scholarly journals Promoter or enhancer activation by CRISPRa rescues haploinsufficiency caused obesity

2017 ◽  
Author(s):  
Navneet Matharu ◽  
Sawitree Rattanasopha ◽  
Lenka Maliskova ◽  
Yi Wang ◽  
Aaron Hardin ◽  
...  
Keyword(s):  
Therapeutic Strategy ◽  
Gene Dosage ◽  
Regulatory Elements ◽  
Adeno Associated Virus ◽  
Wide Range ◽  
Obesity Phenotype ◽  
Altered Gene ◽  
Functional Copy ◽  
Crispr Activation

AbstractHaploinsufficiency, having only one functional copy of a gene, leads to a wide range of human disease and has been associated with over 300 genes. Here, we tested whether CRISPR activation (CRISPRa) could rescue a haploinsufficient disease in vivo. Haploinsufficiency of Sim1, a transcription factor involved in the leptin pathway, results in severe obesity in humans and mice. CRISPRa targeting of either the Sim1 promoter or its ~270kb distant hypothalamic enhancer using transgenic mice, rescued the obesity phenotype in Sim1 heterozygous mice. Interestingly, despite using a ubiquitous promoter for CRISPRa, Sim1 was upregulated only in tissues where the promoter or enhancer are active, suggesting that cis-regulatory elements can determine CRISPRa tissue-specificity. To further relate this to therapy, we injected CRISPRa adeno associated virus into the hypothalamus, leading to reversal of the obesity phenotype. This therapeutic strategy could be used to rescue numerous diseases resulting from altered gene dosage.

scholarly journals CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency

Science ◽  
2018 ◽  
Vol 363 (6424) ◽  
pp. eaau0629 ◽  
Author(s):  
Navneet Matharu ◽  
Sawitree Rattanasopha ◽  
Serena Tamura ◽  
Lenka Maliskova ◽  
Yi Wang ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Gene Dosage ◽  
Adeno Associated Virus ◽  
Endogenous Gene ◽  
Gene Copies ◽  
Wide Range ◽  
Obesity Phenotype ◽  
Potential Strategy ◽  
Altered Gene ◽  
Functional Copy

A wide range of human diseases result from haploinsufficiency, where the function of one of the two gene copies is lost. Here, we targeted the remaining functional copy of a haploinsufficient gene using CRISPR-mediated activation (CRISPRa) in Sim1 and Mc4r heterozygous mouse models to rescue their obesity phenotype. Transgenic-based CRISPRa targeting of the Sim1 promoter or its distant hypothalamic enhancer up-regulated its expression from the endogenous functional allele in a tissue-specific manner, rescuing the obesity phenotype in Sim1 heterozygous mice. To evaluate the therapeutic potential of CRISPRa, we injected CRISPRa-recombinant adeno-associated virus into the hypothalamus, which led to reversal of the obesity phenotype in Sim1 and Mc4r haploinsufficient mice. Our results suggest that endogenous gene up-regulation could be a potential strategy to treat altered gene dosage diseases.

scholarly journals A Limited Number of Transducible Hepatocytes Restricts a Wide-Range Linear Vector Dose Response in Recombinant Adeno-Associated Virus-Mediated Liver Transduction

2002 ◽  
Vol 76 (22) ◽  
pp. 11343-11349 ◽  
Author(s):  
Hiroyuki Nakai ◽  
Clare E. Thomas ◽  
Theresa A. Storm ◽  
Sally Fuess ◽  
Sharon Powell ◽  
...  
Keyword(s):  
Dose Response ◽  
Transgene Expression ◽  
Linear Increase ◽  
Factor Ix ◽  
Adeno Associated Virus ◽  
Linear Dose ◽  
Human Factor Ix ◽  
Wide Range ◽  
High Doses

