CRISPR based therapeutics: a new paradigm in cancer precision medicine.

BACKGROUND: Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems are the latest addition to the plethora of gene-editing tools. These systems have been repurposed from their natural counterparts by means of both guide RNA and Cas nuclease engineering. These RNA-guided systems offer greater programmability and multiplexing capacity than previous generation gene editing tools based on zinc finger nucleases and transcription activator like effector nucleases. CRISPR-Cas systems show great promise for individualization of cancer precision medicine. MAIN BODY: The biology of Cas nucleases and dead Cas based systems relevant for in vivo gene therapy applications has been discussed. The CRISPR knockout, CRISPR activation and CRISPR interference based genetic screens which offer opportunity to assess functions of thousands of genes in massively parallel assays have been also highlighted. Single and combinatorial gene knockout screens lead to identification of drug targets and synthetic lethal genetic interactions across different cancer phenotypes. There are different viral and non-viral (nanoformulation based) modalities that can carry CRISPR-Cas components to different target organs in vivo. CONCLUSION: The latest developments in the field in terms of optimization of performance of the CRISPR-Cas elements should fuel greater application of the latter in the realm of precision medicine. Lastly, how the already available knowledge can help in furtherance of use of CRISPR based tools in personalized medicine has been discussed.

Identification and Characterization of Circular Intronic RNAs Derived from Insulin Gene.

Circular RNAs (circRNAs) are a large family of noncoding RNAs that have emerged as novel regulators of gene expression. However, little is known about the function of circRNAs in pancreatic ß-cells. Here, transcriptomic analysis of mice pancreatic islet RNA-sequencing data identified 77 differentially expressed circRNAs between mice fed with a normal diet and a high-fat diet. Surprisingly, multiple circRNAs were derived from the intron 2 of the preproinsulin 2 (Ins2) gene and are termed as circular intronic (ci)-Ins2. The expression of ci-Ins2 transcripts in mouse pancreatic islets, and ßTC6 cells were confirmed by reverse transcription PCR, DNA sequencing, and RNase R treatment experiments. The level of ci-Ins2 was altered in ßTC6 cells upon exposure to elevated levels of palmitate and glucose. Computational analysis predicted the interaction of several RNA-binding proteins with ci-Ins2 and their flanking region, suggesting their role in the ci-Ins2 function or biogenesis. Additionally, bioinformatics analysis predicted the association of several microRNAs with ci-Ins2. Gene ontology and pathway analysis of genes targeted by miRNAs associated with ci-Ins2 suggested the regulation of several key biological processes. Together, our findings indicate that differential expression of circRNAs, especially ci-Ins2 transcripts, may regulate ß-cell function and may play a critical role in the development of diabetes.

Prolonged exposure to insulin might cause epigenetic alteration leading to insulin resistance.

Glucose homeostasis is maintained by insulin. Insulin resistance is caused by multiple factors including hereditary factors and diet. The molecular mechanism underlying insulin resistance (IR) is not completely understood. Hyperinsulinemia often precedes insulin resistance and Type 2 diabetes. We had previously shown that prolonged exposure of insulin-responsive cells to insulin in the absence of high levels of glucose led to insulin resistance. In the present study, we show that the underlying cause for the impaired insulin signalling is the defective PI3K/AKT pathway. The observed insulin resistance is likely due to epigenetic alterations, as it can be maintained for several generations even when insulin is not provided, and epigenetic modifiers can reverse it. We also show that liver cell line (BRL-3A) developed impaired insulin signalling upon prolonged exposure to insulin in the absence of high levels of glucose. Transcriptomic analysis of the insulin-sensitive and resistance cells uncover altered signalling networks involved in chromatin remodelling, Rho GTPases, and ubiquitination. Furthermore, trimethylation of histone H3 at lysine 4 (H3K4me3) is increased in insulin-resistant cells. We extended these studies to mice, and show that mice injected with low doses of insulin when fasting develop insulin resistance with impaired glucose tolerance and increased HOMA-IR index. Altogether, these findings suggest that dysregulated synthesis of insulin in the absence of glucose stimulus could lead to epigenetic alterations that may ultimately result in insulin resistance.

Hepatocyte-specific Epidermal Growth Factor Receptor Deletion Promotes Fibrosis but has no Effect on Steatosis in Fast-food Diet Model of Metabolic Dysfunction-associated Steatotic Liver Disease.

BACKGROUND & AIMS: Metabolic dysfunction-associated steatotic liver disease (MASLD) has become the most prevalent chronic liver disorder, with no approved treatment. Our previous work demonstrated the efficacy of a pan-ErbB inhibitor, Canertinib, in reducing steatosis and fibrosis in a murine fast-food diet (FFD) model of MASLD. The current study explores the effects of hepatocyte-specific ErbB1 (ie, epidermal growth factor receptor [EGFR]) deletion in the FFD model. METHODS: EGFRflox/flox mice, treated with AAV8-TBG-CRE to delete EGFR specifically in hepatocytes (EGFR-KO), were fed either a chow-diet or FFD for 2 or 5 months. RESULTS: Hepatocyte-specific EGFR deletion reduced serum triglyceride levels but did not prevent steatosis. Surprisingly, hepatic fibrosis was increased in EGFR-KO mice in the long-term study, which correlated with activation of transforming growth factor-ß/fibrosis signaling pathways. Further, nuclear levels of some of the major MASLD regulating transcription factors (SREBP1, PPARγ, PPARα, and HNF4α) were altered in FFD-fed EGFR-KO mice. Transcriptomic analysis revealed significant alteration of lipid metabolism pathways in EGFR-KO mice with changes in several relevant genes, including downregulation of fatty-acid synthase and induction of lipolysis gene, Pnpla2, without impacting overall steatosis. Interestingly, EGFR downstream signaling mediators, including AKT, remain activated in EGFR-KO mice, which correlated with increased activity pattern of other receptor tyrosine kinases, including ErbB3/MET, in transcriptomic analysis. Lastly, Canertinib treatment in EGFR-KO mice, which inhibits all ErbB receptors, successfully reduced steatosis, suggesting the compensatory roles of other ErbB receptors in supporting MASLD without EGFR. CONCLUSIONS: Hepatocyte-specific EGFR-KO did not impact steatosis, but enhanced fibrosis in the FFD model of MASLD. Gene networks associated with lipid metabolism were greatly altered in EGFR-KO, but phenotypic effects might be compensated by alternate signaling pathways.

Application of mass spectrometry based proteomics to understand diabetes: A special focus on interactomics.

Diabetes, a multifactorial disorder is characterized by elevated blood glucose levels resulting from changes in lifestyle, genetic and epigenetic changes or aberrations in proteome. In addition, alterations in post-translational modifications (PTMs) and protein-protein interactions (PPIs) also contribute to the development of diabetes pathogenesis. Recent advances in omics technologies have broadened the perspective for systematic investigation of proteome alterations in understanding the pathogenesis of diabetes. Further, PPIs are central to cellular signaling in all living organisms and deranged PPIs lead to diabetic complications. In this context, affinity purification mass spectrometry (AP-MS) along with diverse bioinformatic approaches has proven to be competent in mapping large-scale PPI networks around the critical players in the glucose homeostasis. In this review, we revisit the application of proteomic approaches in investigating proteome alterations and probing PPI networks for a better understanding of the underlying intricacies of the major signaling pathways in altered glucose homeostasis.