In vivo self-assembled siRNA: a new modality for combination therapy of ulcerative colitis

Abstract Given the complex nature of ulcerative colitis (UC), combination therapy targeting multiple pathogenic genes and pathways of UC may be required. Unfortunately, current therapeutic strategies based on independent chemical compounds or monoclonal antibodies are not applicable for combination therapy of UC. Here, we developed a synthetic biology strategy that integrates the naturally existing exosome-circulating system with artificial genetic circuits for self-assembly and delivery of multiple siRNAs for the combination therapy of UC. Intravenous injection of genetic circuit (in the form of DNA plasmid) designed for inhibition of TNF-α, B7-1 and integrin α4 successfully reprogrammed the host liver to direct the self-assembly of TNF-α, B7-1 and integrin α4 siRNA into secretory exosomes. The multitargeted genetic circuit could rapidly relieved intestinal inflammation and exert a synergistic therapeutic effect against UC through suppressing the proinflammatory cascade in colonic macrophages, inhibiting the costimulatory signal to T cells and blocking T cell homing to sites of inflammation. More importantly, we designed an AAV-driven genetic circuit to induce substantial and lasting inhibition of TNF-α, B7-1 and integrin α4. Overall, this study established a feasible combination therapeutic strategy for UC, which is superior to the conventional biological therapies requiring.

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In vivo self-assembled small RNAs as a new generation of RNAi therapeutics

Cell Research ◽

10.1038/s41422-021-00491-z ◽

2021 ◽

Author(s):

Zheng Fu ◽

Xiang Zhang ◽

Xinyan Zhou ◽

Uzair Ur-Rehman ◽

Mengchao Yu ◽

...

Keyword(s):

Self Assembly ◽

Direct Injection ◽

Sirna Delivery ◽

Genetic Circuit ◽

Genetic Circuits ◽

Rnai Therapeutics ◽

Two Stages ◽

New Generation ◽

Rnai Therapy

AbstractRNAi therapy has undergone two stages of development, direct injection of synthetic siRNAs and delivery with artificial vehicles or conjugated ligands; both have not solved the problem of efficient in vivo siRNA delivery. Here, we present a proof-of-principle strategy that reprogrammes host liver with genetic circuits to direct the synthesis and self-assembly of siRNAs into secretory exosomes and facilitate the in vivo delivery of siRNAs through circulating exosomes. By combination of different genetic circuit modules, in vivo assembled siRNAs are systematically distributed to multiple tissues or targeted to specific tissues (e.g., brain), inducing potent target gene silencing in these tissues. The therapeutic value of our strategy is demonstrated by programmed silencing of critical targets associated with various diseases, including EGFR/KRAS in lung cancer, EGFR/TNC in glioblastoma and PTP1B in obesity. Overall, our strategy represents a next generation RNAi therapeutics, which makes RNAi therapy feasible.

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SULFASALAZINE ATTENUATES ULCERATIVE COLITIS IN RATS VIA DOWNREGULATION OF MIRNA-31, METALLOPROTEINASE-3 AND HIGH MOBILITY GROUP BOX1

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2021v13i8.41775 ◽

2021 ◽

pp. 74-80

Author(s):

SYLVIA A. BOSHRA

Keyword(s):

