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Elevated fasting blood glucose (FBG) is associated with increased risks of developing type 2 diabetes (T2D) and cardiovascular-associated mortality. G6PC2 is predominantly expressed in islets, encodes a glucose-6-phosphatase catalytic subunit that converts glucose-6-phosphate (G6P) to glucose, and has been linked with variations in FBG in genome-wide association studies. Deletion of G6pc2 in mice has been shown to lower FBG without affecting fasting plasma insulin levels in vivo. At 5 mM glucose, pancreatic islets from G6pc2 knockout (KO) mice exhibit no glucose cycling, increased glycolytic flux, and enhanced glucose-stimulated insulin secretion (GSIS). However, the broader effects of G6pc2 KO on ß-cell metabolism and redox regulation are unknown. Here we used CRISPR/Cas9 gene editing and metabolic flux analysis in ßTC3 cells, a murine pancreatic ß-cell line, to examine the role of G6pc2 in regulating glycolytic and mitochondrial fluxes. We found that deletion of G6pc2 led to â¼60% increases in glycolytic and citric acid cycle (CAC) fluxes at both 5 and 11 mM glucose concentrations. Furthermore, intracellular insulin content and GSIS were enhanced by approximately two-fold, along with increased cytosolic redox potential and reductive carboxylation flux. Normalization of fluxes relative to net glucose uptake revealed upregulation in two NADPH-producing pathways in the CAC. These results demonstrate that G6pc2 regulates GSIS by modulating not only glycolysis but also, independently, citric acid cycle activity in ß-cells. Overall, our findings implicate G6PC2 as a potential therapeutic target for enhancing insulin secretion and lowering FBG, which could benefit individuals with prediabetes, T2D, and obesity.
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Diabetes Mellitus Tipo 2 , Glucose-6-Fosfatase , Glucose , Células Secretoras de Insulina , Animais , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Estudo de Associação Genômica Ampla , Glucose/metabolismo , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/enzimologia , Camundongos , Camundongos Knockout , OxirreduçãoRESUMO
The high potential of siRNAs to silence oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, divalent lipid-conjugated siRNAs are optimized for in situ binding to albumin to improve pharmacokinetics and tumor delivery. Systematic variation of the siRNA conjugate structure reveals that the location of the linker branching site dictates tendency toward albumin association versus self-assembly, while the lipid hydrophobicity and reversibility of albumin binding also contribute to siRNA intracellular delivery. The lead structure increases tumor siRNA accumulation 12-fold in orthotopic triple negative breast cancer (TNBC) tumors over the parent siRNA. This structure achieves approximately 80% silencing of the anti-apoptotic oncogene MCL1 and yields better survival outcomes in three TNBC models than an MCL-1 small molecule inhibitor. These studies provide new structure-function insights on siRNA-lipid conjugate structures that are intravenously injected, associate in situ with serum albumin, and improve pharmacokinetics and tumor treatment efficacy.
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Antineoplásicos , Neoplasias de Mama Triplo Negativas , Humanos , RNA Interferente Pequeno , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Linhagem Celular Tumoral , Inativação Gênica , Lipídeos/química , Albuminas/genéticaRESUMO
Fluorescent RNA-based biosensors are useful tools for real-time detection of molecules in living cells. These biosensors typically consist of a chromophore-binding aptamer and a target-binding aptamer, whereby the chromophore-binding aptamer is destabilized until a target is captured, which causes a conformational change to permit chromophore binding and an increase in fluorescence. The target-binding region is typically fabricated using known riboswitch motifs, which are already known to have target specificity and undergo structural changes upon binding. However, known riboswitches only exist for a limited number of molecules, significantly constraining biosensor design. To overcome this challenge, we designed a framework for producing mammalian cell-compatible biosensors using aptamers selected from a large random library by Capture-SELEX. As a proof-of-concept, we generated and characterized a fluorescent RNA biosensor against L-dopa, the precursor of several neurotransmitters. Overall, we suggest that this approach will have utility for generating RNA biosensors that can reliably detect custom targets in mammalian cells.
