-tumor’ state, thereby preventing T cell and adaptive immune cells’ activation, leading to lower survival and increased tumor burden. NCI T32 (5T32CA059366-27)

PO-02-246 PPARΓ ACTIVATION MEDIATES CARDIAC PATHOLOGIES IN TWO MOUSE MODELS OF ARRHYTHMOGENIC CARDIOMYOPATHY (ACM) AND ITS THERAPEUTIC IMPLICATIONS

Using ACM patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we showed that abnormal peroxisome proliferator-activated receptor-gamma (PPARγ) activation on normal adult PPARα-mediated metabolic backgrounds in CMs resulted in pathological hallmarks of ACM. PPARγ inhibitors rescue all ACM pathologies effects (Nature 2013) but may carry systemic side effects. Here, we study the pathogenic role of PPARγ activation in 2 ACM mouse models and test therapeutic potentials of 2 angiotensin II-receptor blockers (ARBs) commonly used to treat cardiomyopathies: Valsartan and Losartan. Valsartan has minimal activity yet Losartan has strong activity for PPARγ gene activation, respectively. We used 2 established ACM mouse models in this study: 1) mice with tamoxifen (TAM)-inducible and CM-specific (by αγMHC-Cre) double knockout (DKO) of plakoglobin (JUP) and βcatenin (MCB 2012) that developed ACM pathologies at 2-3 months only after TAM injection; and 2) mice with CM-specific and homozygous knockout of JUP (JUP KO, Hum Mol Genet 2011) that develop ACM pathologies rapidly in 1-2 months after birth. Feeding DKO mice with 0.1 mg/ml rosiglitazone (Rosi, a PPARγ activator) for 2 weeks (wks) after TAM injection accelerated the decrease in left ventricular ejection fraction (LV EF) (from 67.4% to 27.1%) within 2-3 weeks after feeding Rosi. For JUP KO mice at 8-9 wks after birth, the pathological hearts showed hyperactivated PPARγ (at both mRNA and protein levels) and elevated apoptotic gene expressions (3-fold increases in caspase 3 and BAX/BCL2 ratios). Feeding both ACM mouse models with 0.2 mg/ml Valsartan for 6 wks reduced PPARγ proteins and apoptotic genes (BAX/BCL2 ratios) by 2-3 folds. In JUP KO mice, Valsartan prevented LV EF deterioration yet losartan at equivalent doses led to early death and reduced LV EF. Our study results support strongly that abnormal PPARγ activation in ACM CMs plays a key pathogenic role in both human iPSC-CM-based and 2 mouse models of ACM. An ARB with PPARγ agnostic activities can worsen ACM pathologies and only ARB with minimal PPARγ agonistic activity can prevent ACM disease progression. These results have significant implications in avoiding using any drug with PPARγ agnostic activities to treat human ACM.

Albumin Deficiency Reduces Hepatic Steatosis and Improves Glucose Metabolism in a Mouse Model of Diet-Induced Obesity.

Serum albumin facilitates the transport of free fatty acids (FFAs) from adipose tissue to other organs. It was not known if impeding this process could protect from hepatic steatosis and metabolic dysfunction in obesity. We tested whether albumin knockout (Alb-/-) mice would exhibit a reduction in plasma FFA concentration, reduced hepatic lipid accumulation, and improved glucoregulation as compared to wild-type (WT) mice. Male homozygous albumin knockout mice (Alb-/-) and WT controls were fed a low-fat diet (LFD) or high-fat diet (HFD). Alb-/- mice exhibited a similar body weight gain and body composition as WT on both diets. Despite HFD-induced obesity, Alb-/- mice were protected from various comorbidities. Compared to WT mice on the HFD, Alb-/- exhibited lower plasma FFA levels, lower blood glucose levels during glucose tolerance and insulin tolerance tests, and lower hepatic steatosis and inflammation. Alb-/- mice on HFD also exhibited elevated expression of multiple genes in the liver and adipose tissues, such as peroxisome proliferator-activated receptor α in both tissues, as well as glucose transporter-4 and adiponectin in adipose tissues. The results indicate that albumin's FFA transport function may be involved in the development of hepatic lipid accumulation and dysregulated glucose metabolism in obesity.