Ulcerative Colitis ◽

Inflammatory Mediators ◽

Intestinal Inflammation ◽

High Mobility ◽

Mucosal Healing ◽

High Mobility Group ◽

Albino Rats ◽

Tnf Α ◽

The Impact

Objective: This study targets the inhibition of inflammatory mediators and the enhancement of gastrointestinal mucosa healing in ulcerative colitis in rats through sulfasalazine. Methods: Twenty four female albino rats were divided into 3 groups: normal control, colitis group (rats received 5% dextran sodium sulfate (DSS) in their drinking water for 7 d), sulfasalazine group (500 mg/kg/day was administrated orally one week ahead of DSS and parallel with its administration). The impact of sulfasalazine on intestinal inflammation was investigated via estimation of some inflammatory mediators, namely; serum Leucine rich α 2 Glycoprotein (LRG) as well as colon cAMP, Myloperoxidase (MPO) and TNF-α using ELISA technique as well as gene expression of Trefoil Factor 3 (TFF3), High mobility group box1 (HMGB1), Nuclear factor kappa B (NF-κB) and metalloproteinase-3 (MMP3) and miRNA-31 levels using RT-PCR. Results: Sulfasalazine substantially decreases the release of LRG, MPO and TNF-α and the expression of HMGB1, NF-κB, MMP3, TFF3 and miRNA31 at p≤ 0.05 compared to colitis group in vivo. Moreover, Sulfasalazine significantly increases the colonic cAMP at p≤ 0.05 in groups of rats treated with DSS. Conclusion: Sulfasalazine has a protective effect on inflammatory bowel disease causing mucosal healing within the gastrointestinal tract. Additional studies are warranted to explore the molecular mechanism of sulfasalazine in ulcerative colitis and its clinical application.

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The serotonin reuptake transporter is reduced in the epithelium of active Crohn’s disease and ulcerative colitis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00244.2020 ◽

2020 ◽

Vol 319 (6) ◽

pp. G761-G768

Author(s):

Jonas Woll Jørandli ◽

Silje Thorsvik ◽

Helene Kolstad Skovdahl ◽

Benedikt Kornfeld ◽

Siri Sæterstad ◽

...

Keyword(s):

Ulcerative Colitis ◽

Crohn’S Disease ◽

Intestinal Epithelium ◽

Serotonin Reuptake ◽

Tryptophan Hydroxylase ◽

Intestinal Inflammation ◽

Serotonin Synthesis ◽

Crohn’S Colitis ◽

Tnf Α ◽

Serotonin Reuptake Transporter

The serotonin reuptake transporter is potently reduced in inflamed areas of Crohn’s ileitis, Crohn’s colitis, and ulcerative colitis. The changes are localized to the intestinal epithelium and can be induced by TNF-α. The serotonin synthesis through tryptophan hydroxylase 1 is unchanged. This regulation is suggested as a mechanism underlying the increased extracellular serotonin levels associated with intestinal inflammation.

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Evaluation of Anti-Colitis Effect of KM1608 and Biodistribution of Dehydrocostus Lactone in Mice Using Bioimaging Analysis

Plants ◽

10.3390/plants9091175 ◽

2020 ◽

Vol 9 (9) ◽

pp. 1175

Author(s):

Sullim Lee ◽

Sang-Back Kim ◽

Jaemin Lee ◽

Jimin Park ◽

Sungyoul Choi ◽

...

Keyword(s):

Zingiber Officinale ◽

Complex Nature ◽

Marker Compound ◽

Tnf Α ◽

Optical Fluorescence ◽

Inflammatory Bowel ◽

Dehydrocostus Lactone ◽

Natural Medicines

Inflammatory bowel disease (IBD) is a chronic relapsing disorder modulated by numerous factors. Recent failures of drugs targeting single factors suggest that multitargeting drugs could be useful for the treatment of IBD. Natural medicines may be an alternative option for the treatment of IBD, owing to the complex nature of the disease. However, most natural medicines have poor in vitro and in vivo translational potential because of inadequate pharmacokinetic study. KM1608, a mixture of the medicinal plants Aucklandia lappa, Terminalia chebula, and Zingiber officinale, was examined for its anti-colitis effects and biodistribution using bioimaging. Dehydrocostus lactone, as a marker compound, was analyzed to assess the biodistribution of KM1608. KM1608 significantly attenuated the disease activity of dextran sodium sulfate-induced colitis in mice and suppressed inflammatory mediators such as myeloperoxidase, proinflammatory cytokines (TNF-α and IL-6), and the Th2-type cytokine IL-4 in the colon. Optical fluorescence imaging revealed that KM1608 was distributed in the intestinal area as a target organ. Collectively, our findings suggest that KM1608 is a potential therapeutic formulation for IBD.