ABSTRACT Recombinant adeno-associated virus (rAAV) vectors are promising vehicles for achieving stable liver transduction in vivo. However, the mechanisms of liver transduction are not fully understood, and furthermore, the relationships between rAAV dose and levels of transgene expression, total number of hepatocytes transduced, and proportion of integrated vector genomes have not been well established. To begin to elucidate the liver transduction dose response with rAAV vectors, we injected mice with two different human factor IX or Escherichia coli lacZ-expressing AAV serotype 2-based vectors at doses ranging between 4.0 × 108 and 1.1 × 1013 vector genomes (vg)/mouse, in three- to sixfold increments. A 2-log-range linear dose-response curve of transgene expression was obtained from 3.7 × 109 to 3.0 × 1011 vg/mouse. Vector doses above 3.0 × 1011 vg/mouse resulted in disproportionately smaller increases in both the number of transduced hepatocytes and levels of transgene expression, followed by saturation at doses above 1.8 × 1012 vg/mouse. In contrast, a linear increase in the number of vector genomes per hepatocyte was observed up to 1.8 × 1012 vg/mouse concomitantly with enhanced vector genome concatemerization, while the proportion of integrated vector genomes was independent of the vector dose. Thus, the mechanisms that restrict a wide-range linear dose response at high doses likely involve decreased functionality of vector genomes and restriction of transduction to fewer than 10% of total hepatocytes. Such information may be useful to determine appropriate vector doses for in vivo administration and provides further insights into the mechanisms of rAAV transduction in the liver.

scholarly journals Adeno-associated virus-mediated gene delivery promotes S-phase entry-independent precise targeted integration in cardiomyocytes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yasuaki Kohama ◽  
Shuichiro Higo ◽  
Yuki Masumura ◽  
Mikio Shiba ◽  
Takumi Kondo ◽  
...  
Keyword(s):  
Direct Injection ◽  
Adeno Associated Virus ◽  
Systemic Injection ◽  
Guide Rna ◽  
Human Cardiomyocytes ◽  
Wide Range ◽  
Targeted Integration ◽  
Induced Pluripotent

Abstract Post-mitotic cardiomyocytes have been considered to be non-permissive to precise targeted integration including homology-directed repair (HDR) after CRISPR/Cas9 genome editing. Here, we demonstrate that direct delivery of large amounts of transgene encoding guide RNA (gRNA) and repair template DNA via intra-ventricular injection of adeno-associated virus (AAV) promotes precise targeted genome replacement in adult murine cardiomyocytes expressing Cas9. Neither systemic injection of AAV nor direct injection of adenovirus promotes targeted integration, suggesting that high copy numbers of single-stranded transgenes are required in cardiomyocytes. Notably, AAV-mediated targeted integration in cardiomyocytes both in vitro and in vivo depends on the Fanconi anemia pathway, a key component of the single-strand template repair mechanism. In human cardiomyocytes differentiated from induced pluripotent stem cells, AAV-mediated targeted integration fluorescently labeled Mlc2v protein after differentiation, independently of DNA synthesis, and enabled real-time detection of sarcomere contraction in monolayered beating cardiomyocytes. Our findings provide a wide range of applications for targeted genome replacement in non-dividing cardiomyocytes.

scholarly journals KDM5 inhibition offers a novel therapeutic strategy for the treatment of KMT2D mutant lymphomas

Blood ◽  
2021 ◽  
Author(s):  
James Andrew Heward ◽  
Lola Koniali ◽  
Annalisa D'Avola ◽  
Karina Close ◽  
Alison Yeomans ◽  
...  
Keyword(s):  
Therapeutic Strategy ◽  
Cell Signalling ◽  
Loss Of Function ◽  
H3k4 Methylation ◽  
Altered Expression ◽  
Bcl2 Family ◽  
Expression Of Genes ◽  
Altered Gene

Loss-of-function mutations in KMT2D are a striking feature of the germinal centre (GC) lymphomas, resulting in decreased H3K4-methylation and altered gene expression. We hypothesised that inhibition of the KDM5 family, which demethylates H3K4me3/me2, would re-establish H3K4-methylation and restore the expression of genes repressed upon loss of KMT2D. KDM5-inhibition increased H3K4me3 levels and caused an anti-proliferative response in vitro, which was markedly greater in both endogenous and CRISPR-edited KMT2D mutant DLBCL cell lines, while tumour growth was inhibited in KMT2D mutant xenografts in vivo. KDM5-inhibition reactivated both KMT2D-dependent and -independent genes, resulting in diminished B-cell signalling and altered expression of BCL2 family members, including BCL2 itself. KDM5-inhibition may offer an effective therapeutic strategy for ameliorating KMT2D loss-of-function mutations in GC-lymphomas.