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Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Animais , RNA/química , Aptâmeros de Nucleotídeos/química , Técnica de Seleção de Aptâmeros , Biblioteca Gênica , Corantes , Mamíferos/genética , Mamíferos/metabolismoRESUMO
Glucose, a primary fuel source under homeostatic conditions, is transported into cells by membrane transporters such as glucose transporter 1 (GLUT1). Due to its essential role in maintaining energy homeostasis, dysregulation of GLUT1 expression and function can adversely affect many physiological processes in the body. This has implications in a wide range of disorders such as Alzheimer's disease (AD) and several types of cancers. However, the regulatory pathways that govern GLUT1 expression, which may be altered in these diseases, are poorly characterized. To gain insight into GLUT1 regulation, we performed an arrayed CRISPR knockout screen using Caco-2 cells as a model cell line. Using an automated high content immunostaining approach to quantify GLUT1 expression, we identified more than 300 genes whose removal led to GLUT1 downregulation. Many of these genes were enriched along signaling pathways associated with G-protein coupled receptors, particularly the rhodopsin-like family. Secondary hit validation confirmed that removal of select genes, or modulation of the activity of a corresponding protein, yielded changes in GLUT1 expression. Overall, this work provides a resource and framework for understanding GLUT1 regulation in health and disease.
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Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Glucose , Humanos , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Células CACO-2 , Glucose/metabolismo , Transporte BiológicoRESUMO
The high potential for therapeutic application of siRNAs to silence traditionally undruggable oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, siRNAs were optimized for in situ binding to albumin through C18 lipid modifications to improve pharmacokinetics and tumor delivery. Systematic variation of siRNA conjugates revealed a lead structure with divalent C18 lipids each linked through three repeats of hexaethylene glycol connected by phosphorothioate bonds. Importantly, we discovered that locating the branch site of the divalent lipid structure proximally (adjacent to the RNA) rather than at a more distal site (after the linker segment) promotes association with albumin, while minimizing self-assembly and lipoprotein association. Comparison to higher albumin affinity (diacid) lipid variants and siRNA directly conjugated to albumin underscored the importance of conjugate hydrophobicity and reversibility of albumin binding for siRNA delivery and bioactivity in tumors. The lead conjugate increased tumor siRNA accumulation 12-fold in orthotopic mouse models of triple negative breast cancer over the parent siRNA. When applied for silencing of the anti-apoptotic oncogene MCL-1, this structure achieved approximately 80% MCL1 silencing in orthotopic breast tumors. Furthermore, application of the lead conjugate structure to target MCL1 yielded better survival outcomes in three independent, orthotopic, triple negative breast cancer models than an MCL1 small molecule inhibitor. These studies provide new structure-function insights on optimally leveraging siRNA-lipid conjugate structures that associate in situ with plasma albumin for molecular-targeted cancer therapy.
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Introduction: Short interfering RNAs (siRNAs) are potent nucleic acid-based drugs designed to target disease driving genes that may otherwise be undruggable with small molecules. However, therapeutic potential of siRNA in vivo is limited by poor pharmacokinetic properties, including rapid renal clearance and nuclease degradation. Backpacking on natural carriers such as albumin, which is present at high concentration and has a long half-life in serum, is an effective way to modify pharmacokinetics of biologic drugs that otherwise have poor bioavailability. In this work, we sought to develop albumin-binding aptamer-siRNA chimeras to improve the bioavailability of siRNA. Methods: A Systematic Evolution of Ligands through Exponential Enrichment (SELEX) approach was used to obtain modified RNA-binding aptamers, which were then fused directly to siRNA via in vitro transcription. Molecular and pharmacokinetic properties of the aptamer-siRNA chimeras were subsequently measured in vitro and in vivo. Results: In vitro assays show that albumin-binding aptamers are stable in serum while maintaining potent gene knockdown capabilities in the chimera format. In vivo, the absolute circulation half-life of the best-performing aptamer-siRNA chimera (Clone 1) was 1.6-fold higher than a scrambled aptamer chimera control. Conclusions: Aptamer-siRNA chimeras exhibit improved bioavailability without compromising biological activity. Hence, this albumin-binding aptamer-siRNA chimera approach may be a promising strategy for drug delivery applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00718-y.
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When compartmentally mislocalized within cells, nucleic acids can be exceptionally immunostimulatory and can even trigger the immune-mediated elimination of cancer. Specifically, the accumulation of double-stranded DNA in the cytosol can efficiently promote antitumor immunity by activating the cGAMP synthase (cGAS) / stimulator of interferon genes (STING) cellular signaling pathway. Targeting this cytosolic DNA sensing pathway with interferon stimulatory DNA (ISD) is therefore an attractive immunotherapeutic strategy for the treatment of cancer. However, the therapeutic activity of ISD is limited by several drug delivery barriers, including susceptibility to deoxyribonuclease degradation, poor cellular uptake, and inefficient cytosolic delivery. Here, we describe the development of a nucleic acid immunotherapeutic, NanoISD, which overcomes critical delivery barriers that limit the activity of ISD and thereby promotes antitumor immunity through the pharmacological activation of cGAS at the forefront of the STING pathway. NanoISD is a nanoparticle formulation that has been engineered to confer deoxyribonuclease resistance, enhance cellular uptake, and promote endosomal escape of ISD into the cytosol, resulting in potent activation of the STING pathway via cGAS. NanoISD mediates the local production of proinflammatory cytokines via STING signaling. Accordingly, the intratumoral administration of NanoISD induces the infiltration of natural killer cells and T lymphocytes into murine tumors. The therapeutic efficacy of NanoISD is demonstrated in preclinical tumor models by attenuated tumor growth, prolonged survival, and an improved response to immune checkpoint blockade therapy.