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IFN-γ and TNF-α decrease serotonin transporter function and expression in Caco2 cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00470.2006 ◽

2007 ◽

Vol 292 (3) ◽

pp. G779-G784 ◽

Cited By ~ 61

Author(s):

Kevin F. Foley ◽

Cristen Pantano ◽

Allison Ciolino ◽

Gary M. Mawe

Keyword(s):

Ulcerative Colitis ◽

Irritable Bowel Syndrome ◽

Conditioned Medium ◽

Human Lymphocytes ◽

Protein Levels ◽

Tnf Α ◽

Ifn Γ ◽

5 Ht Uptake ◽

Irritable Bowel

Recent studies have shown that mucosal serotonin (5-HT) transporter (SERT) expression is decreased in animal models of colitis, as well as in the colonic mucosa of humans with ulcerative colitis and irritable bowel syndrome. Altered SERT function or expression may underlie the altered motility, secretion, and sensation seen in these inflammatory gut disorders. In an effort to elucidate possible mediators of SERT downregulation, we treated cultured colonic epithelial cells (Caco2) with conditioned medium from activated human lymphocytes. Application of the conditioned medium caused a decrease in fluoxetine-sensitive [3H]5-HT uptake. Individual proinflammatory agents were then tested for their ability to affect uptake. Cells were treated for 48 or 72 h with PGE2 (10 μM), IFN-γ (500 ng/ml), TNF-α (50 ng/ml), IL-12 (50 ng/ml), or the nitric oxide-releasing agent S-nitrosoglutathione (GSNO; 100 μM). [3H]5-HT uptake was then measured. Neither PGE nor IL-12 had any effect on [3H]5-HT uptake, and GSNO increased uptake. However, after 3-day incubation, both TNF-α and IFN-γ elicited significant decreases in SERT function. Neither TNF-α nor IFN-γ were cytotoxic when used for this period of time and at these concentrations. These two cytokines also induced decreases in SERT mRNA and protein levels. By altering SERT expression, TNF-α and IFN-γ could contribute to the altered motility and expression seen in vivo in ulcerative colitis or irritable bowel syndrome.

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Carrier-free, self-assembled pure drug nanorods composed of 10-hydroxycamptothecin and chlorin e6 for combinatorial chemo-photodynamic antitumor therapy in vivo

Nanoscale ◽

10.1039/c7nr03129g ◽

2017 ◽

Vol 9 (38) ◽

pp. 14347-14356 ◽

Cited By ~ 49

Author(s):

Yan Wen ◽

Wei Zhang ◽

Ningqiang Gong ◽

Yi-Feng Wang ◽

Hong-Bo Guo ◽

...

Keyword(s):

Combination Therapy ◽

Self Assembly ◽

Chlorin E6 ◽

Antitumor Therapy ◽

Carrier Free ◽

Pure Drug ◽

Self Assembled

Carrier-free nanodrug HCPT/Ce6 NRs were prepared through a simple self-assembly approach for chemo-photodynamic combination therapy of tumors in vivo.

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Study on the mechanism of Indigo naturalis and its two main compounds (indigo and indirubin) in the treatment of ulcerative colitis based on TLR4/MyD88/NF-κB pathway and intestinal microorganisms

10.21203/rs.3.rs-31454/v1 ◽

2020 ◽

Author(s):

Qi-yue Yang ◽

Ya-nan He ◽

Le-le Ma ◽

Run-chun Xu ◽

Nan Li ◽

...