scholarly journals KDM5 inhibition offers a novel therapeutic strategy for the treatment of KMT2D mutant lymphomas

2020 ◽  
Author(s):  
James A Heward ◽  
Lola Konali ◽  
Annalisa D’Avola ◽  
Karina Close ◽  
Alison Yeomans ◽  
...  
Keyword(s):  
Therapeutic Strategy ◽  
Cell Receptor ◽  
Loss Of Function ◽  
H3k4 Methylation ◽  
Altered Expression ◽  
Bcl2 Family ◽  
Expression Of Genes ◽  
Altered Gene

AbstractLoss-of-function mutations in KMT2D are a striking feature of the germinal centre (GC) lymphomas, resulting in decreased H3K4 methylation and altered gene expression. We hypothesised that inhibition of the KDM5 family, which demethylates H3K4me3/me2, would re-establish H3K4 methylation and restore the expression of genes repressed upon loss of KMT2D. KDM5-inhibition increased H3K4me3 levels and caused an anti-proliferative response in vitro, which was markedly greater in both endogenous and CRISPR-edited KMT2D mutant DLBCL cell lines, whilst tumour growth was inhibited in KMT2D mutant xenografts in vivo. KDM5-inhibition reactivated both KMT2D-dependent and -independent genes, resulting in diminished B-cell receptor signalling and altered expression of BCL2 family members, including BCL2 itself, allowing it to synergise with agents targeting these pathways. KDM5-inhibition may offer an effective therapeutic strategy for ameliorating KMT2D loss-of-function mutations in GC-lymphomas.Statement of significanceWe detail a novel way of reverting the effects of loss-of-function mutations in the histone methyltransferase KMT2D by inhibiting the KDM5 demethylase family, increasing levels of H3K4me3 and restoring expression of KMT2D regulated genes.

Abstract 119: Cardiac Transcription Factor EB Sumoylation Deficiency Exacerbates Age-associated Reduction In Autophagy

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Heng Ma ◽  
Le Zhang ◽  
Lu Yu ◽  
Yan Li
Keyword(s):  
Transcription Factor ◽  
Therapeutic Strategy ◽  
Transcriptional Regulator ◽  
Contractile Dysfunction ◽  
Post Translational Modification ◽  
Hairpin Rna ◽  
Adeno Associated Virus ◽  
Transcription Factor Eb

Background: Aging-dependent decline of autophagy contributes to cardiac dysfunction and ischemic intolerance. Transcription factor EB (TFEB) is a master transcriptional regulator of the autophagy-lysosome pathway. The present study aimed to characterize the role of TFEB in the autophagic decrease with aging. Methods and Results: We analyzed age-associated autophagic changes in male C57BL/6 young (4-6 mo) and aged (22-24 mo) mice. The results demonstrated that TFEB expressed predominantly as a SUMOylated form in cardiomyocyte nuclei and this SUMOylation of TFEB declined in aged heart associated with autophagy reduction. Interestingly, SUMOylation of TFEB was unaffected by rapamycin. Rapamycin induced translocation of TFEB into nucleus but lower level of nuclear TFEB in aged hearts than that seen in young hearts (P<0.05). SUMO1 downregulation by adeno-associated-virus-mediated small hairpin RNA (rAAV9-shSUMO1) significantly reduced nuclear TFEB levels (P<0.05), depressed cardiac autophagy and accelerated cardiomyocyte contractile dysfunction with worse hypoxia/reoxygenation (H/R) injury (all P<0.05). Therefore, impaired SUMOylation decreased nuclear TFEB during aging. By contrast, SUMO1 restitution significantly augmented nuclear SUMOylated TFEB with enhanced autophagy and ultimately reduced infarct size in aged heart. However, knockdown of cardiac TFEB blocked the protective effect of upregulation of SUMO1 in aged hearts, resulted in decline of autophagy and worse in vivo I/R injury. Conclusions: The present study newly demonstrates that SUMOylation is a critical post-translational modification that regulates cardiac TFEB. Impaired SUMOylation of TFEB aggravates decline of autophagy in the senescent heart. Targeting SUMO1 may provide a novel therapeutic strategy for the treatment of aging-related loss of cardioprotection.