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DNA , Sistemas de Liberação de Medicamentos , Nanopartículas , Nucleotidiltransferases , Animais , Feminino , Humanos , Camundongos , Neoplasias do Colo/terapia , Citocinas/biossíntese , Citocinas/genética , DNA/administração & dosagem , DNA/síntese química , DNA/farmacologia , DNA/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Endossomos/fisiologia , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Mamárias Experimentais/terapia , Melanoma Experimental/terapia , Proteínas de Membrana/fisiologia , Camundongos Endogâmicos C57BL , Nanopartículas/administração & dosagem , Nanopartículas/uso terapêutico , Neoplasias/imunologia , Nucleotidiltransferases/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Tionucleotídeos/farmacologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
INTRODUCTION: The generation of affinity reagents that bind native membrane proteins with high specificity remains challenging. Most in vitro selection paradigms utilize different cell types for positive and negative rounds of selection (where the positive selection is against a cell that expresses the desired membrane protein and the negative selection is against a cell that lacks the protein). However, this strategy can yield affinity reagents that bind unintended membrane proteins on the target cells. To address this issue, we developed a systematic evolution of ligands by exponential enrichment (SELEX) scheme that utilizes isogenic pairs of cells generated via CRISPR techniques. METHODS: Using a Caco-2 epithelial cell line with constitutive Cas9 expression, we knocked out the SLC2A1 gene (encoding the GLUT1 glucose transporter) via lipofection with synthetic gRNAs. Cell-SELEX rounds were carried out against wild-type and GLUT1-null cells using a single-strand DNA (ssDNA) library. Next-generation sequencing (NGS) was used to quantify enrichment of prospective binders to the wild-type cells. RESULTS: 10 rounds of cell-SELEX were conducted via simultaneous exposure of ssDNA pools to wild-type and GLUT1-null Caco-2 cells under continuous perfusion. The top binders identified from NGS were validated by flow cytometry and immunostaining for their specificity to the GLUT1 receptor. CONCLUSIONS: Our data indicate that highly specific aptamers can be isolated with a SELEX strategy that utilizes isogenic cell lines. This approach may be broadly useful for generating affinity reagents that selectively bind to membrane proteins in their native conformations on the cell surface.
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Drug delivery to the central nervous system (CNS) is generally hindered by the selectivity of the blood-brain barrier (BBB). However, there is strong evidence that the integrity of the BBB is compromised under certain pathological conditions, potentially providing a window to deliver drugs to injured brain regions. Recent studies suggest that caveolae-mediated transcytosis, a transport pathway suppressed in the healthy BBB, becomes elevated as an immediate response to ischemic stroke and at early stages of aging, where it may precede irreversible neurological damage. This article reviews early-stage caveolar transcytosis as a novel and promising drug delivery opportunity. We propose that albumin-binding and nanoparticle approaches have the potential to leverage this window of transcellular BBB disruption for trafficking therapeutic agents into the CNS.
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Cells make decisions and fate choices based in part on cues they receive from their external environment. Factors that affect the interpretation of these cues include the soluble proteins that are present at any given time, the cell surface receptors that are available to bind these proteins, and the relative affinities of the soluble proteins for their cognate receptors. Researchers have identified many of the biological motifs responsible for the high-affinity interactions between proteins and their receptors, and subsequently incorporated these motifs into biomaterials to elicit control over cell behavior. Common modes of control include localized sequestration of proteins to improve bioavailability and direct inhibition or activation of a receptor by an immobilized peptide or protein. However, naturally occurring biological motifs often possess promiscuous affinity for multiple proteins and receptors or lack programmable actuation in response to dynamic stimuli, thereby limiting the amount of control they can exert over cellular decisions. These natural motifs only represent a small fraction of the biological diversity that can be assayed by in vitro selection strategies, and the discovery of "artificial" motifs with varying affinity, specificity, and functionality could greatly expand the repertoire of engineered biomaterial properties. This minireview provides a brief summary of classical and emerging techniques in peptide phage display and nucleic acid aptamer selections and discusses prospective applications in the areas of cell adhesion, angiogenesis, neural regeneration, and immune modulation.