Keyword(s):

Ulcerative Colitis ◽

Gut Microbiota ◽

Intestinal Inflammation ◽

Activity Index ◽

Disease Activity Index ◽

Pathological Section ◽

Inflammatory Cytokine Production ◽

Tnf Α ◽

Indigo Naturalis ◽

Inflammation Cytokines

Abstract Background: Indigo naturalis is a natural dye extracted from plants and has a good anti-inflammatory effect. Clinical studies have shown that it can improve ulcerative colitis (UC), but the active constituents and the mechanism are unclear. Methods: The anti-UC activity of Indigo naturalis and its two main compounds (indigo and indirubin) were investigated in dextran sulfate sodium (DSS)-induced UC mice. Indigo naturalis, indigo and indirubin were administrated to DSS-induced UC rats by oral gavage for 1 weeks. The anti-UC effect was evaluated by pathological section, inflammatory cytokine production, western blotting, and gut microbiota analysis via 16S rRNA sequencing. Results: Indigo naturalis, indigo and indirubin can improve the UC induced by DSS. Their effect intensity is Indigo naturalis > indirubin > indigo based on disease activity index, body weight, colon length and pathological section. Indigo naturalis, indigo and indirubin also decrease the expression of NF-κB，TLR4 and MYD88 proteins, thus reducing the level of related inflammation cytokines (IL-1β, IL-6 and TNF-α) both in serum and tissue. In addition, Indigo naturalis and indigo improved symptoms of gut microbial disturbance, and decreased Firmicutes/Bacteroidetes ratio and the significantly increased probiotics such as Lactobacillus. Indirubin has little effect on the regulation of gut microbial. Conclusions: Indigo naturalis could attenuate the DSS-induced UC in mice, by means of ameliorating intestinal inflammation, improving intestinal mucosa, and regulating the disturbed gut microbiota. Indigo and indirubin could also attenuate the DSS-induced UC in mice, but their comprehensive effect is not as good as Indigo naturalis.

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Transcriptome profiling of ulcerative colitis mouse model suggests biomarkers and therapeutic targets for human colitis

10.1101/2020.08.12.225458 ◽

2020 ◽

Author(s):

Reza Yarani ◽

Oana Palasca ◽

Nadezhda T. Doncheva ◽

Christian Anthon ◽

Bartosz Pilecki ◽

...

Keyword(s):

Gene Expression ◽

Ulcerative Colitis ◽

Mouse Model ◽

High Throughput ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Unknown Etiology ◽

Small Rna Sequencing ◽

Complex Nature

1.AbstractBACKGROUND & AIMSUlcerative colitis (UC) is an inflammatory bowel disorder with unknown etiology. Given its complex nature, in vivo studies to investigate its pathophysiology is vital. Animal models play an important role in molecular profiling necessary to pinpoint mechanisms that contribute to human disease. Thus, we aim to identify common conserved gene expression signatures and differentially regulated pathways between human UC and a mouse model hereof, which can be used to identify UC patients from healthy individuals and to suggest novel treatment targets and biomarker candidates.METHODSTherefore, we performed high-throughput total and small RNA sequencing to comprehensively characterize the transcriptome landscape of the most widely used UC mouse model, the dextran sodium sulfate (DSS) model. We used this data in conjunction with publicly available human UC transcriptome data to compare gene expression profiles and pathways.RESULTSWe identified differentially regulated protein-coding genes, long non-coding RNAs and microRNAs from colon and blood of UC mice and further characterized the involved pathways and biological processes through which these genes may contribute to disease development and progression. By integrating human and mouse UC datasets, we suggest a set of 51 differentially regulated genes in UC colon and blood that may improve molecular phenotyping, aid in treatment decisions, drug discovery and the design of clinical trials.CONCLUSIONGlobal transcriptome analysis of the DSS-UC mouse model supports its use as an efficient high-throughput tool to discover new targets for therapeutic and diagnostic applications in human UC through identifying relationships between gene expression and disease phenotype.