Evaluation of 99mTc-Label led Vitamin K4 as an Agent for Testicular Imaging

1991 ◽  
Vol 30 (01) ◽  
pp. 35-39 ◽  
Author(s):  
H. S. Durak ◽  
M. Kitapgi ◽  
B. E. Caner ◽  
R. Senekowitsch ◽  
M. T. Ercan
Keyword(s):  
Soft Tissue ◽  
Room Temperature ◽  
Normal Subjects ◽  
Left Testis ◽  
Wide Range ◽  
Activity Ratios ◽  
The Right ◽  
Radioactivity Levels

Vitamin K4 was labelled with 99mTc with an efficiency higher than 97%. The compound was stable up to 24 h at room temperature, and its biodistribution in NMRI mice indicated its in vivo stability. Blood radioactivity levels were high over a wide range. 10% of the injected activity remained in blood after 24 h. Excretion was mostly via kidneys. Only the liver and kidneys concentrated appreciable amounts of radioactivity. Testis/soft tissue ratios were 1.4 and 1.57 at 6 and 24 h, respectively. Testis/blood ratios were lower than 1. In vitro studies with mouse blood indicated that 33.9 ±9.6% of the radioactivity was associated with RBCs; it was washed out almost completely with saline. Protein binding was 28.7 ±6.3% as determined by TCA precipitation. Blood clearance of 99mTc-l<4 in normal subjects showed a slow decrease of radioactivity, reaching a plateau after 16 h at 20% of the injected activity. In scintigraphic images in men the testes could be well visualized. The right/left testis ratio was 1.08 ±0.13. Testis/soft tissue and testis/blood activity ratios were highest at 3 h. These ratios were higher than those obtained with pertechnetate at 20 min post injection.99mTc-l<4 appears to be a promising radiopharmaceutical for the scintigraphic visualization of testes.

Increased Thromboxane Biosynthesis in Essential Thrombocythemia

1995 ◽  
Vol 74 (05) ◽  
pp. 1225-1230 ◽  
Author(s):  
Bianca Rocca ◽  
Giovanni Ciabattoni ◽  
Raffaele Tartaglione ◽  
Sergio Cortelazzo ◽  
Tiziano Barbui ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Activation ◽  
Essential Thrombocythemia ◽  
Therapeutic Strategy ◽  
Low Dose ◽  
Thrombotic Risk ◽  
Dose Aspirin ◽  
Gender And Age ◽  
Low Dose Aspirin

SummaryIn order to investigate the in vivo thromboxane (TX) biosynthesis in essential thromboeythemia (ET), we measured the urinary exeretion of the major enzymatic metabolites of TXB2, 11-dehydro-TXB2 and 2,3-dinor-TXB2 in 40 ET patients as well as in 26 gender- and age-matched controls. Urinary 11-dehydro-TXB2 was significantly higher (p <0.001) in thrombocythemic patients (4,063 ± 3,408 pg/mg creatinine; mean ± SD) than in controls (504 ± 267 pg/mg creatinine), with 34 patients (85%) having 11-dehydro-TXB2 >2 SD above the control mean. Patients with platelet number <1,000 × 109/1 (n = 25) had significantly higher (p <0.05) 11 -dehydro-TXB2 excretion than patients with higher platelet count (4,765 ± 3,870 pg/mg creatinine, n = 25, versus 2,279 ± 1,874 pg/mg creatinine, n = 15). Average excretion values of patients aging >55 was significantly higher than in the younger group (4,784 ± 3,948 pg/mg creatinine, n = 24, versus 2,405 ± 1,885 pg/mg creatinine, n = 16, p <0.05). Low-dose aspirin (50 mg/d for 7 days) largely suppressed 11-dehydro-TXB2 excretion in 7 thrombocythemic patients, thus suggesting that platelets were the main source of enhanced TXA2 biosynthesis. The platelet count-corrected 11-dehydro-TXB2 excretion was positively correlated with age (r = 0.325, n = 40, p <0.05) and inversely correlated with platelet count (r = -0.381, n = 40, p <0.05). In addition 11 out of 13 (85%) patients having increased count-corrected 11-dehydro-TXB2 excretion, belonged to the subgroup with age >55 and platelet count <1,000 × 1099/1. We conclude that in essential thrombocythemia: 1) enhanced 11-dehydro-TXB2 excretion largely reflects platelet activation in vivo;2) age as well as platelet count appear to influence the determinants of platelet activation in this setting, and can help in assessing the thrombotic risk and therapeutic strategy in individual patients.