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An Autonomous In Vivo Dual Selection Protocol for Boolean Genetic Circuits

Artificial Life ◽

10.1162/artl_a_00160 ◽

2015 ◽

Vol 21 (2) ◽

pp. 247-260 ◽

Cited By ~ 3

Author(s):

David Beneš ◽

Petr Sosík ◽

Alfonso Rodríguez-Patón

Keyword(s):

Rational Design ◽

Selection Process ◽

Logic Gates ◽

Genetic Circuit ◽

Evolutionary Engineering ◽

Genetic Circuits ◽

E Coli ◽

Search And Selection ◽

Selection Of

Success in synthetic biology depends on the efficient construction of robust genetic circuitry. However, even the direct engineering of the simplest genetic elements (switches, logic gates) is a challenge and involves intense lab work. As the complexity of biological circuits grows, it becomes more complicated and less fruitful to rely on the rational design paradigm, because it demands many time-consuming trial-and-error cycles. One of the reasons is the context-dependent behavior of small assembly parts (like BioBricks), which in a complex environment often interact in an unpredictable way. Therefore, the idea of evolutionary engineering (artificial directed in vivo evolution) based on screening and selection of randomized combinatorial genetic circuit libraries became popular. In this article we build on the so-called dual selection technique. We propose a plasmid-based framework using toxin-antitoxin pairs together with the relaxase conjugative protein, enabling an efficient autonomous in vivo evolutionary selection of simple Boolean circuits in bacteria (E. coli was chosen for demonstration). Unlike previously reported protocols, both on and off selection steps can run simultaneously in various cells in the same environment without human intervention; and good circuits not only survive the selection process but are also horizontally transferred by conjugation to the neighbor cells to accelerate the convergence rate of the selection process. Our directed evolution strategy combines a new dual selection method with fluorescence-based screening to increase the robustness of the technique against mutations. As there are more orthogonal toxin-antitoxin pairs in E. coli, the approach is likely to be scalable to more complex functions. In silico experiments based on empirical data confirm the high search and selection capability of the protocol.

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A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly In Vivo

10.1101/109306 ◽

2017 ◽

Author(s):

Khalid K. Alam ◽

Kwaku D. Tawiah ◽

Matthew F. Lichte ◽

David Porciani ◽

Donald H. Burke

Keyword(s):

Self Assembly ◽

Living Cells ◽

Rna Aptamers ◽

Genetic Circuits ◽

Fluorescent Reporter ◽

Indirect Measures ◽

Reporter Proteins ◽

Circuit Function

AbstractRNA-RNA assembly governs key biological processes and is a powerful tool for engineering synthetic genetic circuits. Characterizing RNA assembly in living cells often involves monitoring fluorescent reporter proteins, which are at best indirect measures of underlying RNA-RNA hybridization events and are subject to additional temporal and load constraints associated with translation and activation of reporter proteins. In contrast, RNA aptamers that sequester small molecule dyes and activate their fluorescence are increasingly utilized in genetically-encoded strategies to report on RNA-level events. Split-aptamer systems have been rationally designed to generate signal upon hybridization of two or more discrete RNA transcripts, but none directly function when expressed in vivo. We reasoned that the improved physiological properties of the Broccoli aptamer enable construction of a split-aptamer system that could function in living cells. Here we present the Split-Broccoli system, in which self-assembly is nucleated by a thermostable, three-way junction RNA architecture and fluorescence activation requires both strands. Functional assembly of the system approximately follows second order kinetics in vitro and improves when cotranscribed, rather than when assembled from purified components. Split-Broccoli fluorescence is digital in vivo and retains functional modularity when fused to RNAs that regulate circuit function through RNA-RNA hybridization, as demonstrated with an RNA Toehold switch. Split-Broccoli represents the first functional split-aptamer system to operate in vivo. It offers a genetically-encoded and nondestructive platform to monitor and exploit RNA-RNA hybridization, whether as an all-RNA, stand-alone AND gate or as a tool for monitoring assembly of RNA-RNA hybrids.

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