Role of Methylglyoxal in Diabetic Cardiovascular and Kidney Diseases: Insights from Basic Science for Application into Clinical Practice

2018 ◽  
Vol 24 (26) ◽  
pp. 3072-3083 ◽  
Author(s):  
Sowndramalingam Sankaralingam ◽  
Angham Ibrahim ◽  
MD Mizanur Rahman ◽  
Ali H. Eid ◽  
Shankar Munusamy
Keyword(s):  
Therapeutic Strategy ◽  
Kidney Diseases ◽  
Vascular Dysfunction ◽  
Dicarbonyl Compound ◽  
Renal Complications ◽  
Lysine Residues ◽  
Patients With Diabetes ◽  
Prevalence Of Diabetes Mellitus

Background: The incidence and prevalence of diabetes mellitus are increasing globally at alarming rates. Cardiovascular and renal complications are the major cause of morbidity and mortality in patients with diabetes. Methylglyoxal (MG) - a highly reactive dicarbonyl compound – is increased in patients with diabetes and has been implicated to play a detrimental role in the etiology of cardiovascular and renal complications. Derived from glucose, MG binds to arginine and lysine residues in proteins, and the resultant end products serve as surrogate markers of MG generation in vivo. Under normal conditions, MG is detoxified by the enzyme glyoxalase 1 (Glo1), using reduced glutathione as a co-factor. Elevated levels of MG is known to cause endothelial and vascular dysfunction, oxidative stress and atherosclerosis; all of which are risk factors for cardiovascular diseases. Moreover, MG has also been shown to cause pathologic structural alterations and impair kidney function. Conversely, MG scavengers (such as N-acetylcysteine, aminoguanidine or metformin) or Nrf2/Glo1 activators (such as trans-resveratrol / hesperetin) are shown to be useful in preventing MG-induced cardiovascular and renal complications in diabetes. However, clinical evidence supporting the MG lowering properties of these agents are limited and hence, need further investigation. Conclusion: Reducing MG levels directly using scavengers or indirectly via activation of Nrf2/Glo1 may serve as a novel and potent therapeutic strategy to counter the deleterious effects of MG in diabetic complications.

Ethnomedicinal uses, Phytochemistry and Pharmacological Aspects of the Genus Berberis Linn: A Comprehensive Review

2020 ◽  
Vol 23 ◽  
Author(s):  
Roohi Mohi-ud-din ◽  
Reyaz Hassan Mir ◽  
Prince Ahad Mir ◽  
Saeema Farooq ◽  
Syed Naiem Raza ◽  
...  
Keyword(s):  
Chemical Constituents ◽  
Pharmacological Activities ◽  
Traditional Use ◽  
Comprehensive Review ◽  
Wide Range ◽  
Anti Cancer ◽  
Comprehensive Survey ◽  
Ethnomedicinal Uses

Background: Genus Berberis (family Berberidaceae), which contains about 650 species and 17 genera worldwide, has been used in folklore and various traditional medicine systems. Berberis Linn. is the most established group among genera with around 450-500 species across the world. This comprehensive review will not only help researchers for further evaluation but also provide substantial information for future exploitation of species to develop novel herbal formulations. Objective: The present review is focussed to summarize and collect the updated review of information of Genus Berberis species reported to date regarding their ethnomedicinal information, chemical constituents, traditional/folklore use, and reported pharmacological activities on more than 40 species of Berberis. Conclusion: A comprehensive survey of the literature reveals that various species of the genus possess various phytoconstituents mainly alkaloids, flavonoid based compounds isolated from different parts of a plant with a wide range of pharmacological activities. So far, many pharmacological activities like anti-cancer, anti-hyperlipidemic, hepatoprotective, immunomodulatory, anti-inflammatory both in vitro & in vivo and clinical study of different extracts/isolated compounds of different species of Berberis have been reported, proving their importance as a medicinal plant and claiming their traditional use.

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